Livermore, CA 94550, USA ABSTRACT

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1 Adaptive optics widefield microscope corrections using a MEMS DM and Shack-Hartmann wavefront sensor Oscar Azucena, 1 Xiaodong Tao, 1 Justin Crest, 2 Shaila Kotadia, 2 William Sullivan, 2 Donald Gavel, 4 Marc Reinig, 4 Scot Olivier 5, Joel Kubby 1 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, th 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 We demonstrated the used of an adaptive optic system in biological imaging to improve the imaging characteristics of a wide field microscope. A crimson red fluorescent bead emitting light at 650 nm was used together with a Shack- Hartmann wavefront sensor and deformable mirror to compensate for the aberrations introduce by a Drosophila embryo. The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the structure of the sample to be resolved (510 nm). The use of the crimson beads allow for less photobleaching to be done to the science object of the embryo, in this case our GFP model (green fluorescent beads), and allows for the science object and wavefront reference to be spectrally separated. The spectral separation allows for single points sources to be used for wavefront measurements, which is a necessary condition for the Shack-Hartmann Wavefront sensor operation. Keywords: Shack-Hartmann Wavefront Sensor, fluorescent microscopy, biological imaging, Adaptive Optics, Drosophila Melanogaster 1. INTRODUCTION The telescope and the microscope have allowed scientists to study the universe and world we live in [1]. Both the microscopes and the telescopes suffer from optical aberrations created by changes in the index of refraction in the optical path. Dunn and others have studied the changes in the index of refraction inside biological tissues [2]. Their results indicate that structures with large changes in the index of refraction have large contrast ratios as long as they are near the surface of the biological sample. These changes in the index of refraction degrade the contrast ratio for objects much deeper in the tissue. The effect is much worse for samples with a lot of fine structures, since they introduce high order aberrations in the images. Schwertner et al. measured the specimen-induced aberrations for a range of typical biological samples [3]. Their results indicate that the Zernike mode representation is a useful tool for describing these aberrations. Their results also indicate that lower order aberrations are more pronounced then higher order ones, and that spherical aberrations dominate overall. Adaptive optics (AO) is a method used in the telescope for improving astronomical images. Babcock first introduced the idea of improving astronomical seeing by compensating for the atmosphere induced aberrations [4]. His proposal was to measure the deviations of the rays from all parts of the mirror and feed that information back so as to locally correct for the deviations. While the idea was scientifically sound, it had a few minor technical complications and it was not put into action until 20 years later when the first real-time AO system was used for national defense applications [5]. AO might have been conceived for the purpose of improving astronomical imaging, but other scientist soon realized how important the technology was for other areas of research. In particular, vision science is one of those fields where AO has enlightened curious researchers. The first major obstacle in adapting AO to vision science was to find a reasonable source for measuring the wavefront. The first Shack-Hartmann Wave-Front (SHWF) sensor measurements for vision science were realized by Junzhong Liang by imaging a laser spot onto the retina [6]. A few years later Liang et al. finally constructed the first closed-loop AO system for vision science [7]. MEMS Adaptive Optics V, edited by Scot S. Olivier, Thomas G. Bifano, Joel A. Kubby, Proc. of SPIE Vol. 7931, 79310J 2011 SPIE CCC code: X/11/$18 doi: / Proc. of SPIE Vol J-1

2 The idea for using adaptive optics for microscopes is relatively new and a lot of work is still needed. Most adaptive optics microscopes 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. Instead, most AO microscopy systems have corrected the wavefront by optimizing a signal received at a photo-detector by using a hill-climbing algorithm [8]. 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. Booth described some of the difficulties associated with the utilization of a Shack-Hartmann wavefront sensor (SHWS) in AO microscopy [8]. Most of these difficulties can be overcome if a suitable fluorescent point source could be found. Beverage and others found a suitable method for measuring the wavefront of a microscope objective by using fluorescent microspheres as reference sources [9]. In his research Beverage established that bigger beads (larger then diffraction limited) could be used allowing for more light to measure the wavefront. The size of the beads d bead should be smaller than the diffraction limit of the wavefront sensor when imaged through the microscope objective: λ d o bead = 2.44 = d DLO * N D d 2NAob d / LA D (1) Where λ is the wavelength at which the beads are emitting, NA ob is the Numerical Aperture of the objective, D o is the limiting aperture of the objective, and d LA is the lenslet array pitch. This could also be represented as the diffraction limit of the objective d DLO times the number of sub-apertures across the limiting pupil. Using this technique we can measure the aberration introduced by a biological sample by injecting a fluorescent bead into the sample. In order to reduce the effect of scattered light a field stop can be used. A method for measuring the wavefront in biological samples is to inject a fluorescent bead into the specimen and use its fluorescent light as a reference source for a Shack-Hartmann Wavefront Sensor (SHWS) [10, 11, and 12]. The injection of the fluorescent bead does not cause any significant damage to the live embryo since the needle used for injection is relative small (3-5 microns in diameter) and the embryo is able to repair the perforated membrane [10]. The peak emission of the reference source, a 1 μm diameter crimson bead at 647 nm (Invitrogen, Carlsbad CA), is chosen to be different from the peak of the sample s green fluorescent emission at 510 nm (science object) [11] so that the two signals can be imaged separately. In addition to reducing the impact of photobleaching on the sample, the density of the guide stars can be chosen to be sparse, so that only one guide star will appear in the field of view of the SHWS. The advantage of this method is that it provides a point-like source which is incoherent to the source of illumination, overcoming the disadvantages of using scattered light. This method also provides a direct way of measuring the wavefront as well as the effect of the corrections as the wavefront error can be constantly monitored in the AO system. 2. METHODS Figure 1 shows the design of the adaptive optics wide-field microscope. An AO system was added to the back port of an Olympus IX71 inverted microscope (Olympus Microscope, Center Valley, PA). This allowed use of the side image port for point spread function (PSF) measurements which were compared to the PSF viewed after the AO system to ensure that the AO optical system did not add aberrations. Using a very small pixel camera (flea2 with 4.65 micron pixels, Point Grey, NY) we were able to verify a very close match between the PSF before and after the AO system. The AO system was designed around an Olympus 60X oil immersion objective (Ob) with a numerical aperture of 1.42 and a working distance of 0.15 mm. Lenses L1 and L2 have 180 mm and 85 mm focal lengths, respectively, and are used to image the back pupil of the 60X objective onto the Deformable Mirror (DM) (Boston Micromachines, Boston, MA). The DM has 140 actuators on a square array with a pitch of 400 μm, a stroke of 3.5 μm and a 4.4 mm aperture. Note that 0.5 μm of stroke was lost to AO path compensation and flattening of the deformable mirror. L3 and L4 are 275 mm and 225 mm focal length lenses, respectively, and are used to reimage the back pupil of the objective onto the Shack-Hartmann Wavefront Sensor (SHWS). The system has two illumination and imaging arms, the first is a science arm in which we used a set of filters F1 and F2 (Semrock, Rochester, NY) to redirect a beam from an argon 488 nm laser (Blue Laser) to the objective for excitation of 1 μm green fluorescent beads (Invitrogen, Carlsbad CA) that are placed behind the sample. The light emitted from the green fluorescent beads is imaged by the Green Science Camera (Green SC). Filter F3 (Semrock) is used redirect the HeNe nm laser (Red Laser) through a confocal illuminator (not shown) onto the Proc. of SPIE Vol J-2

3 optical path for excitation of the crimson reference beads [10, 12]. This confocal illuminator allows us to illuminate a single crimson reference bead to create a single diffraction limited spot. The beam splitter (BS) lets 90 percent of the emitted light coming from the crimson reference beads go to the SHWS for wavefront measurement and 10 percent for imaging in the Crimson Science Camera (Crimson SC). The SHWS is composed of a 44x44 element lenslet array (AOA Inc., Cambridge, MA) and a cooled CCD camera (Roper Scientific, Acton, NJ). Fig. 1. Adaptive optics wide-field microscope set up. Deformable Miror (DM), Shack-Hartmann Wavefront Sensor (SHWS), 488 nm laser (Blue Laser), Green flourescent Science Camera (Green SC), HeNe nm laser (Red Laser), Crimson flourescent Science Camera (Crimson SC). L1, L2, L3, L4 are 180, 85, 275, and 225 mm focal length lenses, respectively. Fold mirror D helps to bring the optical path into alignment for the SHWS. Fig. 2. Adaptive optics wide-field microscope set up with Olympus IX71 and Boston Micromachines DM. There are various ways of estimating a wavefront from the Hartmann slopes [7,13]. Two essential pieces of information are needed for this: (1) the phase difference (slopes measurements times sub-aperture size) from each sub-aperture, (2) the geometrical layout of the sub-apertures. The wavefront can then be calculated by relating the slope measurement to the phases at the edge of the sub-aperture in the correct geometrical order. A method for directly obtaining the deformable mirror commands from wavefront sensor measurements is described by Tyson [14]. First a mask with the sub-apertures must be created; this will generate the geometric layout of the sub-apertures in the aperture. The next step is to measure and record the response of all the sub-aperture slope changes while actuating each actuator. The results will be a set of linear equations which shows the response of the wavefront sensor for each actuator commands known as Proc. of SPIE Vol J-3

4 the poke matrix (also known as the actuator influence matrix). The DM commands can then be obtained by solving the following equation: s = Av (2) Where s is an n size vector obtained from the SHWS slope measurements, v is an m size vector with the DM actuator commands, and A is an nxm size poke matrix. In the linear approximation, equation 2 can be pseudoinverted to obtain an estimate of the DM commands matrix. Note that DMs are nonlinear devices, applying a large change in voltage to an actuator will not result in the same change in shape every time, but the matrix given in equation 2 performs well in a close loop system since only very small volt changes occur thus reducing the nonlinear effects. There are various methods for inverting the matrix A including singular value decomposition (SVD). The advantage of using SVD is that the mode space can be directly calculated. The noisier modes, and all the null space modes by default, can then be removed by setting a threshold on the singular value space [13]. Figure 3 shows the poke matrix and its SVD pseudo inverse. The Matrix was obtained using the method described above. Fig. 3; a) Image of real time poke matrix obtained by pushing single DM actuator and gathering wavefront sensor readings for all actuators. b) SVD Pseudo inverse of matrix in a. 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 methanol-free 40% paraformaldehyde. 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 The importance of using an AO system with a Shack-Hartmann Wavefront Sensor is that we can use one source, in this case a crimson bead, to correct for the aberrations introduced by the tissue to make wavefront corrections for features at another wavelength of interest [10, 11, 12]. The images in Figure 2 are of green fluorescent beads that were excited using the 488 nm laser and imaged with the green science camera. In Figure 2(a) the adaptive optics system is off. We Proc. of SPIE Vol J-4

5 can see some detail about structures underneath the embryo but we are not able to resolve the individual beads that make up the clumps of material shown in the image. In Figure 2(b) the AO system had been turned on and we can clearly resolve the individual 1 μm fluorescent beads. Figures 2(c) and 2(d) show cross-sectional profiles along the red lines in Figures 2(a) and 2(b), respectively. These figures show that with the AO system on we can clearly resolve the individual beads, and thus are able to obtain higher resolution structural information. Even though the wavefront aberrations were measured using the crimson beads, the corrections applied to the mirror still improves the image of the green fluorescent beads, which are more than 100 nm apart in wavelength. Fig. 4: Real time AO correction of 1 micron green fluorescent microspheres 20 μm beneath the surface of a fruit fly embryo. 4. CONCLUSIONS We demonstrated the used of an adaptive optic system in biological imaging to improve the imaging characteristics of a wide field microscope. A crimson red fluorescent bead emitting light at 650 nm was used together with a Shack- Hartmann wavefront sensor and deformable mirror to compensate for the aberrations introduce by a Drosophila embryo. The measurement and correction at one wavelength improves the resolving power at a different wavelength, enabling the structure of the sample to be resolved (510 nm). The use of the crimson beads allow for less photobleaching to be done to the science object of the embryo, in this case our GFP model (green fluorescent beads), and allows for the science object and wavefront reference to be spectrally separated. The spectral separation allows for single points sources to be used for wavefront measurements, which is a necessary condition for the Shack-Hartmann Wavefront sensor operation. 5. ACKNOWLEDGMENTS This research was supported by a grant from the California Institute for Regenerative Medicine (Grant Number RT ). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of CIRM or any other agency of the State of California. Oscar Azucena was supported by University of Proc. of SPIE Vol J-5

6 California Systemwide Biotechnology Research & Education Program GREAT Training Grant , Shaila Kotadia and Justin Crest were supported by NIH (GM046409), William Sullivan by the California Institute for Quantitative Biosciences (QB3). 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 Peter Kner from the University of Georgia for his support in this project. REFERENCES 1. Van Helden, A., The Invention of the Telescope, Trans. Am. Phil. Soc. 67, no. 4., pp (1977). 2. A. Dunn and R. Richards-Kortum, Three-dimensional computation of light scattering from cells, IEEE J. Sel. Topics Quantum Electron. 2, pp (1996).M. Schwertner, Specimen-induced distortions in light microscopy, J. Microscopy 228, pp (2007). 3. 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 (2003). 4. Babcock, H. W., The possibility of compensating astronomical seeing, Pub. Astron. Soc. Pac., pp (1953). 5. Hardy, J. W., Adaptive Optics for Astronomical Telescopes, Oxford University Press, New York 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 , (1994). 7. J. Liang, D. R. Williams, D. T. Miller, Supernormal vision and high-resolution retinal imaging through adaptive optics, J. Opt. Soc. Am. A14, pp (1997). 8. M. J. Booth, Adaptive optics in microscopy, Phil. Trans. A, Math Phys. Eng. Sci. 365, pp (2007). 9. 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 (2002). 10. O. Azucena, J. Crest, J. Cao, W. Sullivan, P. Kner, D. Gavel, D. Dillon, S. Olivier, and J. Kubby, Wavefront aberration measurements and corrections through thick tissue using fluorescent microsphere reference beacons, Opt. Express 18, pp (2010). 11. O. Azucena, J. Kubby, J. Crest, J. Cao, W. Sullivan,P. Kner, D. Gavel, D. Dillon, and S. Olivier, Implementation of a Shack-Hartmann Wavefront Sensor for the measurement of embryo induced aberrations using fluorescent microscopy, Proc. SPIE 7209, pp (2009). 12. O. Azucena, J. Crest, J. Cao, W. Sullivan,P. Kner, D. Gavel, D. Dillon, and S. Olivier, J. Kubby, Implementation of adaptive optics in fluorescent microscopy using wavefront sensing and correction, Proc. SPIE 7595, pp. 7950I I- 9 (2010). 13. D. Gavel, Suppressing Anomalous Localized Waffle Behavior in Least Squares Wavefront Reconstructor, Proc. Of SPIE 4839, pp (2003). 14. R. K. Tyson, Principles of Adaptive Optics 2 nd ed, Academic Press, San Diego 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 (2000). Proc. of SPIE Vol J-6