Here we demonstrate an evolution of sequential fluorescence in situ hybridization (seqFISH+). We show that seqFISH+ can image mRNAs for 10,000 genes in single cells—with high accuracy and sub-diffraction-limit resolution—in the cortex, subventricular zone and olfactory bulb of mouse brain, using a standard confocal microscope.
Here, we demonstrate a multiplex single-molecule in situ method, intron seqFISH, that allows the imaging of 10,421 genes at their nascent transcription active sites in single cells.
Nature Communications, 2017
The application of seqFISH to chicken embryos enabled identification of previously unknown populations of neural crest stem cells and neural stem cells occupying distinct spatial niches in the neural crest. This work was a collaboration between the Cai lab and the Bronner lab.
Nature MEthods, 2017
RNA SPOTs profiles the transcriptome without reverse transcription and library prep. SPOTs detects over 10,000 genes in vitro at the single molecule level using seqFISH. Not only is SPOTs highly accurate, it can also avoid strongly expressed housekeeping genes by simply not including probes for those genes.
Biophysical Journal, 2017
Combining CRISPR imaging and seqFISH allowed 12 chromosomal loci to be tracked in live mouse embryonic stem cells. Using a “track first and identify later” approach, all loci were tracked by live imaging and then uniquely identified by seqFISH post-fixation. This method can scale to hundreds of loci per cell.
In this collaboration with the Elowitz lab at Caltech, seqFISH was used to read out information about lineage relationships and dynamic event histories in individual mouse embryonic stem cells within their native spatial context.
Identifying the spatial organization of tissues at cellular resolution from single-cell gene expression profiles is essential to understanding biological systems. Using an in situ 3D multiplexed imaging method, seqFISH, we identify unique transcriptional states by quantifying and clustering up to 249 genes in 16,958 cells to examine whether the hippocampus is organized into transcriptionally distinct subregions.
Nature Methods, 2016
Correlation FISH (corrFISH) is a method to resolve dense temporal barcodes associated with very highly expressed genes in seqFISH experiments. Using corrFISH, we quantified highly expressed ribosomal protein genes in single cultured cells and mouse thymus sections, revealing cell-type-specific gene expression.
Imaging gene expression levels with single-cell resolution in intact tissues is essential for understanding the genetic programs in many systems, such as developing embryos and dynamic brain circuits. However, background due to sample autofluorescence is significantly higher in tissue samples than in cell culture, making it difficult to robustly detect smFISH signals in tissue. Here, we developed a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR).
We showed here that smFISH can be applied to PACT and CLARITY cleared samples. This was a collaboration of the Gradinaru and Cai labs at Caltech.
Molecular Cell, 2014
smFISH measurements in mouse embryonic stem cells revealed distinct states in their transcriptional programs. This was a collaboration of the Elowtiz and Cai labs at Caltech.
Nature methods, 2014
Birth of seqFISH! This is the original seqFISH paper. In this paper, we showed that 12 genes can be multiplexed in single cells by sequentially barcoding transcripts with seqFISH. Since then, the technique has scaled faster than Moore’s law, and in 2017 we showed that over 10,000 genes can be multiplexed by seqFISH.
Nature Methods, 2012
Profiling the transcriptome was hard before seqFISH. We proposed using super-resolution microscopy to resolve all mRNAs in cells. Originally, we wanted to look at protein binding on DNA inspired by Barbara Wold’s work on ChIP-seq, but Arjun Raj convinced us that RNA FISH is very cool.