The transcriptome of a cell contains a wealth of information about the cell’s current state and its recent history. System-wide analyses of the copy number and localization of RNAs in single cells promise to transform our understanding of many areas of biology, such as the origin and consequences of cellular heterogeneity, functions and mechanisms underlying sub-cellular localization of transcripts, and organization of cell types in tissues. Fluorescent in situ hybridization (FISH) based approaches for measuring single-cell gene expression offer high spatial resolution and single molecule sensitivity, but lack throughput in comparison with sequencing based methods.
In the first part of this thesis, we present multiplexed, error robust FISH (MERFISH) for transcriptome imaging in single cells. We labeled each cellular RNA with multiple complementary oligonucleotide probes, which contain additional readout sequences. Each RNA species is encoded with a unique combination of readout sequences. We then identified these RNAs by using successive rounds of hybridization and imaging, with a different fluorescently labeled readout probe each round. This combinatorial labeling scheme allows the number of detectable RNA species to grow exponentially with the number of hybridization rounds, but the effect of readout errors also increases. By utilizing error-robust encoding schemes similar to those common in digital electronics, we are able to match the measured sequence of on/off fluorescence signals from individual RNAs to their assigned barcodes with high efficiency and minimal error.
In the second part of this thesis, we optimize the performance of localization based super-resolution microscopy by conducting a systematic characterization of the photo-switching properties of 26 organic dyes. These properties, which include the photons per switching event, on-off duty cycle, photostability, and number of switching cycles, largely dictate the quality of super-resolution images. Our study identified the top performing dyes in each fluorescent channel, allowing us to perform super-resolution imaging in four colors.
We envision that by combining multi-color super resolution microscopy with our multiplexed RNA imaging approach, we can visualize all the RNA content, the transcriptome, of a cell in situ. The ability to perform spatially-resolved transcriptomic analysis of cells and tissues will dramatically improve our abilities to understand biology and disease. / Chemistry and Chemical Biology
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/17467198 |
| Date | 02 May 2016 |
| Creators | Chen, Kok Hao |
| Contributors | Zhuang, Xiaowei |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation, text |
| Format | application/pdf |
| Rights | open |
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