Single-cell analysis of regions of interest (SCARI) using a photosensitive tag

Abstract

The functional activity and differentiation potential of cells is determined by their interaction with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a trade-off between spatial resolution and cell profiling depth. Here, we developed a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran)-cage coupled to a set of nanobodies allowed high-resolution photo-uncaging of different cell types in areas of interest. Single cell RNA-sequencing of spatially defined CD8 + T cells was used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for analysis of spatially defined cells in diverse biological systems, including clinical samples.

Photoselective sequencing: microscopically-guided genomic measurements with subcellular resolution

“…An emerging strategy is to perform a genomic or epigenomic measurement within particular spatial regions under the guidance of microscopic visualization [10][11][12][13] . For example, laser-capture microdissection and patch pipette aspiration are early implementations of this strategy where the relevant subpopulation of cells is physically excised from the sample prior to downstream sequencing-based analysis [14][15][16] .…”

“…Within complex tissues, cells respond to environmental cues or intercellular signaling through diverse transcriptomic and epigenomic states [7][8][9] . A complete understanding of the interplay between spatial structures and sequence-based information will require new methods that simultaneously measure these properties.An emerging strategy is to perform a genomic or epigenomic measurement within particular spatial regions under the guidance of microscopic visualization [10][11][12][13] . For example, laser-capture microdissection and patch pipette aspiration are early implementations of this strategy where the relevant subpopulation of cells is physically excised from the sample prior to downstream sequencing-based analysis [14][15][16] .…”

Spatiotemporal Investigation of Intercellular Heterogeneity via Multiple Photocaged Probes

“…Recently, by coupling RNA–sequencing and microfluidic‐assisted DNA‐barcoding, a high‐resolution spatial transcriptome in tissue samples was analyzed [8a] . Similarly, ZipSeq [10] and single‐cell analysis of regions of interest (SCARI) [8b] were constructed to label and isolate cells by designed phototriggers. Antibodies are also widely used in mapping protein distribution and coupled with mass spectrometry, which enables multiplex analysis [11] .…”

Antigen-Specific T Cell Detection via Photocatalytic Proximity Cell Labeling (PhoXCELL)

“…Moreover, the lack of spatiotemporal control further rendered these methods difficult to accurately capture transient or specific T cell interactions within primary tissue samples. Meanwhile, photo-decaging techniques with spatiotemporal specificity have been used to dissect intercellular organizations in human tissues, but most of these methods cannot offer accurate and full separation of neighboring cells by unique molecular signatures. , To fulfill the urgent query of spatiotemporal and quantitative detection of antigen-specific T cells, we envisioned that a photocatalytic platform with the aforementioned remote and temporal control capability is highly desirable.…”