Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization

Abstract

The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (216 = 65,536 or 39 = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping

“…This in situ protein analysis method can also be combined with nucleic acids [24][25][26][27][28][29][30][31][32] and metabolic imaging technologies [33] to enable the integrated DNA, RNA, protein and metabolic profiling of single cells in intact tissues. Moreover, a program-controlled microfluidic system [34] together with a standard fluorescence microscope can be easily made into an automatic tissue imaging platform.…”

FISH Going Meso-Scale: A Microscopic Search for Chromatin Domains

“…As a reminiscence of evolution in C-methods, the demand of ever-growing genomic resolution and higher throughput stimulate the development of chromatin imaging toward multiplexing the number of visualized genomic loci together with other nuclear landmarks, increase in the number of cells analyzed, streamlining and unifying of protocol and analysis pipelines. Apart from further expansion of multiplexed sequential FISH ( Xiao et al, 2020 ; Takei et al, 2021 ), recent coupling of Oligopaint probes for targeting genomic loci and fluorescent in situ sequencing (OligoFISSEQ) demonstrated the potential for imaging more targets in fewer rounds of sequencing and with higher resolution, than chromatin tracing and DNA-MERFISH ( Nguyen et al, 2020 ). The gap between imaging-based and sequencing-based methods of spatial genome probing is progressively shrinking.…”

Multiplexed Single-Cell in situ RNA Profiling

“…To keep the pre-decoding probes hybridized to their targets during signal removal, photobleaching has been explored to erase the staining signals ( Figure 1B ) ( Xiao and Guo, 2015 ; Xiao et al, 2020 ). This approach enables the efficient signal elimination while maintaining the RNA integrity.…”

“…To visualize a large number of varied RNA species directly in their original cellular environment, a number of in situ hybridization technologies have been developed. These methods include error-robust fluorescence in situ hybridization (MER-FISH) ( Chen et al, 2015 ), sequential hybridization ( Eng et al, 2019 ) and reiterative hybridization ( Xiao and Guo, 2015 ; Shaffer et al, 2017 ; Mondal et al, 2018b ; Xiao and Guo, 2018 ; Xiao et al, 2020 ). Each cycle of these approaches is mainly composed of three steps ( Figure 1A ).…”