Label-Free Optical Imaging of Chromophores and Genome Analysis at the Single Cell Level
CitationLu, Sijia. 2012. Label-Free Optical Imaging of Chromophores and Genome Analysis at the Single Cell Level. Doctoral dissertation, Harvard University.
AbstractSince the emergence of biology as a quantitative science in the past century, a lot of biological discoveries have been driven by milestone technical advances such as X-ray crystallography, fluorescence microscopy and high-throughput sequencing. Fluorescence microscopy is widely used to explore the nanoscale cellular world because of its superb sensitivity and spatial resolution. However, many species (e.g. lipids, small proteins) are non-fluorescent and are difficult to label without disturbing their native functions. In the first part of the dissertation, we explore using three different contrast mechanisms for label-free imaging of these species – absorption and stimulated emission (Chapter 2), heat generation and diffusion (Chapter 3) and nonlinear scattering (Chapter 4). We demonstrate label-free imaging of blood vessels, cytochromes, drugs for photodynamic therapy, and muscle and brain tissues with three dimensional optical sectioning capability. With the rapid development of high throughput genotyping techniques, genome analysis is currently routinely done genome-wide with single nucleotide resolution. However, a large amount of starting materials are often required for whole genome analysis. The dynamic changes in DNA molecules generate intra-sample heterogeneity. Even with the same genome content, different cells often have very different transcriptome profiles in a functional organism. Such intra-sample heterogeneities in the genome and transcriptome are often masked by ensemble analysis. In this second part of the dissertation, we first introduce a whole genome amplification method with high coverage in sequencing single human cells (Chapter 6). We then use the technique to study meiotic recombinations in sperm cells from an individual (Chapter 7). We further develop a technique that enables digital counting of genome fragments and whole genome haplotyping in single cells (Chapter 8). And we introduce our ongoing efforts on single cell transcriptome analysis (Chapter 9). In the end, we introduce our initial effort in exploring the genome accessibility at the single cell level (Chapter 9). Through the development of techniques probing the single cell genome, transcriptome and possibly epigenome, we hope to provide a toolbox for studying biological processes with genome-wide and single cell resolution.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:9830348
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