91�鶹����

Research of the Shribak lab is focused on development of advanced light microscopy and computation imaging techniques, which should serve as advanced tools for biological discoveries and would answer to the live image challenges. As member of a small team, which was lead by Dr. Inoué and Dr. Shimomura, Michael Shribak contributed to discovery of the polarization fluorescence of GFP crystals (PNAS, 99(7), 4272-4277 (2002)). Together with Dr. Oldenbourg, he developed a scanned-aperture PolScope, which generated a map of birefringence in the 3-dimensional orientation - a world-first (US Patent 6924893). Michael Shribak proposed new computation algorithms, which are currently used in LC-PolScope (US patents 7372567, 7239388, 7202950). He also elaborated on a theory of polarization aberration in light microscopes and polarization rectifiers (Opt. Eng., 41(5), 943-954 (2002)).

The orientation-independent differential interference contrast (OI-DIC) microscope, which was proposed in 2002 (US Patents 7564618, 7233434), allows the bias to be modulated and shear directions to be switched rapidly without mechanically rotating the specimen or the DIC prisms. A set of raw DIC images with orthogonal shear directions and different biases is captured within a second, which then used to compute the high-resolution quantitative phase image. An example of OI-DIC image of the crane fly spermatocyte (full metaphase of meiosis-I) is shown in Fig. 1. The three autosomal bivalent chromosomes are pulled apart at the spindle equator, along with one of the X-Y sex univalents located on the right. The distribution of tubular mitochondria surrounding the spindle and granual chromosome structure is clearly visible.

The new polychromatic polarizing microscopy (PPM) technique, which exploits a vector polarization interference of white light, was proposed in 2009 (US Patent 9625369). Unlike the traditional polarizing microscope, the full spectrum interference colors in the PPM appears even in specimens with low retardance levels of only a few nanometers, which was not possible before. The image brightness shows the phase distribution and the hue depicts the birefringence orientations. Simultaneous direct observation of phase and polarization images is another unique advantage first introduced by the PPM. Fig. 2 demonstrates a PPM image of spatial orientation of neurons in thin section of the mouse midbrain. Neurons oriented at 0-degrees appear green, while those a 45-degrees are purplish-blue and those at -45 degrees are pinkish-red. These colors don’t involve staining or tagging the cells with fluorescent markers: the colors are generated strictly from the light interacting with the physical orientation of each neuron. Not only does this image neurons at single-cell resolution, this image shows how these cells are interconnected. Such information is critical for learning more about how a brain works or about what might be going wrong with those interconnections during Alzheimer’s disease and other neurological conditions.

The both OI-DIC and PPM assemblies are implemented as add-ons, which can be used for extension of functionality of a regular light microscope without its modification. These new imaging modalities allow the users to observe and record unstained living cells with extremely fine details, some of which have never been seen with any other mode of microscopy before. The both assemblies are available for dissemination. They can be manufactured for other users per request.