The other day I had an conversation with someone who had an interesting interpretation of what spatial transcriptomics is. They thought the “spatial” in spatial transcriptomics refered to outer space. They thought scientists were collecting samples from outer space and this was a method to analyze them.
In reality, spatial transcriptomics is a scientific technique used to understand how genes are active in different parts of the body, almost like creating a genetic map. It describes cutting-edge technologies in the field of genomics and molecular biology that allow researchers to analyze the gene expression patterns within tissues or even entire organisms while preserving the spatial context of each individual cell.
Here are eight examples of such spatial transcriptomics platforms, in no particular order, that are in situ hybridization-based. The techniques are still based on probes binding to the transcripts but the method of detection is very diverse.
The links will take you to a freely available copy of the original publication either through ResearchGate or the webpage of the journal.
Femino and colleagues modified Fluorescence in situ hybridization (FISH) and digital imaging microscopy techniques to detect individual RNA molecules. Special probes with multiple fluorochromes were created and calibrated for single molecule detection. The researchers observed points of light in images of hybridized cells, providing data on fluorescent intensities and distances between probes, indicating single messenger RNA molecules.
- RNAscope RNAscope A Novel in Situ RNA Analysis Platform for Formalin-Fixed, Paraffin-Embedded Tissues
This is a technology with a unique tree-like probe design enabling signal amplification and background suppression. RNAscope enables single-molecule resolution while preserving tissue morphology. It can use both chromogenic dyes for bright-field microscopy and fluorescent dyes for multiplex analysis.
This platform is commercially available through ACD
Hybridization chain reaction (HCR) is an innovative nucleic acid detection and amplification method. It involves designing two hairpin-shaped DNA probes, each with a specific loop and arms complementary to a target sequence. When complementary primary probes are hybridized, the hairpin probes bind, initiating a chain reaction. A toehold region is exposed, allowing additional probes to bind sequentially, creating an autocatalytic amplification of the hybridization chain.
This platform is commercially available through Molecular Instruments
Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH) is an imaging base method employing combinatorial labeling and single-molecule fluorescence techniques. In contrast to other low throughput in situ methods this platform is using chemical cleavage for signal removal between imaging rounds, expanding the field of view, and employing multicolor imaging. These enhancements have enabled RNA profiling in over 100,000 human cells, with up to 40,000 cells measured in a single 18-hour session.
This platform is commercially available through Vizgen.
This is a non-barcoded and unamplified cyclic-ouroboros smFISH method (osmFISH) to address limitations in existing techniques. OsmFISH allows linear scaling of targets with hybridization rounds, visualizing RNA molecules as diffraction-limited spots using fluorescently labeled DNA probes. Multiple transcripts are targeted in each round, distinguished by color. The absence of barcoding enables independent analysis of each image, accommodating various expression levels. Optimized for brain tissue sections, osmFISH features a short hybridization time, reduced background via tissue clearing, and a semiautomated system for efficiency. It demonstrates a dynamic range of 1-125 molecules per cell.
- clampFISH ClampFIsH detects individual nucleic acid moleculesusing click chemistry–based amplification
ClampFISH is a method for detecting single nucleic acids in cells and tissues. This methode achieves high specificity and significant signal amplification by forming a ‘C’ configuration around the target sequence through hybridization. After that the probe ends are chemically linked using click chemistry, enhancing the fluorescence signal. This iterative process enables the detection of RNA species using low-magnification microscopy and RNA-based flow cytometry. ClampFISH’s modular design allows multiplexing of RNA and DNA detection, prevents probe detachment in expansion microscopy, and can be applied in tissue samples.
- seqFISH & seqFISH+ Transcriptome-scale super-resolved imaging intissues by RNA seqFISH+
SeqFISH+ is an advanced spatial genomics technique that enables transcriptome-level profiling of cells within their native tissue context. Traditional methods, limited by optical density, struggle to resolve individual mRNA molecules due to their high density in cells. SeqFISH+ addresses this challenge by employing a palette of pseudocolours through sequential hybridization. This approach dilutes mRNA molecules into separate images, enabling their precise localization. By using three fluorescent channels and barcoding genes within each channel, seqFISH+ achieves super-resolution imaging and multiplexing of 10,000 genes in a single cell.
The authors introduced Expansion-Assisted Iterative-FISH (EASI-FISH) in 300-μm-thick brain sections, combining Expansion microscopy (ExM) for high-resolution 3D imaging and an automated data processing pipeline. They applied EASI-FISH to the mouse lateral hypothalamic area (LHA) . The study demonstrates EASI-FISH’s potential in deciphering complex neural organization in important brain regions.
These are only eight examples of the many platforms in spatial transcriptomics and all of them have their strengths and weaknesses. These techniques not only provide valuable insights into cellular functions but also pave the way for groundbreaking discoveries in the field of spatial genomics. And who knows, maybe one day in the future we will apply these techniques or their successors on samples from other planets.
Have you tried any of these techniques? Do you have experience with any other method? Share your experience in the comments.
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