High Throughput Whole Organ Imaging Based on Multifocal Multiphoton Microscope

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1 High Throughput Whole Organ Imaging Based on Multifocal Multiphoton Microscope LBRC researchers: Peter So, Jae Won Cha, Elijah Yew, Vijay Singh External technology collaborators: Prof. Hanry Yu (University of Singapore), Prof. Ki Hean Kim (Poshtech U.), Prof. Daekeun Kim (Dankook University), Prof. Shih- Chi Chen (Chinese University of Hong Kong), Dr. Tim Ragan (Tissuevision, Inc.) Technology Overview Multiphoton excitation fluorescence microscopy has inherent 3D resolution due to the nonlinear dependence of excitation upon the incident light distribution [1, 2]. Excitation region is localized to a femtoliter volume at the focal point of a high numerical aperture objective lens. Multiphoton excitation fluorescence microscopy is routinely used in a variety of tissue imaging applications due to its excellent imaging depth, high resolution, and lower photo- damage. However, one of the practical limitations of multiphoton excitation fluorescence microscopy is its imaging speed, typically up to a few frames per second. While this imaging speed is sufficient in many cases, several classes applications require higher imaging speed. These applications include the measurement of dynamic processes including calcium signaling and action potential propagation [3], high throughput image cytometric study of tissue physiology [4-6], and clinical applications where the effects of physiological motion should be minimized. A particularly promising method is multifocal multiphoton microscopy (MMM) that parallelize the imaging process by generating an array of excitation spots [7, 8]. With a lenslet array or diffractive optical element (DOE), a number of foci are generated simultaneously and scanned together. The size of the whole scanning region corresponds to the sum of the sub- region scanned by each focus. Therefore, the imaging speed can be improved proportionally to the number of foci. One practical limitation for the imaging speed of MMM is available laser power. For typical titanium- sapphire laser with several Watts of output, approximately one hundred foci can be effectively generated for tissue imaging resulting in approximately two orders of magnitude improvement in imaging speed. Biomedical Application Potential Compared with point scanning multiphoton microscope, MMM approach offers up to about one hundred times improvement in imaging speed with negligible loss in signal to noise level [9]. However, with increasing imaging depth and increasing scattering loss, fewer foci can be generated, limited by the total laser power, reducing speed gain. Further, since the MMM approach uses multiple element detectors (such as CMOS camera or multi- anode photomultiplier array), the scattering of emitted photons into several detector elements reduces image signal to noise ratio [10]. Therefore, single focus imaging will offer deeper imaging as compared with MMM approach due to the availability of high laser power and minimal sensitivity to emission photon scattering. In summary, MMM is suitable for substantially higher imaging speed while is more limited in image depth. Nonetheless, LBRC has imaged living neurons with micron level resolution in living mouse brain down to over 300 microns.

2 The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. Ongoing Projects a. Non- descan MMM with multi- anode PMT detector b. Spectral- resolved MMM c. Photon Reassignment of spectral- resolved MMM d. Automated tissue sectioning for whole organ imaging Schematics of Non- descan MMM with MAPMT detector

3 Improvement in aberration reduction

4 The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again. Imaging performance improvement in mouse kidney specimen Background Publications 1. Denk, W., J.H. Strickler, and W.W. Webb, 2- PHOTON LASER SCANNING FLUORESCENCE MICROSCOPY. Science, (4951): p So, P.T.C., et al., Two- photon excitation fluorescence microscopy. Annual Review of Biomedical Engineering, : p Svoboda, K., et al., In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature, (6612): p

5 4. Lee, W.C.A., et al., A dynamic zone defines interneuron remodeling in the adult neocortex. Proceedings of the National Academy of Sciences, (50): p Ragan, T., et al., Two- photon tissue cytometry. Methods in cell biology, : p Kim, K.H., et al., Three- dimensional tissue cytometer based on high- speed multiphoton microscopy. Cytometry Part A, (12): p Bewersdorf, J., R. Pick, and S.W. Hell, Mulitfocal multiphoton microscopy. Opt. Lett., : p Brakenhoff, G.J., et al., Real- time two- photon confocal microscopy using a femtosecond, amplified Ti:sapphire system. J Microsc, (Pt 3): p Bahlmann, K., et al., Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz. OPTICS EXPRESS, (17): p Kim, K., et al., Multifocal multiphoton microscopy based on multianode photomultiplier tubes. OPTICS EXPRESS, (18): p Representative Center Publications 1. Rowlands, C., Yew, E. Y., So, P. T. C., High Speed Multiphoton Microscopy, Biophys. J., In Press. 2. Cha, J. W., Yew, E., Kim, D., Subramanian, J., Nedivi, E., So, P. T. C., Non- descanned Multifocal Multiphoton Microscopy With Multianode Photomultiplier Tube, Biomed. Opt. Express, Submitted. 3. Cha, J. W., Tzeranis, D., Subramanian, J., Yannas, I. V., Nedivi, E., So, P. T. C., Spectral- Resolved Multifocal Multiphoton Microscopy with Multianode Photomultiplier Tube, Optics Express, Submitted. 4. Cha, J. W., Singh, V. R., Kim, K. H., Subramanian, J., Nedivi, E., So, P. T. C., Reassignment of Scattered Emission Photons in Multifocal Multiphoton Microscopy, Opt. Express, Submitted. Synergistic Funding NIH, RO1 EY Singapore- MIT Alliance 2 Singapore- MIT Alliance for Science and Technology Center Hong Kong Research Grant Council