Table 1.
Comparison of different spatial transcriptomics methods.
| Method | Year | Resolution | Probes | Sample | Advantage | Limitation |
|---|---|---|---|---|---|---|
| In situ capture | ||||||
| ST | 2016 | 100 µm | Oligo probes | Fresh-frozen tissue | High throughput | Lower resolution |
| APEX-seq | 2019 | Subcellular (<10 µm) | Enzymatic tagging | Mammalian cells | Captures in situ activity; High temporal resolution | Requires specific enzyme for taggingLimited to cells with enzymatic activity |
| DSP | 2019 | 10 µm | DNA Oligo probes | FFPEFrozen tissue | Profile both RNA and proteinsFlexibility in ROI selection | Limited to predefined panelsSpecialized equipment needed |
| HDST | 2019 | 10-100 µm | Barcoded beads | Fresh-frozen tissue | High resolution Analyze large tissue areas | |
| Slide-seq | 2019 | 100-200 µm | Unique molecular identifiers (UMIs) | Fresh-frozen tissue | High resolutionHigh sensitivity | |
| DBIT-SEQ | 2020 | Subcellular (<10 µm) | Barcoded probes | Fresh-frozen tissue | High resolutionCaptures both mRNA and protein | |
| Seq-Scope | 2021 | Subcellular (<10 µm) | RNA probes | Fresh-frozen tissue | High resolutionHigh compatibility | |
| Slide-seqV2 | 2021 | 10-20 µm | Barcoded probes | Fresh-frozen tissue | Improved resolution over Slide-seq;Can detect low-abundance transcripts | |
| Stereo-seq | 2022 | Subcellular (<10 µm) | Expansion microscopy | Fresh-frozen tissue | 3D imaging capability | |
| Visium HD | 2024 | 55 µm | Bead-based | FFPE, frozen tissue | High throughputAnalyze large tissue areas | |
| Imaging-Based approaches | ||||||
| ISH | ||||||
| FISH | 1969 | 10-20 nm | DNA or RNA probes | Fixed cells or tissues | High specificity for DNA or RNA targets | Small number of targetsCross-hybridization and autofluorescence |
| smFISH | 2011 | Single-molecule | Oligo-dT probes | Fixed or live cells | Visualization of individual mRNA moleculesQuantitative analysis | Limited by the number of fluorophoresComplex imaging required |
| seqFISH | 2014 | Single-cell | Multiplexed probes | Fixed cells | Enables the detection of thousands of RNA molecules in a highly multiplexed manner | Complex image analysisHigher background signal |
| MERFISH | 2015 | Single-cell | Error-robust barcodes | Fixed cells | High multiplexing capabilityError correction | Requires high-quality imaging equipment and expertise |
| osmFISH | 2018 | Single-molecule | Multiplexed probes | Fixed cells | High multiplexingHigh sensitivity | Low signal-to-noise ratioRequire optimization for different samples |
| EEL FISH | 2022 | Single-cell | High-density probes | Fixed cells | High spatial resolutionLarge-scale gene expression analysis | |
| ISS | ||||||
| FISSEQ | 2014 | Subcellular (<10 µm) | Fluorescently labeled probes | Fixed cells | Captures all types of RNA in situSubcellular levels | Lower throughputSmall field of view |
| BaristaSeq | 2018 | Single-cell | Padlock probes | Fresh-frozen tissue | High amplification efficiencySequencing accuracy of at least 97% | Requires specific equipment and expertise |
| STARmap | 2018 | Subcellular (200-300 nm) | Optimized probes | Fresh-frozen tissue | High-resolution 3D intact-tissue sequencing | Complex sample preparationNot suitable for all types of samples |
| BARseq | 2019 | Single neuron level | Viral encoding RNA barcodes | Frozen tissue, fixed tissue | High throughputNeuronal markers | Only for neuron |
| MERSCOPE | 2022 | 100nm | Fluorescently labeled probes | FFPEFrozen tissue | High throughputNo sequencing required | |
| Xenium | 2022 | Subcellular (<10 µm) | Padlock probes | Fresh-frozen tissue | High sensitivity and specificitySupports customized gene panels | |
| Other approaches | ||||||
| LCM | 1996 | Cell-level | None | Frozen or FFPE tissue sections | High precision in isolating specific cell types | Lower throughputLimited by size and structure of the tissue |
| TSCS | 2016 | Single-cell | None | FFPE | Assesses genomic copy number variations | Lower sequencing depth |
| TIVA | 2014 | Single-cell | None | Live tissue | ST analysis in vivoCaptures dynamic gene expression | Limited by the number of photoactivatable tags |
| RAINBOW-seq | 2015 | ~1 micrometer | DNA probes | Fresh-frozen tissue | Enables multi-color imaging and quantitative analysis of mRNA | Complex probe design |