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Project I - Technology Development for Improved Intravital Multiphoton Microscopy


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We are using mouse peritoneum as a model tissue because it is easy to handle and represents a tissue that is relatively difficult to image through. We have constructed a 2f system in which the phase modulator is optically conjugate to the back aperture of a 20x/0.5 NA dipping objective. The PSF from this objective (fluorescent spot in a solution of fluorophore) is then imaged from the side through a coverslip using a 50x/0.55 NA Mitutoyo objective with a 13mm working distance (Figure. I-18). This strategy allows us to directly view aberrations and the effects on them by introduced phase profiles.  Currently, the wavefront surface and its 2p-modulo counterpart are controlled by algorithms written in the IDL image analysis environment. These algorithms will be converted to C/C++ for faster operation necessary for live specimens.  Correction phase profiles are calculated using a simple algorithm that steps through Zernike modes and optimizes for the maximum integrated value from the imaged PSF.  Preliminary results suggest that the PSF can be relatively well corrected through peritoneum (Figure I-18).  We will use this system initially to characterize the types of aberrations that are introduced by a variety of biological tissues.  


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