Table 1.

Single-cell technologies to study in situ complex tissue architecture for drug target discovery

Technology	Approach	Tissue	Advantages	Disadvantages	Resolution	Platform	Ref.
Proteomics
Co-detection by indexing (CODEX)	Tissues are stained with oligo-conjugated antibodies and are specifically detected by reporters that are imaged in cycles with the use of a standard microscope	FFPE; fresh frozen tissue	Reagents available for custom conjugating of antibodies Non-destructive staining procedure that allows morphological analysis after image acquisition	Multiple rounds of imaging with long acquisition time Limited commercially available antibody panels	Subcellular	Akoya	145
Multiplexed ion beam imaging (MIBI)	Tissues are stained with antibodies conjugated to heavy metals and are imaged using a specialized mass cytometer	FFPE; fresh frozen tissue	–	Long acquisition time	Subcellular	IonPath	146
Imaging mass cytometry (IMC)	Tissues are stained with antibodies conjugated to heavy metals and imaged using an atmospheric laser ablation chamber interfaced to a mass cytometer	FFPE; fresh frozen tissue	Large selection (>100) of validated antibodies	–	Subcellular	Fluidigm	147
Transcriptomics
Spatial transcriptomics	Tissues are attached to slides that contain barcoded probes capable of capturing RNA from the permeabilized sample cDNA synthesis occurs on the slide and is subsequently used for sequencing	FFPE; fresh frozen tissue	–	–	50–100 μm	Visium 10× Genomics	148
Multiplexed error-robust fluorescence in situ hybridization (MERFISH)	Uses combinational labelling with sequential imaging and error-robust barcoding of individual mRNAs	Fresh frozen tissue	High-throughput, single-cell resolution with up to 1 cm2 of tissue imaged per single run High multiplexing power that measures thousands of transcripts Subcellular localization of transcripts Can detect low-expression genes with single-molecule sensitivity	Molecular crowding of signal and possible spatial overlapping of signal Lengthy imaging workflow Depends on a finite number of hybridized probes to known mRNA sequences	Subcellular	Vizgen	149
Slide-seq	A monolayer of DNA-barcoded beads placed on a slide are set to capture the RNA from tissue placed onto that slide	Fresh frozen tissue	–	Typically detects a low number of genes Incompatible with FFPE	Cellular (~10 μm)	NR	150
RNAscope	Novel in situ hybridization assay for detection of target RNAs within intact cells or tissue Based on proprietary probe design (‘ZZ’ oligonucleotide probe pairs) to amplify target-specific signals but not background noise from non-specific hybridization	FFPE; fresh frozen tissue	Low background noise with a single RNA molecule level of sensitivity within intact cells The probe design (based on short target regions) allows successful hybridization of partially degraded RNA (degraded-sample compatible) Suitable when the target cannot be detected by antibodies (commercial antibodies unavailable, targets are low abundance, extracellular targets)	Typically detects a low number of targeted transcripts	Cellular	Bio-Techne	151
Proteomics and transcriptomics
Digital spatial profiling	The tissue slide is stained with fluorescence labelled reagents to select a region of interest Tissues are also stained with a panel of proteins or RNA targets of interest by using specialized UV-cleavable oligo-barcodes attached to either a target complementary sequence (transcriptomics) or a target antibody (proteomics) The oligos are cleaved from the region of interest and counted for digital quantification	FFPE; fresh frozen tissue	Non-destructive staining procedure for tissues Quantitative expression data	Limited markers (three) for visualization	Regions of interest comprising many cells	NanoString	152
Sequential fluorescence in situ hybridization (SeqFISH)	Sequential rounds of fluorescent in situ hybridization and imaging	Fresh frozen tissue	Multiplexing (>10,000 molecules) Multiomics capability No quantification bias caused by the use of reverse transcription Can detect low copy number mRNAs that are undetectable using scRNA-seq or in situ hybridization	Molecular crowding of signal and spatial overlapping of signal	Subcellular	NR	153
Deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq)	Microfluidic barcoding of mRNAs and proteins in tissues on slides is followed by high-throughput sequencing	FFPE	Microfluidic device that requires little microfluidics expertise	Limited resolution, might not ensure single-cell readouts	Cellular (~10 μm)	NR	154

FFPE, formalin-fixed paraffin-embedded; NR, not reported; scRNA-seq, single-cell RNA sequencing.