Uncovering the correspondence between multiscalar anatomical patterning, cellular morphology, electrophysiological function, and cell type defined by transcriptomics is a current challenge in understanding complex organs such as the mammalian brain. Multiplexed fluorescent in situ hybridization (mFISH) can spatially profile the transcriptomic state of cells within intact tissues by tagging single mRNA transcripts with fluorescent labels. These labels can be stripped or silenced and the targets re-probed across multiple rounds, building up a code base of mRNA readout information from fluorescent spot localizations. Ultimately tens or hundreds of genes over thousands of cells can be targeted within intact tissue, yielding precise transcriptomic cell type identification while retaining essential spatial and morphological information of these cells. This method carries single-molecule fluorescence microscopy into a scheme for interrogating single-cell transcriptomic identities in intact tissues. Instrumentation and algorithms for imaging, detection, segmentation, and analysis are translated from single-molecule imaging to this application. We have built a mFISH pipeline aimed at robust and repeatable methods for quantifying the distribution of single mRNA molecules in situ and ultimately mapping the abundance of neuronal cell types in the mouse and human brain. Scaling a single-molecule method to the necessary throughput involves many challenges we have overcome with solutions that should be applicable to the wider single-molecule imaging field. We will present these outcomes along with data from our production pipeline for mouse and human cortex tissue. We also connect the mFISH readout to other experimental methods performed on the same tissue. As an example, use case we have probed the transcriptomic type of cells previously targeted in patch clamp and optogenetic connectivity experiments. Light sheet microscopy is used to interrogate intact brain tissue slices 350 microns thick to identify cell morphology, anatomical location, connectivity, and transcriptomic identity on a cell-by-cell basis. Connecting the results of the patch clamp and optogenetic experiments to those from mFISH yields correspondences between these previously disparate metrics. A second pipeline development for these applications is underway with preliminary results using mFISH to disambiguate transcriptomic cell type in mouse and human neuronal connectomic experiments.

Spatial transcriptomics through single-molecule imaging

2019 Spring National Meeting

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