Bio Photonics - July 2018

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LA S ER S • OPTICS • IMAGIN G • SP E C T R O S CO PY • MICR O SCO PY

Volume 25 • Issue 5

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NEWS 8 • BIOSCAN

BioPhotonics editors curate the most significant headlines for photonics in the life sciences — and take you deeper inside the news. Featured stories include: • Microsensor could help individualize dialysis treatment • AO-LLSM microscope achieves aberration-free imaging • Light-based approach manages chronic neuropathic pain

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18 • RAPIDSCAN

• Luminate NY names Double Helix Optics top winner • UCI biophysicist receives $2M grant for insect control research

FEATURES 24 • DOUBLE-HELIX POINT SPREAD FUNCTION DELIVERS PRECISE EXTENDED-DEPTH MICROSCOPY

by Katie Heiser and Leslie Kimerling, Double Helix Optics LLC A simple modification allows researchers to extract more data in a single image, reducing the number of images needed to fully reconstruct a cell.

28 • LENSLESS CAMERAS MAY OFFER DETAILED IMAGING OF NEURAL CIRCUITRY 32

by Nick Antipa, Grace Kuo, and Laura Waller, University of California, Berkeley New architecture could enable simultaneous monitoring of millions of neurons in 3D space at frame rates limited only by image sensor read times.

32 • FLUORESCENCE MICROSCOPY UNRAVELS MORPHOGENESIS, FUNCTION OF LYMPH SYSTEM THE COVER 3D reconstruction of the nuclear lamina of a HeLa cell. Courtesy of A.K. Gustavsson et al. (2018), Nature Communications, Vol. 9, Issue 1. Cover design by Art Director Suzanne L. Schmidt.

by Marco Arrigoni, Coherent Inc. Researchers are targeting a comprehensive understanding of lymphatic vessels with potential implications for improved treatment.

36 • POINT-OF-CARE OPTICS HELPS HALT THE SPREAD OF INFECTIOUS DISEASES

by Marie Freebody, Contributing Editor Optical technologies offer an accurate, rapid, low-cost approach to diagnosing infectious diseases noninvasively.

COLUMNS AND DEPARTMENTS 5 • EDITORIAL PHOTONICS The technology of generating and harnessing light and other forms of radiant energy whose quantum unit is the photon. The range of applications of photonics extends from energy generation to detection to communications and information processing. BIOPHOTONICS The application of photonic products and techniques to solve problems for researchers, product developers, clinical users, physicians, and others in the fields of medicine, biology, and biotechnology.

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BioPhotonics • July/August 2018 BioPhotonics • February/March

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EDITORIAL

Deepening capabilities

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n our cover story this month, Katie Heiser and Leslie Kimerling of Double Helix Optics write about a device that integrates depth information with increased resolution. For such work, Double Helix was named a top winner of the first Luminate NY awards. The award comes with a $1 million investment and is sponsored by an accelerator program in Rochester, N.Y., designed to speed innovation and time to market. The company’s device modifies a microscope so it can extract more data with highprecision 3D information. The method behind it — the double-helix point spread function — already has found research applications in cell biology and neuroscience. The cover story, “Double-Helix Point Spread Function Delivers Precise ExtendedDepth Microscopy,” begins on page 24. It’s one of three this month that discuss technologies that enable tissue imaging at greater depths and with higher precision, opening the way for more complex understandings in the life sciences. Lensless 3D imaging is the topic of a report by Nick Antipa, Grace Kuo, and Laura Waller of UC Berkeley. The imager produced by Waller’s lab can compute the locations and brightness of a large number of points from a single 2D measurement. It makes it possible to record activity over a large area at sufficiently fast frame rates for imaging neural dynamics. “Lensless Cameras May Offer Detailed Imaging of Neural Circuitry” begins on page 28. The deep-imaging capability of two-photon laser scanning microscopy is outlined in a third story, from Coherent’s Marco Arrigoni. Coupled with the use of triple-transgenic mice, the technology has yielded some breakthrough results for the research team headed by Friedemann Kiefer at the European Institute for Molecular Imaging. The group seeks to sort out the poorly understood intricacies of the lymphatic system, with the goal of lessening surgical trauma. “Fluorescence Microscopy Unravels Morphogenesis, Function of Lymph System” begins on page 32.

BioPhotonics Editorial Advisory Board

Mark A. Anastasio, Ph.D. Professor of Biomedical Engineering Washington University in St. Louis

Stephen A. Boppart, M.D., Ph.D. Bliss Professor of Engineering Electrical and Computer Engineering, Bioengineering and Medicine Beckman Institute for Advanced Science and Technology University of Illinois at Urbana-Champaign

David Benaron, M.D. Professor, Medicine (consulting) Founder, Stanford Biomedical Optics program Stanford University School of Medicine CEO, Spectros Corp.

Aydogan Ozcan, Ph.D. Chancellor’s Professor, Electrical & Computer Engineering Department University of California, Los Angeles HHMI Professor, The Howard Hughes Medical Institute

Elsewhere in the magazine: • Contributing Editor Marie Freebody writes about the progress being made to develop point-of-care devices to diagnose infectious diseases such as pneumonia and tuberculosis. Both diseases remain formidable killers worldwide. Portable, noninvasive screening devices promise to save a lot of lives, particularly in low-resource regions where medical testing can be difficult to access or nonexistent. “Point-of-Care Optics Helps Halt the Spread of Infectious Diseases” begins on page 36. • For our Biopinion this month, Loren Looger of the Janelia Research Campus at the Howard Hughes Medical Institute outlines the advantages of open-source, sharing-driven technology development. This model has advanced life science, especially neuroscience, by leaps and bounds. There is much more government agencies, universities, and investigators can do, Looger writes, to foster ever-more open mechanisms of innovation and dissemination. “Open-source biophotonics is better for everyone” begins on page 6.

Adam Wax, Ph.D. Professor of Biomedical Engineering Duke University Founder and President, Lumedica

Enjoy the issue!

Marcia Stamell [email protected]


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BIOPINION Views on how to advance biophotonics

Open-source biophotonics is better for all BY LOREN LOOGER, HOWARD HUGHES MEDICAL INSTITUTE

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up the idea of nonprofit tool dissemination with colleagues at iophotonics has revolutionized many research fields, with the National Institutes of Health (NIH), they express concern direct effects on human well-being. Obviously, imaging about what a given member of Congress will think if we take requires microscopes and contrast agents. An open-source, jobs away from their state. Meanwhile, runaway reagent and sharing-driven model of technology development and disseminainstrument costs from companies are stressing the NIH budget. tion can foster discoveries at a faster rate than under the status Government agencies also could help address this funding quo. problem by creating a mechanism to disseminate reagents from For the past decade, the Janelia Research Campus at the HowNIH-funded tool-builders to NIH-funded biologists. Currently, ard Hughes Medical Institute (HHMI) has put enormous effort no large-scale, low-cost dissemination mechanism exists. What’s into the development of cutting-edge microscopes and contrast worse, funding skews toward for-profit entities at the expense agents — both are DNA-based tools that can be genetically of nonprofits. Small business innovation research (SBIR) grants delivered and small-molecule reagents that are physically added are relatively easy to get, but nonprofits must compete with to specimens. HHMI, and specifically Janelia, has made open academic entities for NIH Research Project Grants. Companies dissemination a backbone of its tool-development efforts. have their place, of course, but the notion that they are the only Thousands of packages of DNA-based, small-molecule tools or best engines of innovation and dissemination is outdated and have been sent at no cost around the world — often before self-serving. publication — and thousands more through nonprofit organizaUniversities and investigators play their part. Universities tions such as Addgene, based in Cambridge, Mass. Detailed encourage overpatenting to boost revenue and prestige. Investimicroscope plans are available on Janelia’s website, and building gators realize that patents look good on their CV during tenure workshops are held on-site to facilitate knowledge transfer. In reality, few institutions or The Advanced Imaging Center A sharing-driven model of review. people get rich from tool invention — at HHMI (a collaboration with the Gordon and Betty Moore Foundation) technology development has those that do frequently exploit loopsuch as cornering the market on hosts scientists for free to use best-inadvanced life science, particularly holes critical parts or chemicals, creating a class microscopes before commercialization. Janelia patents useful advances neuroscience, by leaps and bounds. monopoly. the intellectual properwhen it makes sense to facilitate Nothing accelerates tool uptake ty Meanwhile, landscape becomes ever-more clutdissemination, but other biophotonics like giving it away for free. tered, hindering innovation through tools simply go into the public domain. the implicit threat of litigation. Patent trolls, nuisance lawsuits, This open-source attitude has advanced life science, particularly and hedge funds make a bad situation worse. Universities and neuroscience, by leaps and bounds. And, from a selfish perspecinstitutes could explicitly reward their faculty and staff for the tive, nothing accelerates tool uptake — and with it name recogcreation and low-barrier dissemination of broadly enabling nition and citations, for example — like giving it away for free. technologies that empower discoveries. Instead of pushing proAt Janelia, we realize our great privilege to be here. But fessors to spin out startup companies, how about low-overhead, everyone can help. Microscopes are sold at high markup, often subsidized startup nonprofits in university incubator space? with proprietary file formats to discourage open-source software There are reasons for optimism. Tool development continues use and to silo customers into single-company loyalty. Smallat breakneck speed. Large-scale, open-source software efforts molecule and DNA-based reagents have historically been sold by are becoming mainstream in the field. Similar efforts in microscompanies at high-gross-margin markup (>95 percent), frequentcopy and reagents could boost innovation, stretch research budly with little reinvestment in research of improved products. gets, and create jobs worldwide. Molecular Probes, of Eugene, Ore., used to have an immense As discoveries pour in, companies could focus on tasks that small-molecule catalog, but a series of corporate takeovers and play to their large economies of scale, such as shepherding profit-wringing has shrunk this to only their cash cows. Many potential drugs through decades-long clinical trials, large-scale useful compounds are currently unavailable to researchers. production, distribution, and other hurdles. Sharing-driven Nonprofits such as Addgene are shaking up the landscape approaches could lead to a brighter future for all involved. of DNA-based reagents by stocking many more than companies have ever done, selling them at near cost, and expanding Meet the author services such as virus distribution. Small-molecule tools are not Loren Looger, Ph.D., is a group leader at the regenerable the way DNA is and require more infrastructure Janelia Research Campus at the Howard Hughes to create and disseminate. But academic research has led to Medical Institute in Ashburn, Va. His research improved reagents and better chemistry, allowing the synthesis areas include the design and optimization of of compounds at a tiny fraction of the price and at higher quality imaging reagents for all fields of biology; than is available from companies. The time is ripe for large-scale email: [email protected]. efforts to make and distribute useful small molecules at cost. Government agencies could offer remedies. When we bring

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Nick Antipa is a Ph.D. student at the University of California, Berkeley. He has a bachelor’s degree in optics from the University of California, Davis and a master’s in optics from the University of Rochester. He is currently researching the incorporation of advanced signal processing algorithms into optical imaging system design. Page 28. Marco Arrigoni is director of strategic marketing at Coherent Inc. His work covers the scientific research markets. Page 32. Regular contributing editor Marie Freebody is a freelance science and technology journalist with a master’s degree in physics, with a concentration in nuclear astrophysics, from the University of Surrey, England. Page 36. Katie Heiser, Ph.D., is a senior research scientist and applications specialist at Double Helix Optics LLC. She has a doctorate in molecular and cell biology with expertise in high-resolution fluorescence microscopy. Page 24. Leslie Kimerling is co-founder and CEO of Double Helix Optics LLC. She has led multiple technology startups from launch to growth. She has a master’s degree in economics from Stanford University and an MBA from the Anderson School of Business at the University of California, Los Angeles. Page 24. Grace Kuo is a Ph.D. student in the department of electrical engineering and computer science at the University of California, Berkeley. She has a bachelor’s degree in electrical engineering from Washington University in St. Louis. Page 28. Loren Looger, Ph.D., is a group leader at the Janelia Research Campus at the Howard Hughes Medical Institute. His research areas include the design and optimization of imaging reagents for all fields of biology. Page 6. Laura Waller, Ph.D., is head of the computational imaging lab at the University of California, Berkeley, which develops new methods for optical imaging. She holds the Ted Van Duzer Endowed Professorship and is a senior fellow at the Berkeley Institute of Data Science, with affiliations in bioengineering, applied sciences, and technology. Page 28.

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BIOSCAN A closer look at the most significant biophotonics research and technology headlines

Microsensor could help individualize dialysis treatment EINDHOVEN, Netherlands — A new microsensor could make it possible to directly monitor and adjust the composition of kidney dialysis fluid (dialysate) — an important step toward customizing dialysis to individual patients. The sensor provides a way to monitor the salt concentrations in dialysate so concentrations can be continuously adjusted. The ability to monitor and adjust salt levels in dialysate could lower side effects, such as heart rhythm disturbance and renal bone

disease, that can be caused by use of a standard dialysate. Researcher Manoj Kumar Sharma of the Eindhoven University of Technology developed a microsystem with a centrally positioned microchannel through which dialysate flows. He covered the walls of the microchannel with photoinduced electron transfer (PET) sensor molecules, which only fluoresce in the presence of a salt. The more salt there is in the dialysate, the stronger the fluorescence. To

Microscope image of the microfluidic device developed by Eindhoven University of Technology researcher Manoj Kumar Sharma. The horizontal stripe is the microchannel, which measures 0.2 mm across. The six other stripes are optic fibers that capture the fluorescent light and lead it to a spectrometer. The 15 dots in the middle are micropillars. Courtesy of Eindhoven University of Technology.

reinforce this effect, Sharma introduced micropillars into the microchannel to cover an even larger surface area with the sensor molecules. A laser light was shined on the microchannel, activating the fluorescence of the sensor molecules. The fluorescence is captured using glass fibers that are connected to the channel in the microsystem, and the light passes through the fibers to a spectrometer for analysis. The laser light, which is of a different wavelength than the fluorescence, is first filtered out. Then, based on the measured intensity of the fluorescence, the sodium concentrations can be read out. It is important to ensure the sensor molecules are not disturbed by other salts so that a pure measurement of the concentration of a specific type of salt (in this case, sodium) is possible. The microfluidic sensor system, which is approximately 5 × 2 cm, is now able to measure sodium accurately and live. Sharma expects he will be able to extend the microsystem with channel sections coated with other PET sensor molecules, which will be sensitive to the other essential salts, such as potassium and phosphate. The technique is relatively inexpensive, stable, and accurate. In addition, the size of the new sensor system could be further reduced to about 1 × 1 cm, facilitating integration into dialysis machines. The technique may also become part of a portable artificial kidney, a solution that could significantly ease the life of kidney disease patients.

AO-LLSM microscope achieves aberration-free imaging cellular environments, under as gentle illumination as possible, with minimal external perturbation. The microscope could create new opportunities to study the diversity of intracellular dynamics, extracellular communication, and collective cell behavior across different cell types, organisms, and developmental stages.

The microscope uses LLSM to sweep an ultrathin sheet of light through a volume of interest while acquiring a series of images for building a high-resolution 3D movie of the dynamics within the cell. The illumination is confined to a thin plane, ensuring regions outside the volume of interest remain unexposed. The parallel collection of fluorescence from

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ASHBURN, Va. — Scientists from Howard Hughes Medical Institute’s (HHMI) Janelia Research Campus have combined lattice light-sheet microscopy (LLSM) with adaptive optics (AO) to capture highresolution 3D movies of cells deep within living systems. The AO-LLSM microscope enables cells to be viewed in their native multi8

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Inside the spinal cord of a zebrafish embryo, new neurons light up in different colors, allowing scientists to track nerve circuit development. Courtesy of T. Liu et al./Science 2018.

across the plane permits low, less perturbative intensities to be used. An AO system maintains the thin illumination of a lattice light sheet as it penetrates within the cell, and a second AO system creates distortion-free images on the illuminated plane from above. By shining a laser through either of the AO system pathways, the researchers can create a bright point of light — a “guide star” — within the region they want to image. AO measures sample-induced distortions to the image of this fluorescent guide star created within the volume and compensates for these distortions by

changing the shape of a mirror to create an equal but opposite distortion. Over large volumes, the distortions change as the light traverses different tissues. Large 3D images can be assembled from a series of subvolumes, each with its own independent excitation and detection corrections. Using the microscopy method, the researchers studied a variety of subcellular events in vivo, including organelle remodeling during mitosis and growth cone dynamics during spinal cord development. Clear delineation of cell membranes allowed the researchers to isolate for indi-

vidual study any cell within the multicellular environment of the intact organism. By doing so, they could compare specific processes across different cell types. According to group leader Eric Betzig, the clarity level provided by the new system allows researchers to computationally disassemble cells in the tissue to focus on the dynamics within a single cell, such as the remodeling of internal organelles during cell division. This level of detail is difficult to see without adaptive optics, Betzig said. In his view, AO is one of the most important areas in microscopy research today, and the lattice light-sheet microscope, which excels at 3D live imaging, provides a suitable platform for its use. The next step for the researchers will be to make the technology affordable and user-friendly. They are working on a next-generation version that should fit on a small desk at a cost within reach of individual labs. The first such instrument will go to Janelia’s Advanced Imaging Center, where scientists from around the world could apply to use it. A guide for those who want to create their own copies of the AO-LLSM microscope will be made available. The research was published in Science (doi:10.1126/science.aaq1392).

Light-based approach manages chronic neuropathic pain ROME — A light-based method that has been tested on mice could be used to manage neuropathic pain. Researchers from the European Molecular Biology Laboratory (EMBL) Rome have identified a specific population of nerve cells in the skin that are responsible for sensitivity to gentle touch. These are also the cells that cause severe pain in neuropathic patients. The researchers developed a lightsensitive chemical that selectively binds to this type of nerve cell. By first injecting the affected skin area with the chemical and then illuminating it with nearinfrared light, the targeted nerve cells retract from the skin’s surface, leading to pain relief. By clipping off the nerve endings with

Hairy skin of a mouse, with the nerve cells responsible for sensitivity to gentle touch (darker green). The neurons are located around the hair follicles (lighter green). Courtesy of R. Dhandapani et al./Nature Communications.


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light, the gentle touch that can cause severe pain in neuropathic patients is no longer felt. The skin is only desensitized to the gentlest touch — other nerve cells in the skin are not affected by the light treatment. To test the technique, the researchers used laser ablation to administer the light therapy to mice affected by neuropathic pain in their limbs. Affected mice typically will quickly withdraw their paw when it is gently touched. After the light therapy, the mice exhibited normal reflexes upon gentle touch. The effect of the therapy lasted for a few weeks, after

which the nerve endings grew back and gentle touch again caused pain. The team also investigated human skin tissue. The overall makeup of the tissue and the specifics of the neurons of interest appeared to be similar to the mouse tissue, indicating that the method could be effective in managing neuropathic pain in humans. Previous attempts to develop drugs to treat neuropathic pain have focused on targeting single molecules. In contrast, the light-based technique targets the small subgroup of neurons causing neuropathic pain.

“We think that there’s not one single molecule responsible; there are many,” said EMBL group leader Paul Heppenstall. “You might be able to succeed in blocking one or a couple, but others would take over the same function eventually. With our new illumination method, we avoid this problem altogether.” The researchers are actively seeking partners to develop the method further, with the hope of one day using it in the EMBL clinic. The research was published in Nature Communications (doi:10.1038/s41467-01804049-3).

Lidar helps Yellowstone manage threatened ecosystem

The Montana State University lidar instrument aims a laser beam downward through a hole in the bottom of the plane. The beam is angled backward so the light’s reflection on the water is deflected away and does not saturate the receiver. Courtesy of Joseph A. Shaw, Montana State University.

technology at Yellowstone Lake in 2004. Data collected during those test flights helped the U.S. National Park Service find previously unknown spawning areas that were then validated with on-the-ground, gill-netting operations. A new system was then designed to provide sufficient optical power at the lowest possible cost. Tests of the new setup, conducted in 2015 and 2016, successfully identified numerous trout groupings. “The key problem we address with this research is the need for a method to find where the invasive lake trout spawn so fisheries biologists can deploy various methods of reducing their population,” said professor Joseph A. Shaw. “There are several other methods being explored for tracking these fish, including acoustic sensing, but an airplane can cover the large lake in a much shorter time than is possible for boats.” He added that the system could be further improved with a technique called pushbroom scanning, in which the laser beam is scanned in a line to cover a wider swath. This would allow scanning of the full lake area more quickly than the single fixed-angle laser used in the current setup. The Montana researchers plan to develop additional tools to help users rapidly translate lidar-generated data into actionable information and to adapt the system for other types of freshwater ecosystems. “We are interested in developing automated fish-detection algorithms and in using this method as a routine tool to help the fisheries biologists in their battle against invasive lake trout,” Shaw said.

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BOZEMAN, Mont. — Lidar could offer a fast, efficient way to locate and capture lake trout, an invasive nonnative fish that is upending the ecosystem in Yellowstone Lake. Thanks to aircraft-mounted lidar, lake managers may be able to hunt for invasive fish across a wider area at lower cost, making more efficient use of the approximately $2 million spent on lake trout control each year. A series of test flights performed by researchers at Montana State University was successful in locating groups of two or more lake trout swimming as deep as 15 m below the surface. The lidar instrument, bolted to a small aircraft, is able to


detect fish in a 5-m swath of water and cover 80 km per hour. The device works by transmitting a short pulse of laser light from the airplane through the air and into the water. The lidar receiver measures backscattered light, which allowed the researchers to pick out fish from the surrounding water. To optimize the setup for use on the lake, they used a green beam laser to penetrate water better than other types of lasers used for lidar applications on the ground. The beam was angled backward so the light’s reflection on the water would be deflected away and not saturate the receiver. The team first tested the use of lidar

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“We also are exploring options for using this lidar, along with multispectral and hyperspectral imaging systems, to monitor river health.” Lidar has been used to track fish in marine ecosystems, but this is the first time it has been used to study fish in lakes, where the water is cloudier. Lake trout prey upon the lake’s native cutthroat trout, which have historically been a key food source for many top predators. This has substantially reduced the food supply for bears, birds, and other animals that cannot prey upon the invasive fish because, unlike the cutthroat trout, lake trout spend most of the year in deep water. Montana State University engineers developed the new instrument for less than $100,000 and optimized it for operation on a single-engine airplane that can be flown for $500 per day. This is a practical solution for ecologists as well as local fishery and water resource managers. The research was published in Applied Optics, a publication of The Optical Society (OSA) (doi:10.1364/AO.57.004111).

Michael Roddewig, a former doctoral student at Montana State University, sets up the lidar in a plane for a flight over Yellowstone Lake. Courtesy of Joseph A. Shaw, Montana State University.

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/ Germany • www.lasos.com


7/9/2018 2:54:40 PM

NIH researchers merge microscopy techniques for faster images BETHESDA, Md. — To create sharper images faster, researchers at the National Institute of Biomedical Imaging and Bioengineering (NIBIB) have blended instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM). The technique — instant TIRF-SIM — allows the researchers to observe rapidly moving objects about 10 times faster than other microscopes at similar resolution. TIRFM has been used in cell biology for decades, but it produces blurry images of small features within cells. In the past, superresolution microscopy techniques have been applied to TIRF microscopes to improve image resolution. However, such attempts have compromised speed, making it difficult to use TIRFM to clearly image rapidly moving objects. To resolve this issue, the researchers modified an iSIM microscope to perform as a TIRF microscope. Developed by the NIBIB lab in 2013, iSIM (a high-speed, superresolution microscope) can capture video at 100 frames per second — more than three times faster than most movies or internet videos. However, iSIM does not have the contrast capabilities TIRF microscopes do. The NIBIB team designed a simple mask that blocked most of the illumination from the iSIM, causing it to mimic the contrast features of a TIRF microscope. This step led to instant TIRF-SIM, a technique that improved the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, enabling frame rates up to 100 Hz over hundreds of time points. The researchers applied instant TIRFSIM to multiple live samples, subsequently achieving rapid, high-contrast super-

The rapid movements of Rab11 particles can be clearly imaged with the new instant TIRF-SIM microscope. Courtesy of Hari Shroff, National Institute of Biomedical Imaging and Bioengineering.

resolution imaging close to the coverslip surface. For example, the researchers were able to follow rapidly moving Rab11 particles near the plasma membrane of human cells. Attached to molecular cargo that are transported around the cell, these particles move so fast that they are blurred when imaged by other microscopes. “Our method improves the spatial resolution of TIRF microscopy without

compromising speed — something that no other microscope can do,” said Hari Shroff, lab chief of NIBIB’s Section on High Resolution Optical Imaging. “We hope it helps us clarify high-speed biology that might otherwise be hidden or blurred by other microscopes,” he said, “so that we can better understand how biological processes work.” The research was published in Nature Methods (doi:10.1038/s41592-018-0004-4).

Optogenetic tool controls voltage-gated calcium channels COLLEGE STATION, Texas — An optogenetic tool, developed by researchers at Texas A&M University, could control voltage-gated calcium (CaV ) channels to enable phototunable modulation of CaV channel activity in excitable mammalian cells. The tool, called optoRGK, provides a way to interrogate the physiological and pathophysiological processes that are mediated by CaV channels. BioPhotonics • July/August 2018


Such channels constitute the major route of calcium entry into a cell and regulate a series of physiological processes. Traditional calcium-channel blockers, widely used to treat cardiovascular disorders, can cause cytotoxicity and off-target effects. The researchers combined genetic strategies with optical techniques to engineer a new class of genetically encoded

inhibitors for CaV channels. They tested this photoswitchable inhibitor in cardiac muscle cells. In the dark, the muscle cells showed rhythmic oscillations of calcium that matched the heart-beating rhythm. “However, upon blue-light illumination, the rhythmic oscillations can be substantially reduced or even terminated,” said Yubin Zhou, an associate

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professor at the Texas A&M Institute of Biosciences and Technology. “Notably, this process is totally reversible after removal of the light source.”

Using this method, the researchers can regulate the activity of excitable cells in the nervous and cardiovascular systems. “The optoRGK toolkit provides a

Voltage-gated Ca2+ channels (CaV) are important therapeutic targets for cardiovascular and neuropsychiatric disorders. Texas A&M University researchers engineered a class of genetically encoded photoswitchable inhibitors for CaV channels to control Ca2+ signals (yellow graphs in round bubbles) and biological activities in excitable cells. The optoRGK can be adapted to suppress cardiac arrhythmia (asterisks), thereby intervening in atrial fibrillation and other cardiovascular disorders by light. Courtesy of Yubin Zhou, Texas A&M University Health Science Center.

unique opportunity to switch off calcium signals in excitable cells,” said researcher Youjun Wang from Beijing Normal University, which collaborated on the study. “Our optogenetic tools can be conveniently applied to control a wide range of physiological processes mediated by voltage-gated calcium channels in multiple biological systems. While traditional voltage-gated calcium-channel blockers lack reversibility, selectivity, and tissue-specificity, optoRGK opens exciting opportunities to intervene in related physiological processes with unprecedented precision,” Zhou said. “We hope that these kinds of studies will eventually lead to a new generation of optogenetic devices for curing cancer and cardiovascular and neurological diseases.” The research was published in Angewandte Chemie (doi:10.1002/ anie.201713080).

Graphene biointerface optically controls heart cells SANTA BARBARA, Calif. — Researchers have developed a technique that allows them to speed up or slow down human heart cells on command by shining a light on the cells and varying the light intensity. The optical stimulation platform does not require genetic modification of cells but instead makes use of the optoelectronic properties of graphene, specifically its ability to efficiently convert light into electricity. Researchers at the University of California San Diego School of Medicine and their collaborators generated heart cells from donated skin cells via an induced pluripotent stem cell (iPSC). These iPSC-derived heart cells were grown on a graphene surface. The researchers found the efficiency of stimulation via the graphene biointerface (G-biointerface) was independent of light wavelength but could be tuned by changing the light intensity. “We were surprised at the degree of flexibility that graphene allows you to pace cells literally at will,” said researcher Alex Savchenko. “You want them to

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beat twice as fast? No problem — you just increase the light intensity. Three times faster? No problem — increase the light or graphene density.” The researchers demonstrated that an all-optical evaluation of use-dependent drug effects in vitro could be enabled using substrate-based G-biointerfaces. Mexiletine, a medication used to treat arrhythmias, was added to the heart cells. Mexiletine is known for being use-dependent; it only has an effect when there is an increase in heart rate. The researchers illuminated the heart cells on graphene with light of different intensities. The faster the heart cells beat, the better the mexiletine inhibited them. Using dispersible G-biointerfaces in vivo, the team performed optical modulation of the heart activity in zebrafish embryos. The cells in the lab grew better on graphene than other on materials, such as glass or plastic. The cells grown on graphene behaved more like cells in the body. They also observed an absence of toxicity as a result of introducing a new material (graphene) to the process.

“It makes us hopeful that we’ll be able to avoid harmful problems later on, as we test various medical applications,” Savchenko said. The graphene/light system could empower numerous fundamental and translational biomedical studies. Potential applications could include testing therapeutic drugs in more biologically relevant systems, developing use-specific drugs, and creating better medical devices. The researchers may eventually apply the new graphene/light system to the search for drugs that specifically kill cancer cells, while leaving healthy cells alone. The researchers also envision using graphene to find opioid alternatives — use-dependent pain medications that would only work when and where a person is in pain, thus reducing systemic effects that could lead to misuse. Lightcontrolled pacemakers could be made of graphene, according to Savchenko, which could be safer and more effective than current models. The research was published in Science Advances (doi:10.1126/sciadv.aat0351).


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Triboluminescence used to test for drug efficacy WEST LAFAYETTE, Ind. — A new instrument, developed by Purdue University researchers, uses triboluminescence (TL) to enable selective detection of trace crystallinity within amorphous solid dispersions. It can be used in the preparation of pharmaceutical formulations to make the drug more efficacious. The instrument can accurately detect in the early stages of drug development whether a pharmaceutical formulation has trace crystalline content that could negatively affect the drug’s stability and bioavailability. The TL instrument measures the light that is emitted when a pharmaceutical powder is crushed. The light that is measured is in direct proportion to the quantity of crystallinity in the pharmaceutical formulation. Crystallinity at levels as low as 140 ppm can be detected. To do this, pharmaceutical powder is placed on a microscope slide and an electric charge is applied to the powder using a solenoid. A photomultiplier tube positioned beneath the slide measures the


optical radiation resulting from the triboluminescence of the powder. The slide is mechanically moved down the line so that new areas of the powder can be probed. “This technique would be more like a

The front side of the triboluminescence instrument developed by Purdue University researchers to crush pharmaceutical formulations to test for trace crystallinity. The red plastic holds a solenoid that strikes the formulation. A motor moves the microscope slide (black box) down the line. A photomultiplier tube beneath the slide measures the optical radiation resulting from the triboluminescence of the compound. Courtesy of Purdue University.

prescreen off an assembly line in a factory where they’re making these drugs,” said researcher Scott Griffin. “They can send a small amount of the material into this instrument for triboluminescence measurements. If they get a positive outcome from the sample, then they can send it off for more rigorous testing.” Other ways to determine crystallinity

The back side of the instrument for testing trace crystallinity in pharmaceutical formulations. The main circuit board consists of a high-power voltage regulator to power the striking solenoid. Courtesy of Purdue University.

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include a second-harmonic generation process that uses a femtosecond pulsed laser, also developed at Purdue. The TL instrument is simpler, according to the researchers. “It boils down to the earliest time point that crystallinity can be detected,” said professor Garth Simpson. “We wanted to have something that would be a simple yes or no assessment that could be done on site. If something fails, it can be taken to a more advanced instrument to get a better sense of quantitative characterization.”

Simpson said the TL instrument also could be used to determine whether changes in the way a drug is produced could cause crystallinity. The researchers are working on a flow cell that will enable similar crystallinity testing in slurries, and they’re exploring what fraction of drug molecules and compounds could be responsive to TL analysis. “New drugs that are coming out are increasingly larger and more hydrophobic, or do not dissolve easily in water,”

Simpson said. “If crystallinity is detected, there’s a good chance that it won’t dissolve in a time frame required to be bioavailable and efficacious.” The researchers have obtained a provisional patent on the device, which is available for licensing from the Office of Technology and Commercialization at Purdue. The research was published in Analytical Chemistry (doi:10.1021/acs. analchem.8b01112).

QCL-based IR microscopy performs rapid cancer diagnosis BOCHUM, Germany — Researchers at Ruhr-Universität Bochum (RUB) have deployed an IR microscope with quantum cascade lasers (QCLs), replacing Fourier transform (FT) with QCL technology. By simplifying the measurement setup through the use of a QCL, the team reduced the time required for analysis from one day to a few minutes. Along with bioinformatical image analysis, the QCL-based IR microscope can perform label-free classification of cancer tissue and can be fully automated. IR imaging has been shown to be a reliable method for tissue classification.

However, the Fourier transform infrared (FTIR) microscopy technique that has been used to date takes a full day to analyze samples. The time required for analysis has hampered the use of IR imaging in clinical settings. QCL-based IR microscopes, in contrast to FTIR microscopes, allow the use of a single frequency. Thereby, within very short measuring times, an overview image can be obtained for selecting the region of interest. It can then be analyzed in detail. The team used QCL-based IR imaging to analyze 110 tissue samples taken

from colorectal cancer patients. The results showed 96 percent sensitivity and 100 percent specificity for this label-free method, as compared to histopathology, which is considered the gold standard in routine clinical diagnostics. “We have … reduced the measurement period by a factor of 160,” said researcher Frederik Großerüschkamp. As a control, the measurements were carried out using two different pieces of equipment, and the analyses were performed by several users with no effect on results. “The method is now very fast and reliable and does not depend on a specific device or a specific user,” said researcher Angela Kallenbach-Thieltges. “This opens up new avenues for automated classification of tissue samples taken directly from the patient.” As a next step, larger studies of unmet clinical needs will be addressed. The researchers believe this could propel IRbased, label-free, automated tissue classification into clinical routines. The new approach could also be used for biomarker research. The RUB team’s work demonstrates that IR imaging is a nondestructive, labelfree, and now rapid technique for tissue classification and biomarker research. “The results of the study give rise to hope that highly precise therapy is within reach,” said researcher Klaus Gerwert, which “will ultimately prove more successful than traditional approaches.” The research was published in Scientific Reports (doi:10.1038/s41598-01826098-w).

Researchers from Ruhr-Universität Bochum (RUB) have developed new methods of cancer diagnostics. Pictured (from left) are Claus Kuepper, Frederik Großerüschkamp, Angela Kallenbach-Thieltges, and Klaus Gerwert. Courtesy of RUB, Marquard.

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7/9/2018 3:11:34 PM

RAPIDSCAN Business and Markets Luminate NY names Double Helix Optics top winner Double Helix Optics was announced as the winner in the first round of the Luminate NY competition. The Boulder, Colo.-based company will receive $1 million in follow-on funding from New York state through the Finger Lakes Forward Upstate Revitalization Initiative. As the award stipulates, Double Helix will commit to establishing operations in Rochester, N.Y., for at least the next 18 months. Double Helix Optics was awarded first place for its SPINDLE, a light engineering technology for integrating depth information with increased resolution into real-world applications. SPINDLE is a 3D nanoimaging module that seamlessly integrates with existing microscopes, cameras, and other optical instruments to turn 2D imaging into 3D information capture. Intelon Optics Inc. was the secondplace winner, receiving $500,000 for its Brillouin Optical Scanner System (BOSS), which uses laser light to measure the stiffness of tissues inside the body. The first commercial and clinical focus is in ophthalmology.

Positive Science was named third-place winner, securing $250,000 for its headmounted eye-tracking and behavioral analysis systems. Fourth place was awarded to Think Biosolution, which received $250,000 for its wearable devices and software plat-

forms, which use QuasaR technology to measure heart rate, respiratory rate, blood oxygen saturation, heart rate variability, temperature, movement, and location. A panel of judges from the optics, photonics, and imaging (OPI) industry and venture capitalist community scored

UCI biophysicist receives $2M grant for insect control research Todd Holmes, professor of physiology and biophysics at the University of California, Irvine (UCI), has been awarded a competitive five-year $2.1 million Outstanding Investigator Award/Maximizing Investigators’ Research Award (MIRA) R35 grant from the National Institute of

General Medical Sciences. It is the first MIRA grant awarded to a UCI investigator. Holmes will use the funding to examine how insect phototransduction can be used to design better light-based insect control strategies.

UCI biophysicist Todd C. Holmes’ research work builds on his team’s recent discovery of two additional ways that insects detect light. Courtesy of Steve Zylius/UCI Strategic Communications.

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Current insect control devices use UV light to attract insects to an electric grid or trap. In contrast to toxic insecticides, which cause considerable health and environmental harm, light-based insect control is very appealing because of its safety and low environmental impact. So far, the discovery science has been conducted in Drosophila fruit flies, the most useful insect model organism for laboratory molecular genetics. Holmes credits the development of CRISPR gene editing as the technology that will enable him to conduct rigorous molecular genetic science on mosquitoes. In recent years, cases of vector-borne diseases, including West Nile, Zika, chikungunya, and dengue fever, have grown explosively in the Western Hemisphere and the U.S. More than 700,000 deaths annually have been reported and account for more than 17 percent of all infectious diseases worldwide. The National Institute of General Medical Sciences is among the U.S. National Institutes of Health, supporting research for disease diagnosis, treatment, and prevention. BioPhotonics • July/August 2018

7/9/2018 3:10:14 PM

Larson named MSA fellow Luminate NY candidates based on their business pitches. The more than 500 attendees at the event also had an opportunity to vote for their favorite company. The Audience Choice award for $10,000 went to Molecular Glasses Inc. for its creation of a new class of materials capable of reducing power consumption in mobile applications while also providing higher display resolution and enabling lower-cost manufacturing processes. Luminate NY, which is administered by NextCorps, is the world’s largest business accelerator for startup firms in the OPI industries. Located in Rochester, it selects 10 promising companies each year and provides comprehensive training and resources to advance their technologies and businesses. Applications are being accepted for Round II of the Luminate 2018 competition now through Sept. 24. Companies earning one of the 10 available slots in the next cohort will receive a minimum investment of $100,000 and major investments of up to $1 million.

David J. Larson, director of scientific marketing for CAMECA Instruments Inc., has been appointed a fellow of the Microscopy Society of America (MSA). The MSA is a nonprofit organization dedicated to the promotion and advancement of techniques and applications of microscopy and microanalysis in all relevant scientific disciplines. Fellows have been conferred the MSA’s Distinguished Scientist Award and are senior distinguished members of the organization, making significant contributions to the advancement of microscopy and microanalysis through a combination of scientific achievements and service. The appointment is restricted annually to 0.5 percent of total MSA membership. Larson was selected Courtesy of CAMECA Instruments Inc. for his “pioneering contributions to the development of atom probe science and technology, especially its application to complex materials systems, and for his many contributions to the Society,” according to the MSA. Larson has a Ph.D. from the University of Wisconsin and is currently the president of the International Field Emission Society. Prior to joining CAMECA, he held staff positions at Seagate Technology and Oak Ridge National Laboratory and was a U.S. National Science Foundation International Research fellow at the University of Oxford. CAMECA is a devleoper and manufacturer of scientific instruments for material micro- and nanoanalysis.

Nuclear medicine, molecular imaging industry group forms

UA engineer inducted into AIMBE

The Society of Nuclear Medicine and Molecular Imaging (SNMMI) Value Initiative Industry Alliance has been established to connect corporate members to the future of nuclear medicine and molecular imaging, and to relevant stakeholders. Value Initiative Industry Alliance corporate members aim to help shape the field’s future by providing funding support, strategic industry guidance, and collaborative knowledge-sharing. The group is led by an advisory committee, which is co-chaired by James Williams, head of Siemens Healthineers Molecular Imaging, and Jonathan Allis, CEO of Blue Earth Diagnostics. The alliance will help implement SNMMI’s Value Initiative, which provides the vision for and forms the basis of the society’s new strategic plan. It focuses on five key domains: quality of practice, R&D, workforce pipeline, advocacy, and outreach. BioPhotonics • July/August 2018


Matthew Becker, who holds the W. Gerald Austen Endowed Chair of Polymer Science and Polymer Engineering at the University of Akron (UA), was inducted into the College of Fellows by the American Institute for Medical and Biological Engineering (AIMBE). The College comprises the top 2 percent of medical and biological engineers in the U.S. for their significant contributions to the field. Becker was honored for developing families of degradable polymers for use in additive manufacturing, regenerative medicine, and drug delivery. He is a widely recognized scholar and innovator in organic polymer chemistry and biomaterials. Becker was elected by peers and members into the College of Fellows, which is among the highest professional distincCourtesy of the University of Akron. tions accorded to medical and biological engineers. Two degradable polymer platforms from Becker’s lab, developed for regenerative medicine and 3D printing, were recently exclusively licensed by 21MedTech from the University of Akron Research Foundation. At its onset, the deal represents one of the most significant licensing arrangements by UA in its 148-year history. Becker’s pioneering research at UA into America’s deadly opioid epidemic was awarded $2 million from the state of Ohio’s Third Frontier Commission. UA is working with 21MedTech and pharmaceutical company Merck & Co. Inc. to commercialize a nonopiate degradable polymer mesh designed to control postsurgical pain, which will help patients avoid potential addiction to pain pills.

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Cellmic patent aims to grow Leti portfolio Leti, a research institute at CEA Tech, and Cellmic LLC, a company that leverages patient health care with smartphones and biophotonics, have joined forces to accelerate the market adoption of lens-free imaging and sensing techniques by growing Leti’s patent portfolio with a core patent from Cellmic. Pioneered by Aydogan Ozcan, who is Chancellor’s Professor at the University of California, Los Angeles and co-founder of Cellmic, and his research group, this patented computational lens-free imaging approach reconstructs detailed images of specimens from their holographic shadows that contain unique 3D information of samples such as tissue sections, blood smears, and cell cultures. Cellmic LLC, a UCLA spinoff, holds some of the core patents of this computational imaging technique. Lens-free microscopy has emerged as a powerful imaging and sensing platform, replacing bulky and expensive optical components found in standard optical microscopy systems with dedicated algorithms. Leti developed a lens-free microscope in 2012. Today, the technology offers an ultrawide field of view, tracking more than 10,000 biological, microscopic objects at a time per image. It provides lab technicians with a cost-effective, highly compact, robust solution. The Cellmic patent complements Leti’s IP portfolio and accelerates ongoing valorization of its lens-free technology for diagnostics, biomedical sensing, and related applications. Leti specializes in miniaturization technologies that enable smart, energy-efficient, secure solutions for industry.

Zebra Medical announces algorithm approval Health care imaging analytics developer Zebra Medical Vision has announced CE (European Conformity) regulatory approval of a new algorithm to be included in its deep learning imaging analytics platform. The algorithm, capable of detecting suspected malignant lesions in mammography scans, is the latest addition to other automated tools announced in the past as part of its artificial intelligence (AI) imaging business model. Others include algorithms that automatically detect brain bleeds, vertebral fractures, coronary artery disease, osteoporosis, and more. The company provides a state-of-theart malignancy detection product. The first version to be released supports 2D hologic devices, and Zebra Medical Vision expects to add support for vendors and 3D support during the course of 2019. The algorithm broadens the AI1 (All-InOne) Imaging Analytics package, which has already analyzed over one million scans in more than five countries. Zebra Medical Vision uses deep learning to develop next-generation products and services for the health care industry.

Lensar system receives FDA clearance Ocular laser developer Lensar Inc. has been granted 510(k) clearance from the U.S. FDA for its LENSAR laser system with Streamline IV, expanding the platform’s capabilities to include the creation of the corneal pockets and flaps used in ophthalmic procedures treating presbyopia. With the new indications, the LENSAR laser system now supports surgeons offering the latest presbyopic inlay devices to patients struggling with the loss of near-vision caused by aging. The presbyopia procedure features of the LENSAR laser system with Streamline IV include a new curved contact patient interface device that enables the creation of corneal pockets and flaps without compromising patient comfort.

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7/9/2018 3:10:19 PM

KARL STORZ launches blue light bladder cancer treatment Following the approval of a supplemental new drug application and premarket approval supplement from the U.S. Food and Drug Administration, KARL STORZ Endoscopy-America Inc. has launched the Photodynamic Diagnosis (PDD) Blue Light Flexible Video Cystoscopy system for enhanced detection of nonmuscle invasive bladder cancer (NMIBC). The FDA approval extends the indication for Blue Light Cystoscopy with Cysview (BLCC) to include use of the new KARL STORZ PDD video cystoscope. It also includes an expanded indication for the repetitive use of Cysview within the same patient and the identification of carcinoma in situ, one of the most challenging types of bladder cancer to detect. These new indications greatly increase the treatment possibilities of such therapy, which has rapidly become the standard of care for bladder cancer

The Photodynamic Diagnosis (PDD) Blue Light Flexible Video Cystoscopy completes the two-part system, which also includes rigid blue light cystoscopy. Blue Light Cystoscopy with Cysview enables cancerous tumors to fluoresce in a bright pink color, improving tumor visibility and enhancing florescence-guided resection. Courtesy of KARL STORZ.

treatment at major medical institutions across the U.S. Bladder cancer affects more than 708,000 patients in the U.S. Treatment is difficult because NMIBC tumors can look similar to normal healthy tissue and can be missed or incompletely removed.

$12.6B

— projected size of the global medical lasers market by 2023, according to a report published by Allied Market Research Five years ago, our July/August issue highlighted the fiber optics market and the role fiber optics plays in medical device manufacturing. As diagnostic and treatment technologies evolve and increasingly turn to light, medical device manufacturers increasingly turn to fiber optic components and systems to deliver that light to tissues and organs. A feature article, “Fiber Optics’ Versatility Helps Market Grow,” focused on different companies that reported continued growth in the biomedical market, as fiber optics can take various forms for a range of biomedical applications. It is this versatility that gives fiber optics a huge advantage. As new and better imaging methods are born, photonics-based technologies are moving in on the market.

2013

‘The market is only going to get bigger. New technologies will inspire applications we can’t even imagine right now, and new diagnostic challenges will drive the market to continue to innovate. Also, I believe you’ll see much more specificity in diagnostics sensing as fiber optic systems evolve.’ — Rob Morris, marketing operations manager at Ocean Optics in Dunedin, Fla.



Patients have a high probability of cancer recurrence, and in 2018 it is estimated that there will be 17,240 patient deaths caused by bladder cancer. The PDD video cystoscopy completes the two-part system, which also includes rigid blue light cystoscopy. The flexible and rigid systems are designed to improve the visualization and resection of deadly NMIBC tumors. The new system expands upon the rigid platform, enabling comfortable, anesthesia-free examinations to confirm suspected lesions from a previous cystoscopy and for ongoing monitoring of NMIBC. The FDA approval is based on results of a large Phase III study using the KARL STORZ blue-light-enabled rigid, flexible cystoscopes and blue light video system. KARL STORZ Endoscopy-America Inc. is an affiliate of KARL STORZ SE & Co. KG, a developer of reusable endoscope technology.

Certara acquires Analytica Laser Drug development consultant company Certara LP has acquired Analytica Laser, a consultancy for medicine and health technology. Analytica Laser employs quantitative methodologies and proprietary software to study and predict realworld outcomes for drug-value assessment. Leveraging evidence-based practices, the company integrates analytics, advanced pharmacoepidemiology, pharmaco-economics, modeling and data science expertise, public health intelligence, and extensive experience to advise on pricing and market access strategy. Certara aims to optimize drug development and improve health outcomes. Analytica Laser seeks to provide high-level scientific evidence of real-world value of medicines and health technologies.

$3B

— projected size of the global optical imaging market by 2024, according to MarketWatch Inc. 21

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SAEHi deploys Biotricity’s Bioflux mobile cardiac monitor The San Antonio Endovascular & Heart Institute (SAEHi) — a practice specializing in diagnosing, treating, managing, and researching diseases and disorders of the heart and vascular system — has deployed the Bioflux mobile cardiac telemetry solution from health care technology company Biotricity Inc. SAEHi will leverage the real-time, high-precision mobile device to assist in the diagnosis of cardiac arrhythmias, en-

hance patient outcomes, improve patient compliance, and curb health care costs. The Bioflux system is designed to be a complete solution for cardiac monitoring and diagnosis. It consists of the Bioflux device, proprietary software, and a 24/7 monitoring center that merges with physicians’ existing platforms and workflows. Unlike traditional cardiac monitoring solutions, Bioflux extends the support a patient receives at a care facility into the

Nanopositioning systems developer Physik Instrumente GmbH & Co. KG (PI) has named Thomas Bocher marketing segment head for microscopy and life sciences. Bocher previously held product and segment manager positions with Carl Zeiss Microscopy GmbH and Bruker BioSpin GmbH. He also served as chairman of the ISO/TC172/SC5 committee for international standardization in the fields of light microscopy and endoscopy. PI is a manufacturer of piezo systems, hexapods, and instrumentation for precision motion control.

Brad Reynolds has joined LED technology developer CoolLED Ltd., and he will be responsible for field sales management across the East Coast of the U.S. He comes to CoolLED after years of service in sales with an Olympus distributor, a Leica distributor, and as direct sales representative for Carl Zeiss and Leica Microsystems in New England. He received a bachelor’s degree in biology with a focus on marine ecology from the University of Connecticut. CoolLED designs and manufactures LED illumination systems for researchers and clinicians using the latest LED technology.

Courtesy of CoolLED Ltd.

Courtesy of Bruker BioSpin GmbH.

Health monitoring technology provider Raytelligence has added applied machine learning researcher Jens Lundström to its team. Lundström will work on the company’s eHealth offering. He has a doctorate in information technology with a focus on artificial intelligence for health technology. Previously, he was an assistant professor at Halmstad University in Sweden. Raytelligence offers sensor and cloud solutions for the monitoring of respiration, heart rate, positioning, and motion.

Courtesy of Raytelligence.

PEOPLE IN THE NEWS

Baby sleep monitor developer Nanit secures $14M in funding Nanit, a smart baby monitor developer that uses computer vision technology to help baby sleep development, has secured $14 million in Series B financing led by Jerusalem Venture Partners (JVP). With additional participation from existing investors Upfront Ventures, RRE Ventures, Vulcan Capital, and Vaal Investment Partners, this latest round of funding brings the company’s financing total to nearly $30 million. It will be used to expand its team of world-class computer vision and machine learning engineers and scale production to meet growing retail demand domestically and abroad. Nanit combines computer vision, machine learning, and advanced camera sensors to measure a baby’s sleep cycle by providing actionable insights that lead to improved sleep for the entire family. 22


patient’s home. The device monitors a patient’s electrocardiogram in near real time, constantly analyzing and collecting data on the device and periodically uploading to the cloud via embedded cellular technology. “Bioflux helps health care professionals like SAEHi leverage advanced technology to transmit data as it happens,” said Bartlomiej Gora, clinical research coordinator at the Institute. “This not only facilitates data analysis and integration into our workflow, but also allows the patient to be diagnosed at home and to feel safer. At the end of the day, we are improving our patient-consumer outcomes because Bioflux provides actionable data and feedback.” Biotricity is a medical technology company focused on delivering remote biometric monitoring solutions to the medical and consumer markets.

PicoQuant opens Chinese app center Optoelectronics developer PicoQuant GmbH has opened Application Center China, a showroom for time-resolved fluorescence test measurement technology based in China’s Optical Valley. The new facility will serve potential customers all over the country, providing the opportunity to perform test measurements with their own samples on PicoQuant’s time-resolved fluorescence spectrometers and microscopes. The center is equipped with the fully automated, high-end, modular FluoTime 300 spectrometer featuring the most currently available accessories. In the near future, the MicroTime 200 time-resolved, singlemolecule-sensitive confocal fluorescence microscopy platform will also be available for customer test measurements. PicoQuant’s product portfolio encompasses picosecond-pulsed diode lasers and LEDs, photon counting instrumentation, fluorescence lifetime spectrometers, FLIM and FCS upgrade kits for laser scanning microscopes, and time-resolved confocal and superresolution microscopes.


7/9/2018 3:10:25 PM

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Double-Helix Point Spread Function Delivers Precise Extended-Depth Microscopy A simple modification allows researchers to extract more data in a single image, reducing the images needed to fully reconstruct a cell.

BY KATIE HEISER AND LESLIE KIMERLING, DOUBLE HELIX OPTICS LLC

L

Figure 1. Superresolution image of microtubules in a Cos7 cell. Conventional 2D reconstruction (simulated, left) double helix 3D reconstruction (right). Depth encoded in color, scale at bottom. Top panel scale bars = 10 μm; bottom panel scale bars = 2 μm. Courtesy of Double Helix Optics LLC.

ing or damage to the sample. Recent developments in the field have pushed these limitations. A major development is superresolution imaging. This enables researchers to break the diffraction barrier and visualize subcellular structures well below the conventional 200-nm limit1. The highest resolution methods are a family of techniques known as single-molecule localization microscopy (SMLM)1. Although SMLM enables high-precision imaging of 10 to 20 nm in the lateral dimension, it typically lacks axial (Z) resolution, especially near focus. One method to extract axial information uses an astigmatic lens to distort the point spread function, enabling extraction of a limited amount of 3D information. Unfortunately, this approach has a depth capability of about 800 nm. Alternative methods developed to obtain highprecision 3D images, such as iPALM and multifocal imaging, require custombuilt microscopes, optics, and software that may not be accessible to most researchers2. An alternative technique, known as the double-helix point spread function (DH-PSF) offers a solution to this problem by enabling high-depth, highprecision 3D imaging. The DH-PSF modifies the point spread function on the microscope such that, instead of an Airy disc, the image of each point source is in the form of two well-separated lobes. These rotate around their midpoint as the emitter is moved along the axial dimension. The axial dimension of the emitter is encoded in the angle of the two lobes. The center point between them indicates the lateral position3. With this simple modification of the microscope, researchers can extract more

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ight microscopy is a powerful method used in many scientific disciplines, including the life sciences, to visualize finer details in samples. The use of fluorescence enables specific targeting

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and labeling of biomolecules and other chemicals. However, in light microscopy there are trade-offs between the time it takes to acquire an image, the resolution of the resulting image, and photobleach-

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With the addition of high-precision axial information and extended depth of field, researchers can obtain more data with more accurate information to directly measure 3D movement.

data with high-precision, 3D information compared to 2D SMLM (Figure 1). The increased depth of the DH-PSF captures more microtubules with high-precision axial information over a larger volume than conventional 2D SMLM. By enabling extraction of more data in a single image, the technique reduces the number of images required to fully reconstruct a cell, while improving the resolution of the image. Using this powerful method, Jain and colleagues were able to describe core structures in RNA stress granules for the first time4. Constituted of RNA and protein, stress granules are subcellular aggregates associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (also known as frontotemporal dementia) (Figure 2a, 2b). Based on biochemical studies, the researchers hypothesized that the stress

Figure 2. Stress granules imaged by superresolution microscopy. Stress granules (green) imaged by structured illumination microscopy in a cell (a). Zoomed inset of in vivo stress granule (b). Biochemical isolate showing smaller substructures (c). 3D models of double helix superresolution imaging showing stress granules (gray) with dense core structures of protein, GFP-G3BP (green, d) and poly(A+) RNA (yellow, e). All scale bars = 0.5 μm. Images reprinted with permission from reference 4.

Figure 3. Human serum albumin (HSA) hops on a fused silica (FS) surface. Representative trajectories for HSA on a modified FS surface; X-, Y-, and Z-axis are 2000 nm (a, b). The insets show the corresponding trace of Z position versus time. Reprinted with permission from reference 6.



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3D Microscopy

APPLIED SCIENTIFIC I N S T R U M E N TAT I O N

Figure 4. TILT3D combines light sheet microscopy, localization microscopy, and light engineering. Single-molecule (SM) localization with double helix (DH) and light sheet (a). Conventional wide-field imaging set up with tetrapod for drift correction and tracking (b). 3D reconstruction of the nuclear lamina of a HeLa cell, depth encoded in color, scale shown on the right (c). PSF: point spread function; LS: light sheet; Epi: Epi-illumination. Scale bars = 3 µm in (a) and (b); 5 µm in (c). Images reprinted with permission from reference 7.

WE CREATE SOLUTIONS Ultra Precise Motion Control: DC Servomotors down to 20 nm, Piezos down to 1 nm. Microscopy: Automation, Modular Microscopes, Complete Light Sheet Systems, and components. OEM: Custom designed systems to user specifications. VISIT US AT: The 3rd EMBO Practical Course on Light Sheet Microscopy Aug. 2nd - 11th • Dresden, Germany The 10th Annual LSFM Conference Aug. 12th - 15th • Dresden, Germany www.asiimaging.com • [email protected] (800) 706-2284 or (541) 461-8181

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granules may contain dense core structures (Figure 2c). However, they were unable to easily visualize or measure the stress granule cores in conventional 2D superresolution imaging. Using double helix light engineering, they were able to visualize, identify, and quantify these core structures in three dimensions (Figure 2d, 2e). Core structures These quantifications demonstrated for the first time that stress granules contain smaller dense core structures in vivo. Identifying the core structures helped researchers modify their prevailing hypothesis on how stress granules form, dissemble, and relate to pathological aggregates. These principles of improved depth and resolution can aid researchers in a range of biomedical and materials applications. In the case of single-particle tracking, using the DH-PSF method improves the depth over which a particle can be tracked, enables 3D measurements, and

improves the duration over which a particle can be observed. Traditional methods of particle tracking, such as total internal reflection fluorescence (TIRF) microscopy have very limited axial depth. Consequently, particles diffusing vertically are rapidly lost to defocus and can therefore be tracked only for a limited amount of time. Additionally, particles in biological and biochemical systems moving in three dimensions means diffusion coefficients can be estimated from the 2D data but not accurately measured. With the addition of high-precision axial information and extended depth of field, researchers can obtain more data with more accurate information to directly measure 3D movement. Wang, Agrawal, and colleagues used DH-PSF technology to improve the signal-to-noise ratio in measurements of chemical interface interactions5. Using the DH-PSF, the researchers were able to track the movement of biomolecules on liquid interfaces6. It was hypothesized based on 2D tracking data that biomolBioPhotonics • July/August 2018

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ecules take hops in the direction perpendicular to the interface before binding. However, these 3D hops had not been visualized or quantified. Wang, Wu, and colleagues demonstrated for the first time that fast 3D molecular hops occurred and, in some cases, flights through the liquid phase were followed by binding at the solid surface (Figure 3)6. In this case, the addition of high-precision axial information was vital to observation of these hopping and flight events. Developments in light With high-precision 3D information, the researchers were able to visualize and measure the effects of long-range electrostatic interactions on biomolecule dynamics. This kinetic information demonstrates how proteins move in different biochemical environments and aid in the development of more efficient pharmaceutical purification processes. Another recent development in light microscopy is the use of planar illumination light, called light sheet or selective plane illumination microscopy (SPIM). Unlike traditional illumination methods, a light sheet illuminates a thin section of the sample that is orthogonal to the detection path. The light sheet reduces background, increases the signal-to-noise ratio, and reduces photobleaching of the sample outside the light sheet. However, conventional light sheet methods do not allow imaging with highnumerical-aperture (NA) objectives close to the coverslip. These high-NA objectives are required for high-resolution, single-molecule imaging methods such as SMLM. To combine the powerful methods of light sheet and high-precision 3D SMLM, Gustavsson and colleagues created a technique called TILT3D7. TILT3D uses a tilted light sheet created with a glass prism to image cells close to the coverslip. The setup alleviates the need to have two high-NA objectives in close proximity or submerged in the sample media. It allows for the typical benefits of light sheet imaging, including reduced photobleaching, and is inexpensive to implement. However, this setup results in an angled illumination plane and a thicker (2-µm) light sheet. The extended depth of DHPSF enables molecule localization over this thicker illumination volume (Figure 4a). This enables precise localization of the molecules within the angled volume BioPhotonics • July/August 2018


The increased depth of the DH-PSF captures more microtubules with high-precision axial information over a larger volume than conventional 2D SMLM. and accurate reconstruction of the axial dimension. The addition of the DH-PSF for high-precision 3D localization enabled extremely high-depth, high-precision reconstructions with axial stitching. Gustavsson and colleagues also used a second modified point spread function with a very large depth range called the tetrapod PSF. A fiducial marker was imaged with the tetrapod to correct for drift and provide an accurate measurement of the axial position of the sample for axial stitching (Figure 4b). With this optical setup, the researchers were able to image and reconstruct structures throughout whole mammalian cells, such as mitochondria and the nuclear lamina (Figure 4c). By combining light sheet, SMLM, DH-PSF, and the tetrapod PSF, Gustavsson and colleagues created a powerful method with improved 3D spatial resolution, a large axial volume, and reduced photobleaching. This method will enable researchers to more easily visualize subcellular structures throughout the entire cell volume with very high precision. DH-PSF can be implemented on a variety of microscopes, cameras, and experimental setups. Researchers can accomplish this by using a simple add-on module, such as the SPINDLE, which was designed to integrate with commercially available scientific microscopes and cameras. It works by inserting a phase mask chosen from the library of masks optimized for various axial ranges, emission spectra, and applications. For 3D localization microscopy and 3D particle tracking, the SPINDLE can be combined with localization software such as an ImageJ plugin 3DTRAX. This flexibility allows use of existing setups to perform high-precision, high-depth, 3D measurements without expensive modifications. Meet the authors Katie Heiser, Ph.D., is a senior research scientist and applications specialist at Double Helix Optics LLC. She has a doctorate in molecular and cell biology with expertise in high-resolution fluorescence microscopy; email: [email protected]. Leslie Kimerling is co-founder and CEO of Double Helix Optics LLC, a 3D imaging

company based in Boulder, Colo. A serial entrepreneur, she has led multiple technology startups from launch through growth. She holds a master’s degree in economics from Stanford University and an MBA from the Anderson School of Business at UCLA; email: [email protected].

Acknowledgments

Some of this material is based upon work supported by the National Science Foundation under Grant #IIP-1059286 to the American Society for Engineering Education. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the American Society for Engineering Education. The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core. Microscopy was performed on a Nikon Ti-E microscope supported by the Howard Hughes Medical Institute.

References

1. M. J. Rust et al. (2006). Sub-diffractionlimit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods, Vol. 3, Issue 10, pp. 793–796. 2. G. Shtengel et al. (2009). Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc Natl Acad Sci, Vol. 106, Issue 9, pp. 3125–3130. 3. S. R. P. Pavani and R. Piestun (2008). High-efficiency rotating point spread functions. Opt Express, Vol. 16, Issue 5, pp. 3484–3489. 4. S. Jain et al. (2016). ATPase-modulated stress granules contain a diverse proteome and substructure. Cell, Vol. 164, Issue 3, pp. 487–498. 5. D. Wang, A. Agrawal, et al. (2017). Enhanced information content for three-dimensional localization and tracking using the double-helix point spread function with variable-angle illumination epifluorescence microscopy. Appl Phys Lett, Vol. 110, p. 211107. 6. D. Wang, H. Wu, et al. (2017). Threedimensional tracking of interfacial hopping diffusion. Phys Rev Lett, Vol. 119, p. 268001. 7. A. K. Gustavsson et al. (2018). 3D singlemolecule super-resolution microscopy with a tilted light sheet. Nat Commun, Vol. 9, Issue 1, p. 123.

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Lensless Cameras May Offer Detailed Imaging of Neural Circuitry New architecture could enable simultaneous monitoring of millions of neurons in 3D space at frame rates limited only by image sensor read times. BY NICK ANTIPA, GRACE KUO, AND LAURA WALLER, UNIVERSITY OF CALIFORNIA, BERKELEY

U

nderstanding brain function at the scale of individual neurons could open the door for advanced understanding of animal and human behavior and the mechanisms of neurological disorders such as Alzheimer’s. But the size scale of neurons and their extremely large numbers create an extreme imaging challenge. Calcium imaging enables optical monitoring of the action potential of individual neurons, resulting in maps of neural circuitry in living brains. To achieve this, calcium-sensitive fluorescent indicators are introduced into the neurons of a live animal. The indicators change configuration when they bind to calcium, which is released by voltage-gated channels on the cell membrane when a neuron activates. This causes the fluorescent indicators to light up temporarily when the neuron fires. Recording many optical images per second results in a complete view of the calcium levels within individual neurons, providing an in-depth look at the electrical activity.

The speed and scale requirements of imaging neural activity brainwide presents significant challenges for designing optical devices and algorithms. Mammalian brains have millions to billions of neurons that are tens of micrometers in size and distributed over a relatively large volume. A mouse brain, for example, is approximately 1 cm in diameter and can contain up to 75 million neurons. Until recently, neuroscientists have relied on techniques that either image a small region of the brain at high resolution1,2 or monitor the entire brain with resolution significantly less detailed than neuron scale, such as through functional magnetic resonance imaging (fMRI)3. Furthermore, for an animal to move freely while being monitored, the optical devices need to be small enough to implant without excessively encumbering the animal’s movement. To image large numbers of neurons, the devices must have a very large field of view (FOV). The systems also should be able to distinguish neurons that are axially aligned but at

distinct depths. Using coded aperture imaging techniques, a new class of lensless computational imaging systems may enable compact, implantable systems capable of optically monitoring extremely large numbers of neurons in vivo. Light-field microscopes using specialized algorithms have been used for fast 3D neural activity monitoring4. However, these large benchtop microscopes are difficult to miniaturize and implant while achieving whole-brain FOV5. For example, a 2.5×, 0.08-NA objective can image a 10-mm mouse brain with 4-µm resolution. But such an objective is typically more than 30 mm in diameter and 40 mm long — much too bulky to attach to a freely moving mouse. Some strides have been made in miniaturizing objectives by using gradient index lenses6. These systems have an FOV of much less than a millimeter — far from a whole-brain scale. For certain experiments, this may not be a limitation. However, for implanting into living animals, traditional lens-based approaches

Figure 1. The DiffuserCam lensless imaging architecture consists of a diffuser placed in front of a 2D image sensor. When an object is placed in front of the diffuser, its volumetric information is encoded into a single 2D measurement. The system is designed so that calibration is simple and needs to be performed only once. Borrowing tools from the field of compressed sensing, the 3D image is reconstructed by solving a sparsity-constrained optimization problem.

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Images courtesy of Optica/Nick Antipa and Grace Kuo.


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Figure 2. Volumetric information is encoded in pseudorandom patterns called caustics. As shown in the top panel, the mask is chosen so that the caustic pattern shifts as a point in the scene moves laterally, a key feature that enables imaging large volumes efficiently. The bottom panel shows how the pattern varies with depth. Because the pattern is pseudorandom, each point in 3D produces a unique and identifiable pattern.

Instead of a lens, a single thin optical element is placed between the sample and the sensor. The optic is designed such that each point within the volume casts a unique and identifiable pattern. will always run into fundamental physical constraints. In an effort to mitigate this, a more compact imaging architecture is needed. In a traditional lens-based imaging system, a point in the sample maps to a point within the camera body. In contrast, lensless imagers do not rely on lenses to form the image. Instead, a single thin optical element is placed between the sample and the sensor7,8. The optic is designed such that each point within the volume casts a unique and identifiable pattern on the sensor, similar to coded aperture. It is then possible to compute the locations and brightness of a large number of points within the sample, all from a single 2D measurement. The benefits of this are twofold. First, the architecture offers a path to BioPhotonics • July/August 2018


extremely small cameras — the mask is typically less than 1 mm thick, and the FOV is limited primarily by the sensor dimensions. A large FOV can be realized simply by using a larger chip, which does not necessitate increasing the system thickness. Second, lensless architecture can capture depth information for samples close to the mask. The technology could enable simultaneous monitoring of millions of neurons in 3D space at frame rates limited only by image sensor read times. This is more than fast enough to keep up with neuron dynamics. Recently, researchers at the University of California, Berkeley demonstrated a lensless camera (DiffuserCam) that images in 3D8 (Figure 1). It uses a pseudorandom smooth phase mask, called a diffuser, to encode 3D intensity infor-

mation onto a 2D image sensor. A point source creates a pattern of high-contrast, high-frequency caustics on the sensor. This phenomenon is similar to the pattern on the bottom of a swimming pool on a sunny day. With an appropriately chosen mask, each caustic pattern is unique to a location in 3D space. A single point then can be located easily, given knowledge of all possible caustic patterns the mask can produce. However, when multiple light sources are present, their subsequent caustics will superimpose on the sensor. It is then necessary to computationally disentangle the 3D location and brightness of all sources. This reconstruction is not always possible because of the extremely large number of possible locations for each light source. However, when imaging objects that possess a mathematical property known as sparsity — the object can be expressed using very few nonzero values — it is possible to recover the intensity distribution over a large 3D volume, all from a single 2D measurement. A scene consisting of multiple fluorescent sources is an example of a sparse 29 29

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Lensless Camera

Figure 3. Reconstruction of fluorescent resolution target, tilted at an angle to the imaging system (a). 3D image of a small plant recorded by the DiffuserCam, rendered from multiple angles (b). In both cases, the 3D structure is recovered from a single 2D measurement. The corresponding 2D raw data is shown for each panel (upper-right insets).

scene. Most of the space is dark because it is devoid of fluorophores, with light only radiating from the sources. The dark regions can be represented using zeros. Nonzero values are only needed to describe the intensity at voxels containing sources. The entire scene can be represented sparsely. For this method to work, the caustics created by any point within an FOV must be known. To reduce the number of possible patterns, a weak diffuser is used with an aperture. This leads to systematic behavior of the caustics. As the point moves laterally, the caustics simply translate across the sensor (Figure 2, top panel). By measuring just the on-axis caustics at a given depth, the off-axis patterns can be easily computed. Varying patterns As the point changes depth (for example, moves closer), the caustics approximately magnify, encoding depth information of the source (Figure 2, bottom panel). At a given depth, moving the point source laterally simply shifts the caustics across the sensor. This makes it easy to deduce all off-axis patterns at that depth from a single on-axis measurement, accelerating both computation and calibration (Figure 2, top panel). The entire system can be calibrated by recording an image of a point source at each depth within the volume of interest, resulting in calibration sets containing only a few hundred images. In contrast, 30


When imaging objects that possess a mathematical property known as sparsity, it is possible to recover the intensity distribution over a large 3D volume, all from a single 2D measurement. if every point in the volume needed to be measured, it would require hundreds of millions of images — nearly a petabyte of storage — to complete calibration. Using this approach, an image of a sparse 3D object containing tens of millions of voxels can be computed from a single 1.3-MP 2D exposure (Figure 3). The frame rate is limited by the camera exposure, as this method can image a 3D volume using a single 2D image. Thus, it is possible to record activity over a large area at sufficiently fast frame rates for imaging neural dynamics. But there remains work to be done. For lensless cameras to image small objects such as neurons, the object must be placed close to the mask. But this presents a challenge for lensless cameras: Exhaustive calibration is not an option, so most lensless cameras are designed so that the system can be completely characterized using a tractable number of measurements. For refractive masks, one way to do this is to impose a minimum object distance. This causes rays at the mask to behave paraxially, enabling both efficient calibration and image reconstruc-

tion. Unfortunately, it also limits the magnification and resolution of the final system to macroscale scenes. To push the magnification and resolution to neuron scales, new masks and algorithms are being designed in tandem so that high ray angles can be used while maintaining algorithmic efficiency. Currently, most lensless cameras make the assumption that light emitted by a point will travel in a straight line until it hits the camera, but inside the brain this assumption starts to break down. When emitted light interacts with other cell bodies and organelles, the light is scattered in unknown directions. Light emitted near the surface of the brain probably won’t interact with scatterers before detection. In this regime, the current models hold well. But imaging neurons deeper in the brain requires the light to travel farther through tissue, and it will likely hit many scatterers on the way, reducing contrast and resolution. One fix for this problem is to use imaging models that account for scattering. Including accurate scattering models in BioPhotonics • July/August 2018

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the reconstruction algorithm will ensure the best possible image. And since scattering causes a reduction in contrast, small amounts of noise can have a large impact on the reconstruction quality, even with accurate models. Another method is to use longer wavelengths of light, which are less affected by scattering. The most widely used calcium indicator, GCaMP, emits green light. New red indicators have been developed9 that suffer less from scattering, which permits deeper imaging into the brain. The next leap in neural imaging will necessitate time-resolved simultaneous measurement of the electrical dynamics of extremely large numbers of neurons. Calcium imaging has already proven a valuable technique in meeting these demands. Lensless cameras may offer the ability to see the complex dynamics that calcium imaging brings to light. Meet the authors

Nick Antipa is a Ph.D. student at UC Berkeley. He has a bachelor’s degree in optics from UC Davis and a master’s in optics from

the University of Rochester. He is currently researching the incorporation of advanced signal processing algorithms into optical imaging system design; email: nick.antipa@ eecs.berkeley.edu. Grace Kuo is a Ph.D. student in the Department of Electrical Engineering and Computer Science at UC Berkeley. She has a bachelor’s sington University in St. Louis; email: gkuo@ eecs.berkeley.edu. Laura Waller, Ph.D., leads the Computational Imaging Lab at UC Berkeley, which develops new methods for optical imaging, with optics and computational algorithms designed jointly. She holds the Ted Van Duzer Endowed Professorship and is a senior fellow at the Berkeley Institute of Data Science, with affiliations in bioengineering and applied sciences and technology; email: waller@ berkeley.edu.

References

1. F. Helmchen and W. Denk (2005). Deep tissue two-photon microscopy. Nat Methods, Vol. 2, Issue 12, pp. 932-940. 2. F. Helmchen et al. (2001). A miniature head-mounted two-photon microscope: high-resolution brain imaging in freely moving animals. Neuron, Vol. 31, Issue 6, pp. 903-912.

3. S. Ogawa et al. (1990). Brain magnetic resonance imaging with contrast dependent on blood oxygenation. Proc Natl Acad Sci U.S.A., Vol. 87, Issue 24, pp. 9868-9872. 4. N.C. Pégard et al. (2016). Compressive light-field microscopy for 3D neural activity recording. Optica, Vol. 3, Issue 5, pp. 517-524. 5. O. Skocek et al. (2018). High-speed volumetric imaging of neuronal activity in freely moving rodents. Nat Methods, Vol. 15, Issue 6, pp. 429-432. 6. K.K. Ghosh et al. (2011). Miniaturized integration of a fluorescence microscope. Nat Methods, Vol. 8, Issue 10, pp. 871-878. 7. J.K. Adams et al. (2017). Single-frame 3D fluorescence microscopy with ultraminiature lensless FlatScope. Science Advances, Vol. 3, Issue 12, p. e1701548. 8. N. Antipa et al. (2018). DiffuserCam: lensless single-exposure 3D imaging. Optica, Vol. 5, Issue 1, pp. 1-9. 9. H. Dana et al. (2016). Sensitive red protein calcium indicators for imaging neural activity. eLife, Vol. 5, p. e12727.

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Fluorescence Microscopy Unravels Morphogenesis, Function of Lymph System Researchers are targeting a comprehensive understanding of lymphatic vessels with potential implications for improved treatment. BY MARCO ARRIGONI, COHERENT INC.

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he lymphatic system is an underinvestigated part of the vascular system. A team led by Friedemann Kiefer at the European Institute for Molecular Imaging (EIMI) at the University of Münster in Germany is aiming to change that in ongoing studies of the formation and function of lymphatic vessels in lab mice. The researchers use an arsenal of fluorescent microscopy techniques to probe these vessels and unravel the genetic control of formation, function, and repair. Multiphoton microscopy, often using triple-transgenic mice, is proving to be a critical tool in these studies because it is the only high-resolution method that can image at subcellular resolution deep

within live and in vitro tissue samples. The circulatory system in mammals, and most importantly in humans, has been exhaustively studied and characterized. Yet the lymphatic system remains poorly understood at many levels. In major surgery, this limited understanding means lymphatic vessels are routinely excised or damaged inadvertently. This surgical damage is a leading cause of lymphedema, in which a patient’s limb can painfully swell, become fibrotic, and lose function. Normal function of pressurized blood flow means that the fluid component of blood (plasma) leaks out of the capillaries in every tissue. The plasma is collected in tiny blind vessels that are connected

to larger collection vessels. Now called lymph, the fluid flows through nodes in this system of vessels where the fluid is screened for pathogens and where cancer and infections often manifest as a result. Valves in the vessels ensure unidirectional flow, and movement is driven by smooth-muscle contraction in the larger vessel walls and secondary pressure from nearby arterial pumping. The lymph then re-enters the arterial-venal system at two interconnections below the clavicles (collar bones). Even larger lymph vessels may be in a collapsed state, which, together with the fact that lymph is a clear liquid, explains why lymph vessels are often unseen and thus damaged during surgeries. “We are trying to fill in the huge

Figure 1 (above). Kiefer team members Nils Kirschnick (left) and Michael Kuhlmann at the multiphoton microscope equipped with both a titanium:sapphire laser (Chameleon Vision) (closest to men) and one of the next-generation lasers based on ytterbium fiber (Chameleon Discovery) (far right). Courtesy of Kiefer group/EIMI Münster.

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Figure 2. The vessel wall of a mouse carotid artery, showing en face (a) and orthogonal views (b) obtained through deep imaging. The cyan structures are collagen fibers (via second-harmonic generation detection), the red are endothelial cell interfaces labeled with Alexa 647, and the green shows nuclei of the smooth muscle cells labeled with Cyto41. Courtesy of Dominic Depke/EIMI Münster.

gaps in the understanding of the lymph system,” Keifer said. “This includes morphogenesis — how the vessels are first created in an embryo — to normal function at the cellular level; the consequences of hypoxia, or depleted oxygen; and how the development of both lymph and blood vessels are affected by tumors, strokes, arteriosclerosis, trauma, and aging. It’s basic biological research, but with obvious potential for improvements in medical treatments and outcomes.” In a recent landmark publication1, the researchers first revealed how mammalian (murine) lymph vessels are derived from venous endothelial cells during embryo development. Surprisingly, they found that the process is initiated by BioPhotonics • July/August 2018

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In major surgery, the limited understanding of the lymphatic system means that vessels are routinely excised or damaged inadvertently. delamination and cell migration from the cardinal vein, rather than from sprouting, which might be expected intuitively. Kiefer’s group uses several fluorescence microscopy techniques in its studies. These include conventional wide-field epifluorescence microscopy, confocal microscopy, 3D reconstruction microscopy, and selective plane illumination-based ultramicroscopy (often called light-sheet microscopy). All of these play valuable roles in imaging tissue samples and early-

stage murine (mouse) embryos. But none of them have the ability to image within a live animal or thick tissue sample. For deeper images, the group exclusively uses multiphoton excitation: specifically, two-photon laser scanning microscopy (2P-LSM). As an excitation source, they use either a tunable titanium:sapphire (Ti:sapphire) laser (Coherent Chameleon) or one of the latest ytterbium-fiber-based (Yb-fiber) tunable one-box laser sources (Coherent Discovery) (Figure 1). 33 33

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Fluorescence Microscopy

Minimizing Laser Downtime

Figure 3. The multicolor capacity of the Kiefer lab setup. Mixtures either of Cos1 cells expressing different fluorescent proteins — enhanced green florescent protein (eGFP) (green) and mOrange2 (yellow) (a); mOrange2 (yellow) and mCherry (red) (c); and mOrange2 (yellow) and mPlum (cyan) (d) — or a single Cos1 cell expressing two fluorescent proteins in different compartments— mClover3 (cytoplasmic, green) and mRuby3 (nuclear, red) (b). Panels a and b were excited with 820-nm (eGFP, mClover3) and 1100-nm (mFruit, mRuby3); panels c and d with 1100-nm (mFruit) only. Scale bars = 20 µm. Courtesy Nils of Kirschnick and Abel Pereira da Graca/EIMI Münster.

The researchers need deep-imaging capability for many of their studies. “With early-stage embryos, some of the developing vessels are almost at the surface,” Keifer said. “But when we look at the effects of strokes or arteriosclerosis in adult mice through a cranial window or tumors through a skinfold cavity, or even tumor tissue samples from biopsies, we sometimes need a penetration depth of hundreds of microns.” Two- and three-photon excitation of fluorescence using a tightly focused NIR ultrafast laser presents several advantages. Fluorescence is only excited at the narrow beam waist, delivering 3D resolution and eliminating out-of-focus background noise. In addition, compared to visible wavelengths, attenuation of the laser beam is much lower for the NIR wavelengths needed for two-photon excitation, enabling deeper penetration. Just as 34

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important, live samples can be subjected to prolonged and repeated exposure with minimized photodamage because most of the NIR laser light is not absorbed. High power allows deeper imaging, which is why the newest laser in the Kiefer lab is of the Yb-fiber type; it produces higher power at wavelengths well beyond 1 µm and supports the imaging of long-wavelength fluorophores at greater depth. A carotid artery example (Figure 2) from the group’s parallel studies of blood vessels dramatically demonstrates this deep-imaging capability. The specimen was excited at 830 nm and 1100 nm simultaneously. The cyan structures are collagen fibers — using second-harmonic generation (SHG) excited at 1100 nm — around the arterial vessel, together with smooth muscle cells of the media (green nuclei stained by Cyto41). Similar to all blood vessels, the

In many of Friedemann Kiefer’s experiments, it is necessary to repeatedly image the same sample at different intervals over a period ranging from a few hours to several months. This requires long-term laser stability and high uptime. “Even in a small animal like a mouse, arteriosclerosis develops over weeks,” he said. “And some tumors develop over an even longer time frame. In a worst-case scenario, if we lose the ability to image in a certain time window, then the entire experiment is lost, often together with a valuable tripletransgenic mouse, which we have to euthanize.” In next-generation lasers such as the one Kiefer used, laser manufacturers maximize data throughput and minimize unscheduled downtime in two ways. First, the laser is designed to deliver industrial reliability by being tested to destruction multiple times during design and prototyping using rigorous highly accelerated life test (HALT) protocols. In production, it is tested beyond its performance specifications before shipping from the factory using highly accelerated stress screening (HASS) methods. Second, the laser’s onboard smart self-diagnostics and control systems support remote service via an internet link, rather than waiting a week or two for a service technician to visit. To date, Kiefer’s group has had three minor issues with reduced output power. In each case, laser performance was restored to specification the next day, preventing any loss of experiment. innermost lining of the carotid artery is formed by a single-cell layer of endothelial cells, which were stained here with an Alexa 647-coupled PECAM-1 antibody (red). The red staining indicates the interendothelial cell-cell junctions. “The carotid wall that is imaged in this maximum-intensity projection corresponds to approximately 80 µm,” Kiefer BioPhotonics • July/August 2018

7/9/2018 3:05:00 PM

Figure 4. 2P-LSM images of a transgenic mouse expressing the tdTomato protein under the control of a promoter that drives expression predominantly in lymphatic vessels. A lymphatic collector vessel (red) within the muscle of the diaphragm; green fluorescence is generated by SHG of muscle fibers (a). Mesenterial lymph (orange) and blood (blue) vessels (b). Scale bars = 100 µm. Courtesy of Nils Kirschnick/EIMI Münster.

said. “With normal epifluorescence or even confocal microscopy, it is absolutely unthinkable to even penetrate the collagen fibers of the elastic layer of a large arterial vessel. Here, we go through the elastic fibers, then the smooth muscle layer underneath all the way to the lining endothelium. In the 3D rendering, we basically show a slanted cut through the vessel and include only a very few layers in this image. Otherwise, the highly detailed picture becomes so congested, people might not recognize any of the structures we are talking about.” Another reason for choosing the Yb-fiber-based laser was its broad tuning range and fast tuning speed. These features are required because the researchers typically want to image two different fluorescent proteins (and often a third fluorescent dye) near simultaneously, where these fluorophores are chosen to have well-separated excitation/emission spectra to maximize contrast. They also sometimes detect SHG signals, which can image collagen fibers without any staining. This fast multiwavelength capability is needed to take full advantage of the rare (and valuable) triple-transgenic mice bred by the researchers. “Using established protocols, an mFruit gene such as mOrange2 is linked to a promoter gene for lymphatic vessel proteins,” BioPhotonics • July/August 2018

718BI_Lymph.indd 35

Live samples can be subjected to prolonged and repeated exposure with minimized photodamage because most of the NIR laser light is not absorbed. Kiefer said. “However, there is no single protein target that is a unique marker for lymph vessels, so we have to use a combination of two proteins, like mOrange2 and tdTomato. And we often use even a third marker, for example UnaG (a protein derived from Japanese eel), which fluoresces when it binds to bilirubin and allows us to map hypoxia.” In addition to fluorescent labeling, the team also breeds mice with individual genes selectively deleted, which is essential for some of the studies in embryonic development of blood and lymph vessels and also in disease models. For example, they use mice lacking the apolipoprotein E (ApoE) gene, which causes them to have high plasma serum cholesterol levels and to develop spontaneous arteriosclerotic lesions. Kiefer stresses that all the animals are humanely treated according to the legal regulations of the German Animal Welfare Act. Some recent images acquired with 2P-LSM from the ongoing study show the structure of lymphatic vessels in differ-

ent organs (Figure 4). Specifically, these images from the diaphragm and mesentery of a mouse show lymphatic vessels that are unmistakably identified by the fluorescent red and orange proteins they specifically express, with no additional staining procedure. The lymphatic system is poorly understood at virtually every level, yet its disruption by disease and surgical trauma has functional and painful consequences for many people. Multiphoton microscopy is proving to be a powerful tool for researchers to shed light on the exact mechanisms by which this occurs. Meet the author

Marco Arrigoni is a director of strategic marketing at Coherent Inc. He covers the scientific research markets; email: marco.arrigoni@ coherent.com.

Reference

1. Hägerling et al. (2013). A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy. EMBO J, Vol. 32, Issue 5, pp. 629-644.

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7/9/2018 3:05:03 PM

Point-of-Care Optics Helps Halt the Spread of Infectious Diseases Optical technologies offer an accurate, rapid, and low-cost approach to diagnosing infectious diseases noninvasively. BY MARIE FREEBODY, CONTRIBUTING EDITOR

E

CapsoCam Plus capsule endoscope contains four cameras that capture high-resolution images at a maximum 20 fps. Courtesy of CapsoVision.

Through advanced optical technology, CapsoCam Plus captures a full 360° view of the gastrointestinal tract, providing physicians with more detailed imaging and a larger vertical field of view. Courtesy of CapsoVision.

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718BI_FeatInfectious.indd 36

arly diagnosis is the critical starting point to a better outcome for a patient. It allows effective treatment to start earlier, giving the patient the best possible prognosis. Prompt diagnosis also reduces the potential spread of infection, significantly lowering health care costs. Medical experts and health care companies around the world recognize the value of developing ways to diagnose infectious diseases noninvasively at the point of care (POC), particularly in developing countries and rural areas where medical screening can be nonexistent. Many believe that readily available optical componentry that can be scaled up for volume production provides the best route to success. “I’ve worked on optical spectroscopybased noninvasive diagnostics for over 20 years, and application of my experience in this arena to infectious diseases was an opportunity that presented itself in 2014,” said John Maynard, vice president of product management at Avisa Pharma. “I was intrigued by the potential of a rapid, noninvasive, POC test to improve detection of lung infections and to better target antibiotics to treat these infections.” Prior to joining Avisa Pharma, Maynard co-founded and was vice president of technology at VeraLight Inc. in Albuquerque, N.M., which developed POC, noninvasive, optical measurement instruments to quickly detect undiagnosed prediabetes and diabetes. When he joined Avisa Pharma in 2014, he began to focus his expertise on commercializing a rapid, POC breath test for diagnosing pulmonary infections. Lung infections such as pneumonia are particularly deadly among the very young, very old, and patients with compromised immune systems. According to the World Health Organization (WHO), pneumonia kills 1.8 million children under the age of 5 every year. It kills more children than any other illness, in every


7/9/2018 3:02:36 PM

This laser-based breathalyzer comprises a single-use disposable cough sample collection tube and a bio-optical sensor with a patented biochemical coating formulated to react with the TB bacilli. Courtesy of Rapid Biosensor Systems.

region of the world. In spite of its huge toll, relatively few global resources are dedicated to tackling this killer. Currently, the gold standard for detecting the cause of a pneumonia infection is to culture a sputum sample and see what grows. This process is highly dependent on getting a good sample that specifically comes from the part of the lung with the infection. But transporting and preparing the sputum for culture without contamination is challenging. And 24 to 72 hours are required to grow the cultures before a pathologist can determine the causative pathogens. “The long time to an answer means doctors must treat patients empirically because they don’t know the cause of the infection,” Maynard said. “In contrast, the Avisa breath test for certain virulent urease respiratory pathogens measures the entire lung and does not require sputum, delivering a result in 10 minutes.” The test works by detecting labeled CO2 emitted by bacteria that metabolize

in urea in the breath of patients. First, a patient provides a baseline breath sample to establish their natural ratio of 13CO2 (the label) to 12CO2. Then, a nebulized mist of 13C urea is delivered to the lungs. In normal health, a person exhales around 20 times more 12CO2 than 13CO2, but if the patient’s lungs are infected with virulent bacteria that metabolize in urea, there will be relatively more 13CO2. Measuring the difference in the ratio of 13CO2 to 12CO2 in the baseline and postnebulized samples can determine if a lung infection is caused by urease, a virulence factor found in several pathogenic bacteria. The Avisa instrument uses an NIR vertical-cavity surface-emitting laser (VCSEL) that is scanned over the 13CO2 and 12CO2 absorption peaks using wavelength modulation spectroscopy. From this, the difference in the ratios, known as the delta over baseline, can be determined. The entire procedure takes less than 10 minutes and provides the doctor

‘I was intrigued by the potential of a rapid, noninvasive, point-of-care test to improve detection of lung infections and to better target antibiotics to treat these infections.’ — John Maynard, Avisa Pharma



Tuberculosis is one of the world’s most serious public health problems, killing more adults than any other single infectious disease. U.K.-based Rapid Biosensor Systems has developed a laserbased breathalyzer that detects a specific antigen in a bid to contain the growing prevalence of this “old” disease. Courtesy of Rapid Biosensor Systems.

with immediate information as to whether the pneumonia is caused by a virulent urease pathogen to allow better informed decisions on antibiotic use and choice. “Urease pathogens tend to be more virulent than the more common Streptococcus bacteria that cause pneumonia and require powerful, broad-spectrum antibiotics for effective treatment,” Maynard said. “Today, most doctors use empiric therapy to prescribe antibiotics for pneumonia, resulting in overuse that can lead to unnecessary hospitalizations, increasing antimicrobial resistance, and changes to the patient’s gut microbiome that can have longer-term health implications, including secondary C. difficile infection and metabolic changes.” Despite long regulatory review processes associated with any medical technology, Maynard said that several cost-effective optical-based devices that diagnose disease noninvasively could come to market in the next five years. 37 37

7/9/2018 3:02:41 PM

Point-of-Care Optics

Tuberculosis breathalyzer One such example is for detecting tuberculosis (TB). U.K.-based Rapid Biosensor Systems Ltd. has developed a laser-based breathalyzer that detects a specific antigen — as opposed to a byproduct of the disease. This specificity, the company says, is over 95 percent accurate and could help to contain the spread of the disease, which many thought was in decline. According to the WHO, previously common diseases such as TB have recently re-emerged, driven by various social factors, and pose a significant threat to world health. TB is spread through the air by an infected cough, sneeze, or spit. There are 8.8 million active cases diagnosed each year, and almost 2 million of those infected die. Despite effective drugs against TB, it remains one of the world’s most serious public health problems, killing more adults than any other single infectious disease. The problem has been made worse because new strains of multidrug-resistant TB have added further pressure on health care systems. “There was, and still is, an unmet medical need for a very fast, low-cost, noninvasive test to detect active TB at the POC,” said Dennis Camilleri, CEO of Rapid Biosensor Systems. “The WHO had predicted this need because if TB can be screened quickly, while people wait two minutes for a result, then the positives can

be treated sooner, thus minimizing the spread of this killer disease and ultimately reducing health costs.” The Breath Analyser unit from Rapid Biosensor Systems comprises a singleuse disposable cough collection tube in

Avisar laser spectrometer for measurement of 13 CO2 to 12CO2 ratio in exhaled breath collected with the AvisarLink, which is currently in clinical trials. Courtesy of Avisa Pharma.

Pneumonia patient breathing through AvisarLink nebulizer and breath collection kit. The AV-U13 drug is delivered to the lungs as a nebulized mist using the Aerogen Solo mesh nebulizer mated to the Aerogen Ultra adapter. Exhaled breath is sampled from the chamber attached to the Ultra adapter at the baseline and postnebulization. Courtesy of Avisa Pharma.

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which the sample is made up of the fine liquid droplets produced by coughing. After being nebulized using a 0.9 percent saline solution, the patient coughs into the collection tube. At the bottom of it is a bio-optical sensor with a biochemical

‘The emerging market economies need low-cost medical tests for infectious diseases if they are to reduce mortality, but there must be a willingness from governments to implement medical screening at the point of care.’ — Dennis Camilleri, Rapid Biosensor Systems Ltd.


7/9/2018 3:02:45 PM

coating formulated to react with the TB bacilli. A push-and-twist action seals the aerosol sample in the tube, causing the droplets to collect on the biosensor. “With breath-based antigen tests, the actual infection antigen/bacilli in a cough sample is being detected and not a byproduct of TB,” Camilleri said. “In our case, we use [an] evanescent wave optical sensor with a proprietary biocoating that detects the change in optical fluorescence signal caused by the presence of the TB bacilli.” With its portable, easy-to-use, disposable, and speedy TB Breathalyser, Rapid Biosensor Systems hopes that uptake in developing countries, in particular, will help to contain further spread of the disease. As ever, cost is the biggest challenge. But it’s one that Camilleri hopes will be surmountable should sufficient investment be made available to scale up production. “The emerging market economies need low-cost medical tests for infectious diseases if they are to reduce mortality, but there must be a willingness from governments to implement medical screening at the POC,” he said. “Optical/photonics is a volume business: The higher the volume, the lower the cost of materials, which will meet the cost targets proposed by the WHO for tests of this kind.”

physician’s office once the examination is complete,” said Elina Jaime, product manager at CapsoVision. “The procedure is completely wire-free, which also saves time for the staff and eliminates scheduling bottlenecks while reducing valuable exam room time.” The capsule is approximately the size of a vitamin pill and can be swallowed with a few sips of water. With four lateral, onboard cameras that capture highresolution, 360° images, CapsoCam Plus provides a detailed examination of the mucosal surface, including a look at areas behind mucosal folds. “Through advances in optics, we are able to place four high-resolution cameras around the circumference of the capsule to capture a full 360-degree view of the gastrointestinal tract,” Jaime said. “These cameras capture high-resolution images at a maximum frame rate of 20 fps — 5 fps per camera.” Two of the most important features embedded in the capsule are the Smart Motion Sense technology and the onboard storage system. Smart Motion Sense takes

pictures only when the capsule is in motion, which reduces image redundancy and physician review time. The onboard storage capabilities allow the capsule to operate without transmitting radio frequency waves, making it safe for use in patients with implantable devices such as pacemakers and insulin pumps. The capsule can be used for diagnostic purposes and to visualize post-treatment changes in the patient’s digestive tract to assess the effectiveness and success of treatment. Outside the United States, the CapsoCam Plus video capsule system is intended for visualization of the small bowel mucosa in patients ages 2 and above. It may be used as a tool in the detection of abnormalities of the small bowel. In the United States, the CapsoCam Plus video capsule system is intended for visualization of the small bowel mucosa in adults. It may be used as a tool in the detection of abnormalities of the small bowel. [email protected]

Gastrointestinal diseases Infectious diseases within the gastrointestinal tract are notoriously tricky to diagnose, as they are often associated with nonspecific symptoms, such as obscure gastrointestinal bleeding, diarrhea, and bloating. Because of its length and position in the digestive system, the small intestine can be very challenging to reach and visualize, often requiring an invasive procedure during which the patient needs to be sedated. But a new device with four minicameras that can be swallowed by the patient could offer a much less invasive alternative. The CapsoCam Plus capsule endoscope from California-based medical device innovator CapsoVision provides a comprehensive 360° view of the patient’s digestive tract without the need for the patient to be sedated. The procedure itself is pain-free. “Patients don’t have to wear bulky external equipment during the examination, and they don’t have to return to the BioPhotonics • July/August 2018


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7/9/2018 3:02:47 PM

BREAKTHROUGHPRODUCTS ◀ Custom Wavelength Selectors

Custom wavelength selectors (CWS) from Spectrolight Inc. offer cost-effective, off-theshelf options for narrowband transmission filters with custom bandwidth and center wavelength. The CWS series offers a compact optomechanical device with a circular aperture up to 10 mm, whose center wavelength can be set anywhere in the range of 350 to 900 nm, and whose bandwidth can be independently fixed from 1.5 to ~20 nm. Each CWS also features >80 percent in-band transmission efficiency and 10 6 out-of-band extinction, making these devices ideal for demanding uses in research and instrumentation. Typical applications include spectral imaging, machine vision, inspection, cell counting, and fluorescence microscopy for both excitation and image filtering. [email protected]

Microscopy Module ▶

The OptiRecon module for the Xradia Versa 500 series of 3D x-ray microscopes from Carl Zeiss AG allows users to quickly acquire high-quality images. OptiRecon enables users to choose quality and speed parameters for their own use, with a combination of a workflow-based user interface and an efficient proprietary implementation (allows reconstruction of a typical dataset) that delivers results in about three minutes. The technology opens the door to 3D x-ray imaging or computed tomography to a wider range of industrial applications and examination of in situ processes occurring at previously inaccessible timescales. [email protected]

MOS Bio-Optical Sensor ▼

The ULS24 ultralow-light CMOS bio-optical sensor from Anitoa Systems LLC is a single-chip device capable of 3 × 10 −6 lux low-light detection. The ULS24 achieves a high level of sensitivity through the innovation of a temperature-compensated, darkcurrent management algorithm. It features a very small form factor of

Bio Photonics - July 2018

Bio Photonics - July 2018

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