Phase Contrast And Interference Microscopy For Cell Biologists

Author : Zhuo Wang
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (774 download)

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Download or read book Spatial Light Interference Microscopy and Applications written by Zhuo Wang and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Phase contrast microscopy has revolutionized cell biology by rendering detailed images from within live cells without using exogenous contrast agents. However, the information about the optical thickness (or phase) is qualitatively mixed in the phase contrast intensity map. Quantifying optical path-length shifts across the specimen offers a new dimension to imaging, which reports on both the refractive index and thickness distribution with very high accuracy. Here I present spatial light interference microscopy (SLIM), a new optical method, capable of measuring optical path-length changes of 0.3 nm spatially (i.e. point to point change) and 0.03 nm temporally (i.e. frame to frame change). SLIM combines two classic ideas in light imaging: Zernike0́9s phase contrast microscopy and Gabor0́9s holography. The resulting topographic accuracy is comparable to that of atomic force microscopy, while the acquisition speed is 1,000 times higher. I exploit these features and demonstrate SLIM0́9s ability to measure the topography of a single atomic layer of graphene. Using a decoupling procedure for cylindrical structures, I extract the axially-averaged refractive index of semiconductor nanotubes and neurites of a live hippocampal neuron in culture. Owing to its low noise and temporal stability, SLIM enables nanometer-scale cell dynamics. Further, the linear relationship between the cell phase shift and its dry mass enables cell growth measurements in mammalian cells. The SLIM/fluorescence multimodal imaging allows for cell cycle dependent growth measurement, revealing that the G2 phase exhibits the highest growth rate and an exponential trend. Due to the micron-scale coherence length of the illuminating field, SLIM provides high axial resolution optical sectioning. Based on a 3D complex field deconvolution operation, tomographic refractive index distributions of live, unstained cells are obtained. Further, the optical field is numerically propagated to the far-zone and the scattering properties of tissue and cells have been studied. A scattering phase theorem was developed to bridge the gap between scattering and imaging. Other optical degrees of freedom associated with the sample, such as polarization measurement, are also demonstrated. Finally, SLIM renders the refractive index map of unstained histopathology slides to a quantitative color-coded image which is further proved to report onsite the carcinomas for prostate biopsies and calcifications for breast biopsies. The imaging signatures of SLIM report different properties of the tissue and cells compared to the gold standard of stained histopathology, which relies on a subjective practice and is sensitive to variations in the fixation and staining processes. The spatial correlations of refractive index indicate that cancer progression significantly alters the tissue organization. In particular, tissue refractive index exhibits consistently higher variance in prostate tumors than in normal regions. From the refractive index maps, I further obtained the spatially resolved scattering mean free path and demonstrated its direct correlation with tumor presence. I also studied small intestine tissue with amyloid and tonsil tissue with actinomyces. The results show that refractive index is an intrinsic marker for cancer diagnosis.