There had been two transformative technologies in modern systems biology: genomics, which allows all of genes and proteins in an organism to be monitored simultaneously, and single cell biology, which follows a few specific genes in individual cells with high precision in their native micro-environments.  Both techniques are powerful, but have complementary limitations: genomics averages over the heterogeneity and spatial complexity of a cell population, and single cell techniques can only probe a few genes at a time.  Integrating genomics with single cell is the next major challenge in biology. Our lab sets out to synthesize the genomics and single cell approaches.

Single cell in situ profiling by sequential hybridization and barcoding
.   Our lab's current focus is to profile gene expression in single cells via in situ "sequencing" by FISH [link]. To detect individual mRNAs, we use single molecule fluorescence in situ hybridization (smFISH) with 20mer oligonucleotide probes complementary to the mRNA sequence. By putting up to 48 fluorophore labeled probes on an mRNA, single transcripts in cells become readily detectable in situ. We have shown that almost all mRNAs that can be detected are observed by smFISH .

To distinguish different mRNA species, we barcode mRNAs with the FISH probes using sequential rounds of hybridization. During a round of hybridization, each transcript is targeted by a set of FISH probes labeled with a single type of fluorophore. The sample is imaged and the FISH probes are removed by enzymatic digestion. Then the mRNA is hybridized in a subsequent round with the same FISH probes, but now labeled with a different dye (Fig. 1). As the transcripts are fixed in cells, the fluorescent spots corresponding to single mRNAs remain in place during multiple rounds of hybridization, and can be aligned to read out a color sequence. Each mRNA species is therefore assigned a unique barcode. Thus, the number of each transcript in a given cell can be determined by counting the number of the corresponding barcode.

Figure 1. Sequential barcoding FISH (seqFISH). (a) Schematic of sequential barcoding. In each round of hybridization, 24 probes are hybridized on each transcript, imaged and then stripped by DNAse I treatment. The same probe sequences are used in different rounds of hybridization, but probes are coupled to different fluorophores. (b) Schematic of the FISH images of the cell. In each round of hybridization, the same spots are detected, but the dye associated with the transcript changes. The identity of the mRNA is encoded in the temporal sequence of dyes hybridized. (c) Data from 3 rounds of hybridizations on six yeast cells. 12 genes are encoded by 2 rounds of hybridization, with the 3rd hybridization using the same probes as hybridization 1. The boxed regions are magnified in the bottom panels. The matching spots are shown and barcodes are extracted. Different barcodes encode for different transcripts. Note the first and third elements of the barcodes are the same, corresponding to the same probes used in hybridization 1 and 3. Spots without colocalization are due to nonspecific binding of probes as well as mis-hybridization. The number of each barcode corresponds to the abundance of transcripts in single cells.

The sequential FISH (seqFISH) scheme is conceptually akin to sequencing transcripts in single cells with FISH probes. Our method takes advantage of the high hybridization efficiency of FISH (>95% of the mRNAs are detected) and the fact that base pair resolution is usually not needed to identify a transcript. The number of barcodes available with this approach scales as F^N, where F is the number of distinct fluorophores and N is the number of hybridization rounds. With 5 distinct dyes and 3 rounds of hybridization, one can detect 125 unique genes. While in principle the entire transcriptome can be covered by 6 rounds of hybridization (56=15,625), super-resolution microscopy is needed to resolve all of the transcripts in the cell.

Turning single cells into microarrays by super-resolution barcoding. In addition, our lab benefits from super-resolution microscopy to spectrally and spatially barcode different RNAs in individual cells [link].  The basic idea is straight-forward.  Under a super-resolution microscope with a resolution of 10 nm, a typical cell of (10µm)3 can be resolved into 108-109 voxels.  By comparison, there are only ~106 copies of messenger RNA in a single cell.  Thus, there is sufficient room to resolve all of the genomic messages directly in a cell. We can then uniquely identify each message by labeling it with a distinct fluorescent molecular barcode.  When we image a cell labeled this way in a super-resolution microscope, we see the cell lights up like a pointillist painting.  We can then quantitate the abundance of each gene in the cell by counting the number of the corresponding barcodes from the image. 

Figure 2. Super-resolution and combinatorial labeling allow multiplex identification and quantification of individual mRNAs in single cells.  (a-b) Individual molecules are difficult to resolve by conventional microscopy due to the diffraction limit of ~300 nm.  (c)  Super-resolution microscopy allows spatial resolution of individual molecules.  (d) The identity of RNA can be uniquely addressed by a super-resolution barcode.

This imaging based approach preserves the rich spatial cell-to-cell signaling interactions in the tissue, without the laborious and destructive process of physically isolate single cells.  We have demonstrated that over 30 genes, and potentially hundreds of genes, can be multiplexed in this fashion in single cells [link].

We believe that almost every microarray-type experiment can be done single cells using super-resolution barcoding.  Our goal is to directly visualize gene networks and protein interactions in cells, within in their native environment, with advanced microscopy.


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Caltech    last update: 11/10/2014