a scanning microscope with postacquisition ... - OSA Publishing

Letter

Vol. 2, No. 3 / March 2015 / Optica

210

Three-dimensional information from two-dimensional scans: a scanning microscope with postacquisition refocusing capability ALEXANDER JESACHER,1,* MONIKA RITSCH-MARTE,1

AND

RAFAEL PIESTUN1,2

1

Division of Biomedical Physics, Innsbruck Medical University, Müllerstraße 44, 6020 Innsbruck, Austria

2

Department of Electrical and Computer Engineering, University of Colorado, Boulder, Colorado 80309, USA

*Corresponding author: alexander.jesacher@i‑med.ac.at Received 7 October 2014; revised 24 November 2014; accepted 20 January 2015 (Doc. ID 224452); published 27 February 2015

Laser scanning microscopes are helpful tools for the visualization of 3D structures at the submicron scale. By pointwise raster scanning the sample with a tightly focused laser spot, they collect sample information in a sequential manner. We present a scanning microscope that has the ability to record 3D sample information from a single 2D scan. By combining camera detection with double-helix phase engineering in the emission path, the microscope allows for postacquisition refocusing within an axial range of roughly 400 nm with a high-NA lens (NA 1.35). Refocused images are extracted from a single 2D data set by applying different synthetic pinholes, i.e., by integrating over a small predefined area in the image acquired for every sampling point. 3D cross-sectional images of a stained microtubule network within fixed African green monkey kidney cells are presented, demonstrating the capability of the system. The imaging paradigm enables faster data acquisition with potentially lower phototoxic side effects. © 2015 Optical Society of America OCIS codes: (180.6900) Three-dimensional microscopy; (180.5810) Scanning microscopy; (070.6120) Spatial light modulators. http://dx.doi.org/10.1364/OPTICA.2.000210

Confocal scanning microscopy [1] is a powerful imaging technique providing high resolution and optical sectioning. In its standard configuration, it relies on a tight laser focus scanning the sample, a pinhole, and a large area detector collecting the 2334-2536/15/030210-04$15/0$15.00 © 2015 Optical Society of America

sample response, which is either reflected/transmitted light or excited fluorescence. Typically, photomultiplier tubes or avalanche photodiodes are employed as detectors, because of their high speed and dynamic range as well as their excellent noise characteristics. Pixelated image sensors such as CCD or CMOS, on the other hand, have long been unsuitable for low-light confocal microscopy applications, mainly because of the slow and noisy readout process. Nonetheless, the potential advantages of pixelated detectors in terms of signal and resolution were noticed as early as the 1980s [2]. Current advances in sensor arrays make cameras more attractive for scanning microscopy because of their low noise and high frame rate. As a result, recent work has shown experimental implementation of Sheppard’s pixel reassignment microscopy [3] as well as other noteworthy developments based on it [4–10]. The great potential of scanning microscopy with image sensors lies in its combination with point spread function (PSF) engineering, as one is not bound to a fixed physical pinhole but has the freedom to adapt the data processing to the properties of the imaging PSF; i.e., it is possible to define a synthetic pinhole (SP) by reading out only specific pixels, and also to use pixel-specific weights in order to achieve an optimal signal-tonoise ratio. This synthetic pinhole can take arbitrary shape and also vary within a single scan to achieve position-dependent optimal balancing between signal and resolution. The incorporation of PSF engineering with camera-based detection and matched synthetic pinhole postprocessing are key innovations presented in this article. We describe a scanning microscope that is capable of refocusing the sample after the acquisition has been performed, purely by postprocessing the data taken in a single 2D scan. This is achieved by combining camera-based scanning microscopy with doublehelix (DH) PSF engineering [11]. We term our approach RESCH (REfocusing after SCanning using Helical phase

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engineering). The main advantage of RESCH is that information from axial planes adjacent to the nominal focal plane is gathered while the axial resolution is practically maintained. It thus allows us to obtain 3D information of specimens from fewer planar scans. Collecting data from multiple planes in parallel also reduces the overall image acquisition time, reduces phototoxicity, and ensures absolute time synchrony for the data along the z axis. The proposed microscope technique involves minimal modifications to an image scanning microscope [2,3]. A sketch of our setup is shown in Fig. 1(a). The emitted fluorescence is imaged by a scientific CMOS (sCMOS) camera (ORCAFlash4.0 V2), which takes an image of a small region around the focus whenever the sample is moved by a scanning step distance (80 nm in our experiments). The microscope technique can be implemented either with beam or sample scanning as shown here. The main hardware addition in the setup features a DH phase mask in the emission path, which is displayed in this experiment on a spatial light modulator (SLM). Imaging with DH phase masks provides accurate depth estimation over a large axial working range [12] and has so far been used for axial superlocalization in widefield [13,14] and recently also stimulated emission depletion microscopy [15]. We create a DH mask by using the analytic deterministic method described in [11], using nine vortices with an interspacing of 0.4 pupil radii. The mask turns a Gaussian or Airy PSF into a DH, making a small fluorescent particle appear as two “lobes” in the image, which rotate when the particle moves along z or the microscope is refocused by moving the objective lens. The use of such a mask in a scanning microscope has a similar effect: Depending on the axial position of a fluorophore within the excitation focus, its image on the camera consists of two lobes with a specific rotational angle ϕ. A movie consisting of raw data camera images from a 2D RESCH scan through a 3D fluorescent sample is provided in Media 1.


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By applying a synthetic pinhole in the form of two discs matching the lobe positions for a given depth, one thus obtains a sectioned image of the plane defined by ϕ. Applying a pinhole means integrating the signal within the predefined pinhole area and allocating the result to the corresponding sampling point. In this manner, by applying different SPs with varying rotational angles, it is possible to generate an image stack of multiple axial planes from the same data set. This principle is illustrated in Fig. 1(b), which shows two widefield DH images of a 100 nm fluorescent bead, one in focus and the other defocused by 400 nm. The green disc pairs matching the double lobes mark the SPs for extraction of the corresponding image sections. The DH was designed to consist of two compact lobes with a relatively large interspacing (about 1.1 μm in our setup) to enable a good defocus discrimination. In practice, the best positions for the synthetic pinhole pairs were determined by recording a widefield z-stack of a 100 nm fluorescent bead and fitting two Gaussians to each double-lobe image at each axial plane. The pinholes of each pair were then placed at the center positions of the two Gaussians. The mathematical description of image formation in RESCH microscopy is similar to that of a confocal microscope. If we assume stage scanning, the (1D) expression for the measured intensity I m x s at a specific scanning point x s using the synthetic pinhole SPm is ZZ I m x s ρx 0 − x s h1 x 0 h2 x 0 − xSPm xdx 0 dx; (1) where ρ is the sample fluorophore emission (or scattering) density and h1 and h2 are the excitation and emission intensity PSFs, respectively, with h2 being a DH. Each choice of SPm delivers a different image I m , which can also be expressed using a total effective imaging PSF hm incorporating the shapes of h1 and h2 as well as SPm : Z I m x s ρx 0 − x s hm x 0 dx 0 ; (2) with hm defined as hm x SPx h2 xh1 x:

Fig. 1. (a) Sketch of the RESCH microscope. The sample is moved by a piezo scanning stage and the fluorescence generated in the laser focus imaged onto a sCMOS camera. A DH phase mask, placed in a plane that is optically conjugated to the objective back aperture, is displayed on a SLM. A polarizer blocks the light that is not modulated by the SLM. (b) Widefield DH-filtered images of a 100 nm fluorescent bead, with different amounts of defocus. The green discs mark the synthetic pinholes (SPs) for the corresponding defocus values. A movie of RESCH camera raw data is provided in Media 1.

(3)

For a point-like pinhole and with h1 and h2 being equal, hm takes the well-known form h21 , i.e., the PSF of a confocal microscope. We measure hm by performing 3D stacks of a subdiffraction-limit fluorescent bead (100 nm, dark red stained, from the TetraSpeck Fluorescent Microspheres Sampler Kit), which is attached to a glass coverslip and immersed in water. The objective is an Olympus UPLSAPO 60× with a NA of 1.35. The excitation laser is a fiber-coupled iBeam smart from Toptica Photonics, emitting at 640 nm. The results are shown in Fig. 2. The first image column shows sections through the PSF of a standard confocal microscope, which were recorded using Airy-shaped excitation and emission PSFs and data processing with a circular SP (sketched in the top image) of a 0.75 Airy disc diameter (460 nm). The

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SP+1

y x

z x

simulated values in brackets all length values in µm

PSF properties confocal

RESCH SP-1

SP 0

0

-0.18 (-0.18)

0

+0.23 (+0.18)

FWHM in x

0.28 (0.22)

0.28 (0.23)

0.28 (0.22)

0.31 (0.23)

FWHM in y

0.32 (0.26)

0.37 (0.35)

0.33 (0.29)

0.37 (0.35)

FWHM in z

0.72 (0.57)

0.78 (0.57)

0.76 (0.58)

0.73 (0.57)

1

0.35 (0.25)

0.59 (0.68)

0.27 (0.25)

axial shift (rel. to confocal)

max. Intensity (rel. to confocal)

SP+1

Fig. 2. Comparison of point spread functions. First image column: sections through a measured standard confocal PSF. The scale bar is 500 nm. Second to fourth image columns: three RESCH PSFs, all extracted from a single 2D scan with the DH mask in the microscope’s emission path. The PSFs show axial shifts that are determined by the rotational angle of the used pinhole pair. The table contains measured axial positions, widths, and maximal intensities of all shown PSFs. All length values are in μm.

elliptic shape of the PSF in the x–y section is due to the linear polarization of the excitation laser as well as the fact that the emitted fluorescence is polarization filtered by the SLM. The image columns on the right show sections through three RESCH PSFs, which were extracted from a single data set by scanning with the DH mask and then applying pairs of circular pinholes (each circle with a diameter of 0.75 Airy discs) to the obtained data. Depending on the rotational angle of the used pinhole pair, the resulting PSF shows a corresponding axial shift. The table in Fig. 2 compares the axial positions, widths (FWHM), and maximal intensities of the shown confocal and RESCH PSFs. All length values are in μm. The axial positions are the axial centers of mass coordinates of the respective PSFs. The numbers in brackets are results from numerical simulations, which also considered vectorial effects. The fluorescent bead was modeled as an unpolarized emitter.

(a)

-200 nm

in focus

+200 nm

(b)

confocal image

SP 0

confocal stack

SP-1

The table shows that the shapes of the RESCH PSFs are similar to the confocal PSF, with the exception that the RESCH PSFs are wider in the y direction. This is a consequence of the anisotropic shapes of the two lobes. Also noticeable is that almost all measured PSF dimensions are about 25%–30% wider than expected from the simulations, which probably is mainly caused by mechanical vibrations in the system. The useful defocusing range achievable with RESCH is determined by the axial widths of the illumination and DH-shaped excitation PSFs. It is about 200 nm for our system and could be increased by using either an engineered Bessel PSF [16] or simply a smaller NA on the illumination side, combined with an adapted DH PSF on the emission side. We demonstrated the capabilities of our microscope by taking images of stained microtubules (dye: Alexa 647) in fixed African green monkey kidney cells (COS-7). Some results are shown in Fig. 3. Each image shows a region of 8 μm × 8 μm and was sampled in steps of 80 nm. One scan took about a minute. Figure 3(a) compares a confocal image z-stack with data from a single 2D RESCH scan. The structures shown in the images are comparable, although the defocused RESCH

RESCH

pinhole

RESCH

RESCH

confocal

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Fig. 3. Confocal and RESCH images of fluorescent microtubules. (a) First row: confocal image stack, recorded at axial steps of 200 nm; second row: three defocused images, extracted from a single 2D RESCH scan. (b) Confocal (top) and RESCH (bottom) images of another sample region. The RESCH data provide 3D information, revealing structures lying above and below the nominal focal plane (see arrows), while the confocal image does not.

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images are dimmer and less resolved than the confocal ones. However, one has to bear in mind that the confocal image stack required a threefold laser energy dose and recording time. The ability to detect out-of-focus structures is further emphasized in Fig. 3(b), which compares a single confocal image with the corresponding RESCH image. Both were acquired using the same laser energy dose. All images have been normalized to their respective maximum intensities. The RESCH image provides 3D information of the sample, revealing two strings crossing the image below the focal plane (see arrows) and a broader structure (possibly a bundle of strings) crossing the image above (see arrow). The confocal image also shows these structures but does not allow one to infer their axial locations, or even to decide whether the structures are above or below the focal plane. At the same time, the in-focus RESCH image provides a 3D resolution that is comparable to the confocal image. Out-of-focus planes show a minor reduced spatial resolution in one of the transverse directions. Modifications of the DH phase mask design may allow fine-tuning of trade-offs, e.g., between spatial resolutions and signal-to-noise ratios of the in- and out-of-focus plane information. The DH diffraction mask can be implemented with high efficiency phase-only masks that provide mode conversion efficiencies close to 100%. In the experimental demonstration we used a SLM with a lower efficiency of approximately 40% in total (80% for one polarization). In the presented experiments, however, the SLM was used for both the confocal and RESCH data collection to enable a fair quantitative comparison between the two techniques. Advantages of the method are that a 3D specimen can be recorded using fewer 2D scans and is therefore faster compared to other image-based scanning methods and has less phototoxic side effects. While the photon collection efficiency of RESCH is actually reduced for the in-focus plane compared to a confocal microscope, the total efficiency for gathering the full information including the out-of-focus planes is increased. A further advantage is that all the information gathered along the z axis is taken in parallel, i.e., with absolute time synchrony. Processing a single RESCH dataset might also provide enhanced optical sectioning compared to confocal microscopes. The method bears great potential, considering the fast development of high-speed cameras, multi-spot excitation, and the availability of field-programmable gate array frame grabber cards for performing on-the-fly data processing such as pinholing. Using a small number of adequately placed analog photon sensors (such as avalanche photodiodes) as pinhole pairs, the method could also reach high frame rates comparable to those of other single-point scanning microscopes.


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In conclusion, we have presented a scanning microscope that is capable of recording 3D information from a single planar scan. This was achieved by combining image-based scanning with a DH phase mask in the emission path and matched postprocessing involving adapted synthetic pinholes. In the presented configuration, our microscope allows postacquisition refocusing within a range of 200 nm while practically maintaining the sectioning quality of a confocal microscope. FUNDING INFORMATION

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