Stimulated Raman Scattering Imaging of Biomolecules and Single Cell Transcriptome Analysis of Mouse Retina

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Stimulated Raman Scattering Imaging of Biomolecules and Single Cell Transcriptome Analysis of Mouse Retina

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Title: Stimulated Raman Scattering Imaging of Biomolecules and Single Cell Transcriptome Analysis of Mouse Retina
Author: Zhang, Xu ORCID  0000-0003-1764-3662
Citation: Zhang, Xu. 2015. Stimulated Raman Scattering Imaging of Biomolecules and Single Cell Transcriptome Analysis of Mouse Retina. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
Access Status: This work is under embargo until 2019-05-01
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Abstract: Complex information within biological systems is being uncovered at an unprecedented speed thanks to the rapid technical development of a wide variety of research tools, among which imaging and sequencing technologies are attracting big attention in recent years. Optical imaging enables the visualization of the spatial distribution of biomolecules at cellular level, allowing deeper understanding of the structure and dynamics of biological systems. Fluorescence microscopy has contributed greatly to our understanding of these processes, but it relies on the use of fluorescent labels or dyes. These labels may perturb the studied systems especially for imaging small molecules, and the photobleaching problem also limits the long-term biological dynamics observation within living cells. In the first part of this dissertation, we introduce the recent development of Stimulated Raman scattering (SRS) microscopy as a noninvasive imaging technique with superior sensitivity, molecular specificity at video-rate imaging speed. It has superseded coherent anti-Stokes Raman scattering (CARS) microscopy due to the absence of non-resonant background and automatic phase matching. However, SRS imaging has been mostly demonstrated for the visualization of lipid and protein with long vibrational wavenumbers. We extend the detectability of SRS imaging into the crowded fingerprint region with characteristic signatures of more biomolecules such as nucleic acids in live cells (Chapter 2), unsaturated lipid and aromatic amino acid in multiphasic food products (Chapter 3). Noninvasiveness of SRS imaging also brings new opportunities to biomedical applications and we demonstrate its feasibility as a potential pathology diagnostic tool by generating comparable image contrast as golden standard H&E staining in human brain frozen sections (Chapter 4). We further extend SRS imaging to real-time multiband detection using a novel modulation multiplex approach (Chapter 5).
The rapid development of high throughput sequencing technologies has enabled whole genome and transcriptome wide analysis at faster speed and affordable cost, but a large number of cells are often still required for these analyses. However, cell-to-cell variation is significant and may carry important indication to the study of complex wiring in the nervous systems. In this second part of the dissertation, we explore the heterogeneity of retina using a recently developed single cell transcriptome amplification technique based on Multiple Annealing Looping Based Amplification Cycles (MALBAC), which is superior to other single cell techniques with its low amplification bias, high reproducibility rate and low dropout rate. We first classify different retinal cell populations (photoreceptor cells vs. retinal ganglion cells) and closely related subpopulations (different direction selective retinal ganglion cells) (Chapter6). We further study the molecular divergence of an unsolved ON-OFF retina circuit responsible for direction selectivity function. We show that the inhibitory interneurons responsible for this function can be classified into two clusters based on the single cell transcriptome data. This clustering result strongly correlates with the ON-OFF starburst amacrine cells (SACs) based on the immunostaining results of the identified differential genes. The newly reported differential genes can potentially be used as molecular markers for ON-OFF SACs with more validation underway (Chapter 7). These new findings open up more opportunities for the functional studies on the direction-selective circuit in retina.
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