Implementation of adaptive optics in fluorescent ...

Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction Oscar Azucena,1 Justin Crest,2 Jian Cao,2 William Sullivan,2 Peter Kner,3 Donald Gavel,4 Daren Dillon,4 Scot Olivier5, Joel Kubby1 1 Jack Baskin School of Engineering, Univ. of California, Santa Cruz, 1156 High St., Santa Cruz, CA 95064, USA 2 Molecular, Cell, and Developmental Biology, Univ. of California, Santa Cruz, 1156 High St., CA 95064, USA 3 Department of Biochemistry and Biophysics, University of California, San Francisco, 600-16th St., Box 2240, CA 94158, USA 4 Laboratory for Adaptive Optics, University of California, 1156 High St., Santa Cruz CA 95064, USA 5 Physics and Advanced Technologies, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550, USA ABSTRACT Adaptive optics (AO) improves the quality of astronomical imaging systems by using real time measurement of the turbulent medium in the optical path using a guide star (natural or artificial) as a point source reference beacon [1]. AO has also been applied to vision science to improve the view of the human eye. This paper will address our current research focused on the improvement of fluorescent microscopy for biological imaging utilizing current AO technology. A Shack-Hartmann wavefront sensor (SHWS) is used to measure the aberration introduced by a Drosophila Melanogaster embryo with an implanted 1 micron fluorescent bead that serves as a point source reference beacon. Previous measurements of the wavefront aberrations have found an average peak-to-valley and root-mean-square (RMS) wavefront error of 0.77 micrometers and 0.15 micrometers, respectively. Measurements of the Zernike coefficients indicated that the correction of the first 14 Zernike coefficients is sufficient to correct the aberrations we measured. Here we show that a MEMS deformable mirror with 3.5 microns of stroke and 140 actuators is sufficient to correct these aberrations. The design, assembly and initial results for the use of a MEMS deformable mirror, SHWS and implanted fluorescent reference beacon for wavefront correction are discussed. Keywords: Shack-Hartmann Wavefront Sensor, fluorescent microscopy, biological imaging, Adaptive Optics, Drosophila Melanogaster

1.

INTRODUCTION

Changes in the index of refraction due to tissue composition limit the resolving power of biological microscopy [2-4]. This effect is more pronounced in deep tissue imaging where the light travels through many layers of cellular structures including cytoplasm and the plasma membrane. Many important biological processes occur in deep tissue such as stem cell division, neurogenesis and the key developmental events following fertilization [5]. A method that can be used to improve deep tissue imaging is Adaptive Optics (AO). AO is a technique used in telescopes to measure and correct the aberration introduced by turbulence in the optical path [6, 7]. AO has also been applied to vision science to enhance our understanding of the human eye [8, 9]. The idea for using adaptive optics for microscopes is relatively new and a lot of work is still needed [10]. Most adaptive optics microscope systems so far have not directly measured the wavefront due to the complexity of adding a wavefront sensor in an optical system and the lack of a natural point-source reference such as the “guide-star” used in astronomy and vision science. Instead, most AO scanning microscopy systems have corrected the wavefront by optimizing a signal received at a photo-detector using a hill-climbing algorithm [10]. While there is a lot of important research being done in AO microscopy, many of the AO systems are specific to each microscope and a universal method for measuring the wavefront (or the results of the correction algorithm) is not currently available. Marcus Feierabend et al., developed a method of obtaining wavefront measurements in highly scattering samples by using a short pulse of light [11]. The MEMS Adaptive Optics IV, edited by Scot S. Olivier, Thomas G. Bifano, Joel A. Kubby, Proc. of SPIE Vol. 7595, 75950I · © 2010 SPIE · CCC code: 0277-786X/10/$18 · doi: 10.1117/12.846380

Proc. of SPIE Vol. 7595 75950I-1

length of the pulse served to distinguished scattered light coming from focus by using a coherence gating technique. This method produces good measurements but it requires a sample with a relative high scattering coefficient. Booth described some of the difficulties associated with the utilization of a Shack-Hartmann wavefront sensor (SHWS) in AO microscopy [10]. Most of these difficulties can be overcome if a suitable fluorescent point source could be found. Haro et al. developed a technique that utilized the natural fluorescence of lipofuscin to form an incoherent point like source for conventional Shack-Hartmann sensing [12]. Beverage, Shack, and Descour used a fluorescent microsphere to measure the three dimensional point spread function of a 40X objective by measuring the wavefront at the aperture of the objective [13]. A method for measuring and correcting the wavefront aberrations induced by a Drosophila embryo using a Shack-Hartmann wavefront sensor, an imbedded florescent microsphere “reference beacon” and a micro-electromechanical deformable mirror will be presented here. The drosophila embryo is well suited to analysis as it is approximately 200 μm in diameter, rich in cytoplasm and amenable to experimental manipulation.

2. METHODS Figure 1 shows the design of the system used to measure and correct the wavefront aberration introduced by the drosophila embryo. A 40X objectives (Olympus Microscope, Center Valley, PA) with a numerical aperture of 0.75 was used. L1 and L2 are180 and 85 mm focal length lenses that image the back aperture of the objective (plane BP) onto the Deformable Mirror (plane DM) (Boston Micromachines, Boston, MA). The mirror M redirects the light coming off the DM onto the Shack-Hartmann Wavefront Sensor. The lenses L3 and L4 are 275 and 225 mm focal length respectively and serve to reimage the BP plane onto the Lenslet Array plane (LA). The large distance between L4 and the lenslet array allows for the excitation laser (HeNe λ=632nm) to be placed in this area along with the Science Camera (SC). The laser is directed to the optical path via the 45˚ dichroic filter (D) (Semrock, Rochester, NY). During wavefront measurement/correction the laser has a confocal set up that allows for one microsphere to be illuminated at a time. An emission filter (not shown) was also added after D to reduce the effect of scattered laser light by the embryo and allow for the Hartmann Sensor to only see the emission light. By using a 90/10 Beam Splitter (BS) the microsphere can be simultaneously imaged by the Hartmann sensor and the science camera. Immediately following the beam splitter is the wavefront sensor which is composed of a Lenslet Array (LA) (AOA Inc., Cambridge, MA) and a cooled camera (Roper Scientific, Acton, NJ). The lenslet array has 14,432 (44x44) lenses, each with a focal length of 24 mm and diameter dLA of 328 μm. After magnification the back pupil plane of the objective fills a circle of 12 lenslets in diameter at the ShackHartmann Wavefront Sensor (SHWS). Figure 2 shows the actual set-up in the lab with an Olympus inverted microscope to hold the sample and objective lens (model IX71 from Olympus Microscope, Center Valley, PA).

Fig. 1. Microscope set-up with a Deformable Mirror (DM) and a Shack-Hartmann Wavefront Sensor (SHWS). Dichroic filter (D) allows the laser light to be focused onto the sample using a confocal illumination system. Beam Splitter (BS) allows for both the Science Camera (SC) and the SHWS to simultaneously see the fluorescent microsphere.


Olympus IX71 Microscope

Science Camera

SHWS

Laser

L1

Mirror

L3

L4

DM

Fig. 2. Adaptive Optics Microscope set-up with Olympus IX71 inverted microscope and Boston Micromachines DM.

Figure 3 shows the confocal set-up for the excitation laser used to illuminate the fluorescent microspheres inside the fruit fly embryo. This set-up allows us to create a single diffraction limited spot inside the embryo. The focal length of the lens L5, L6, and L7 are 100 mm, 150 mm, and 45 mm respectively. We also introduce a variable pinhole which allows for the spot size to be varied inside the embryo. This set-up is similar to the illumination used in confocal microscopy where sometimes it is require to increase the size of the pinhole to allow more light into the sample. In confocal microscopy the larger pinhole comes with the cost of reducing the resolving power, but it does not really effect the wavefront sensor measurements since the SHWS cannot resolve objects as small as the microscope diffraction limit.

Fig. 3. Confocal laser source set-up. The pinhole size can be varied to create a larger image at the object plane. The lenses are used to the re-image the output of the fiber onto the focus of the microscope.


An important part of measuring an accurate wavefront is the reference source. Astronomical adaptive optics makes use of a laser to create an artificial guide star in the mesospheric sodium layer, 90 km above sea level, which is bright enough to perform adequate wavefront measurements [7]. Powerful and expensive lasers are needed to do so, but the end result is that the adaptive optics system can correct over a much larger portion of the sky relative to the use of “natural guide stars”. The reference source used here to measure the wavefront in our AO microscope set-up is a crimson fluorescent microsphere that is 1 μm in diameter (Invitrogen, Carlsbad, CA) [14]. Sample Preparation: Embryos from the Oregon-R wild-type strain of D. melanogaster were collected for 2 hours on grape juice agar plates at 22°C. These embryos were dechorinated in a 50% bleach solution and transferred to a vial containing 1mL of phosphate-buffered saline (PBS) and 1mL of heptane. Embryos were left at the interface for 45 seconds before addition of 2mL of a formaldehyde solution consisting of 4 parts 37.5% formalin and 5 parts methanolfree 40% para-formaldehyde. These embryos were left in fixative for 25 minutes, at which time all fixative is removed and the embryos are hand devittelinized and stored in PBTA (1x PBS, 1% Bovine Serum Albumin (BSA), 0.05% Triton X-100, 0.02% Sodium Azide) [15]. Following the typical embryo preparation for imaging described above, the embryos were desiccated for 6 minutes. This step helps to maintain a negative pressure inside the embryo and allows for the microsphere solution to stay in the embryo upon injection. The microspheres were diluted in a 1:1000 phosphate-buffered saline solution. A microinjection manipulator and pull-glass capillary tubes were used to inject the solution into the embryo.

3. RESULTS Figure 4 shows the results of the injection process inside the fruit fly embryo. The injection process works very well for live embryos as they have the ability to heal the wound around the injection site. The 1:1000 microsphere solution assures that there will be a microsphere 10 micron around the center of the injection discharge (where the tip of capillary tube discharges).The microspheres spread in a random manner around the discharge site and can usually be found spread throughout the embryo. Higher microsphere concentrations could also be used as the confocal optical set-up shown in Figures 1 and 3 allows for the laser to illuminate one bead at the time.

Microspheres

Injection Site

Fig. 4: Confocal image of injected spheres in fruit fly embryo 40 microns below surface of embryo.

As shown in Figure 4 the microspheres are spread out randomly and there is a possibility to illuminate two beads at the same time, as shown in Figure 5. Since this is a possibility an experiment was set up to determined how much effect the two illuminated beads would have on wavefront sensor measurements. Figure 5 and shows images of two different experiments as seen by the science camera. Figure 5(a) shows the results of illuminating 1 bead and Fig. 5(b) shows the


result of illuminating the two beads at the same time. Note that the bead pointed at by the arrow is the same in both images and the two fluorescent microspheres in Fig. 5(b) were found relative close to each other (5 microns apart). The images obtained from the wavefront sensor camera are displayed in Fig. 6.

Fig. 5. Image a shows a single bead illuminated. Image b shows two beads illuminated (the bead on the bottom left in b and the bead in image a are the same).

From Fig. 6(a) we can see that a single illuminated fluorescent microsphere is indeed diffraction limited as seen by the Shack-Hartmann wavefront sensor. On the other hand, closer inspection of Figure 6(b) shows that two illuminated fluorescent microspheres 5 microns apart are not diffraction limited, but instead show up as a single extended source. Extended sources are not an obstacle for the Shack-Hartmann wavefront sensor and it has been shown that with a robust centroiding algorithm an extended source can actually improve the wavefront sensor measurements [7, 16, 17 and 18].

Fig. 6. Shack-Hartmann wavefront sensor image from the illumination of a single bead in (a) and from two illuminated beads in (b).

Figure 7 shows the result of reconstructing the two wavefronts obtained from the slope measurements shown in Figure 6. The two measurements are virtually identical; there is only a small change in the high frequency components of the


wavefront. In fact, the root-mean-square error between the single bead wavefront measurement and the double bead wavefront measurement is less than five percent of a wavelength.

Fig. 7. Wavefront reconstruction obtained from the slope measurements in Fig. 6. Single illuminated bead (a), double illuminated beads (b).

A reference Hartmann sensor image was obtained to cancel the aberrations introduced by the optical set up, coverslip, and mounting media. The reference image was taken by imaging a single fluorescent bead onto the Shack-Hartmann sensor. The bead was dried onto a glass slide and imaged with the coverslip and mounting media. The image was processed to obtain the location of the Shack-Hartmann spots by using a cross-correlation centroiding algorithm [16]. For each wavefront measurement, a new Shack-Hartmann sensor image was acquired with the sample prepared as describe in the previous section. The new measurement was then processed to determine the displacement of the Hartmann spots (slope measurements) relative to the reference image described above. The slope measurements were finally processed to obtain the wavefront by using a Fast Fourier Transform (FFT) reconstruction algorithm [17]. For each measurement the Peak-to-Valley (PV) and the Root-Mean-Square (RMS) wavefront errors were collected. The wavefront function was also expanded into Zernike’s circle polynomials to determine the relative strength of the different modes [18]. The Zernike polynomials are normalized and indexed as described by Jason Porter et al. [18]. The wavefront measurements were also used to analyze the point spread function (PSF) by taking the Fourier transform of the complex pupil function:

PSF ( x, y ) =

⎧ ⎛ 2π ⎞⎫ FT ⎨ P( x' , y ' ) ∗ exp⎜ i w( x' , y ' ) ⎟⎬ ⎝ λ ⎠⎭ ⎩ FT {P( x' , y ' )}

2

ξ = 0 ,η = 0

2

(1) x y ξ = ,η = λf λf

Where P is one inside the pupil and zero everywhere else, x’ and y’ are the coordinates at the pupil plane, ξ and η are the spatial frequency in the transform domain, x and y are the coordinates at the image plane, w is the wavefront measurement, λ is the wavelength at which the measurement were taken, and f is the focal length of the lens L2. Validation of the wavefront measurements we have described previously [19] can be obtained by correcting the wavefront and thus closing the loop in our system. Figure 8 shows the results of correcting the aberration introduced by the fruit fly embryo. Fig. 8(a) shows the uncorrected image of a fluorescent microsphere 30 microns beneath the surface of a fruit fly embryo. Fig. 8(b) shows the corrected image of the fluorescent bead closing the loop in the AO system. It took 4 steps to close the loop in the AO system. Each correction was done using the light coming from a single fluorescent bead reference beacon to measure the wavefront. The measurement was then applied to the deformable mirror by using a proportional gain of 0.4. Each image has been normalized to its own maximum to clearly show the details of the PSF. The improvement in Strehl was approximately 5X. The relative Strehl ratio S was obtained by


measuring the peak intensity after correction in image 8(b) divided by the peak intensity before correction in image 8(a) using the same integration time ∆t for each, as shown in Equation 2:

S relative =

I peak , e I peak , a

(2)

Fig. 8. Fluorescent microsphere 30 microns beneath the surface of a fruit fly embryo. a) Uncorrected image. b) Corrected image.

4. DISCUSSION & CONCLUSIONS One of the challenges in designing a SHWS is imposed by the amount of light the reference source can provide. Fluorescent microspheres are made out of fluorescent dye and the light emitted is proportional to the radius cubed, thus smaller beads provide less light. The size of the beads should be smaller than the diffraction limit of one subaperture of the Hartmann wavefront sensor. Note that this is larger than the diffraction limit of the microscope aperture by the ratio D (size of the aperture)/dLA(lenslet array diameter). Since the diffraction limit of the microscope is inversely proportional to the numerical aperture (NA) smaller beads are needed for higher numerical aperture systems. Fortunately the light gathered by the objective also increases with increasing NA (light gathering power ~NA2). Thus increasing the wavefront sampling by a factor of 4, increases the size of the microsphere radius by a factor of 2, and the amount of light emitted by a factor of 8. The only way to determine if a microsphere, or any fluorescent source, will work is to image it into a SHWS using the objective, as shown in Figure 1. In order to increase the speed of the AO loop the bead size should be maximized. For the first time we have shown that it is possible to use fluorescent beads as “artificial reference beacons” implanted in a biological sample to close the loop of an adaptive optics imaging system to improve the Strehl ratio by a factor of 5X. The double bead experiment shown in Figures 5,6 and 7 demonstrate that is possible to measure the wavefront coming from an extended source inside a biological tissue. It also demonstrates that by using the confocal setup shown in Figure 3 we can illuminate fluorescent beads dispersed throughout the tissue and obtain a good measurement of the wavefront aberrations. The confocal set up would essentially create a spot that is close to diffraction limited as seen by the SHWS that can be used as a reference source. The light that comes from the out of focus planes would show up as background and does not have any effect of the slope measurements as long as a robust centroiding algorithm is used. An emerging field in adaptive optics is tomography AO, where multiple reference beacons together with multiple SHWS are used. The information from each wavefront sensor is then processed using a reconstructor to acquire a tomographic


image of the changes in the index of refraction in the optical path [7]. One of the advantages of using tomography AO is that it can provide information on the depth dependence variations of the index of refraction in the tissue thus allowing for the AO system to correct for the wavefront aberrations only in the optical path. This technology can also extend the isoplanatic angle by correcting wavefront aberrations that are common to a larger field of view. By depositing multiple fluorescent beads that fluoresce at different wavelength into the biological sample and using multiple wavefront sensors with appropriate emission filters we can also apply the tomographic techniques that have been developed for astronomical AO.

5. ACKNOWLEDGMENTS This work has been partially supported by the National Science Foundation Science and Technology Center for Adaptive Optics, managed by the University of California at Santa Cruz under Cooperative Agreement No. AST 9876783 and by the National Science Foundation Science and Technology Center for Biophotonics, managed by the University of California, Davis, under Cooperative Agreement No. PHY 0120999. Oscar Azucena was supported by University of California Systemwide Biotechnology Research & Education Program GREAT Training Grant 2008-19, Jian Cao and Justin Crest were supported by NIH (GM046409), William Sullivan by the California Institute for Quantitative Biosciences (QB3) and Peter Kner by the Center for Biophotonics Science and Technology. We would like to thank Steve Lane and Sebastian Wachsmann-Hogiu from the NSF Center for Biophotonics Science & Technology (CBST) for lending us their camera. We would also like to thank John Sedat from the University of California, San Francisco for his support in this project.

REFERENCES 1. Van Helden, A., The Invention of the Telescope, Trans. Am. Phil. Soc. 67, pp. 20-21 (1977). 2. A. Dunn, Light scattering properties of cells, Dissertation, University of Texas, Austin http://www.nmr.mgh.harvard.edu/∼adunn/papers/dissertation/node7.html, 1998. 3. M. Schwertner, Specimen-induced distortions in light microscopy, J. Microscopy 228, pp. 97-102 (2007). 4. M. Schwertner, M. J. Booth, M. A.A. Neil & T. Wilson, Measurement of specimen-induced aberrations of biological samples using a phase stepping interferometer, 213, pp. 11-19 (2003). 5. Neil A. Campbell, Jane B. Reece , Biology, Benjamin Cummings, San Francisco. 2002. 6. Babcock, H. W., The possibility of compensating astronomical seeing, Pub. Astron. Soc. Pac., pp. 229-236 (1953). 7. Hardy, J. W., Adaptive Optics for Astronomical Telescopes, Oxford University Press, New York. 1998. 8. Liang J., B. Grim, S. Goelz, and J. F. Bille, Objective measurement of wave aberrations of the human eye with the use of a Hartmann-Shack wavefront sensor, J. Opt. Soc. of Am A 11, pp. 1949-1957, (1994). 9. J. Liang, D. R. Williams, D. T. Miller, Supernormal vision and high-resolution retinal imaging through adaptive optics, J. Opt. Soc. Am. A 14, pp. 2884-2892 (1997). 10. M. J. Booth, Adaptive optics in microscopy, Phil. Trans. A, Math Phys. Eng. Sci. 365, pp. 2829-2843 (2007). 11. Marcus Feierabend, Markus Ruckel, and Winfried Denk, Coherent-gated wavefront sensing in strongly scattering samples, Opt. Lett., 29, pp. 2255-2257 (2004). 12. L. D. S. Haro and J. C. Dainty, Single-pass measurements of the wave-front aberrations of the human eye by use of retinal lipofuscin autofluorescence, Opt. Lett. 24, pp. 61-63 (1999). 13. J. L. Beverage, R. V. Shack, and M. R. Descour, Measurement of the three-dimensional microscope point spread function using a Shack-Hartmann wavefront sensor, J. Microscopy 205, pp. 61-75 (2002). 14. Invitrogen Corporation, Fluorescence SpectraViewer, http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.reg.us.html, Last Accessed: 8/15/2008. 15. Rothwell, W.F., and W. Sullivan, Fluorescent analysis of Drosophila embryos. In Drosophila Prot., W. Sullivan, M. Ashburner, and R.S. Hawley, editors. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY. pp. 141–157 (2000). 16. S. Thomas, T. Fusco, A. Tokovinin, M. Nicolle, V. Michau and G. Rousset, Comparison of centroid computation algorithms in a Shack-Hartmann sensor, MNRAS 371, pp. 323-336 (2006). 17. Lisa A. Poyneer, D. T. Gavel, J. M. Brase, Fast wave-front reconstruction in large adaptive optics systems with use of the Fourier transform, J. Opt. Soc. Am. A, 19, pp. 2100-2111 (2003).


18. J. Porter, H. Queener, J. Lin, K. Thorn, A. Awwal, Adaptive Optics for Vision Science, Wiley-Interscience, New Jersey, 2006. 19. Oscar Azucena, Joel Kubby, Justin Crest, Jian Cao, William Sullivan, Peter Kner, Donald Gavel, Daren Dillon, Scot Olivier, Implementation of a Shack-Hartmann wavefront sensor for the measurement of embryo-induced aberrations using fluorescent microscopy, Proc. SPIE 7209, p. 720906 (2009).
