In general, spatial resolution transcriptomics is mainly divided into two categories: One is an image-based method based on in situ hybridization, in which staining and barcoding RNA in its original position are the key points to achieve multiple detection. The second category is based on in situ capture and sequencing: the spatial information is marked by barcoded primers attached to a solid surface, and then the spatial information is retrieved by high-throughput sequencing. Image-based methods originating from single-molecule RNA in situ hybridization are mainly divided into fluorescence in situ hybridization and fluorescence in situ sequencing. Fluorescence in situ hybridization uses labeled fluorescent probes to determine RNA/DNA abundance in tissues or cells, and the first example of imaging a single RNA species can be attributed to single-molecule fluorescence in situ hybridization (smFISH) [14], which provides absolute quantification of the copy number and localization of RNA molecules in cells. However, due to the limitation of fluorescence signal overlap, it can only be used for imaging low-abundance RNA. Sequential fluorescence in situ hybridization (seqFISH) [15] is a multiplexed smFISH with tens of thousands of distinct transcripts detected. Through continuous rounds of hybridization, imaging, and probe stripping, a single transcript is detected multiple times, which improves the target detection throughput. The existing fluorescence in situ hybridization methods have advantages in spatial resolution and target detection throughput, but they are expensive and time-consuming due to the need to synthesize a large number of fluorescent probes and can only measure known target transcripts.

In contrast, fluorescence in situ sequencing methods use single-base-specific fluorescence hybridization and DNA ligation reactions to determine the sequences of known or unknown transcripts in tissues. The representative technology is in situ sequencing (ISS) [3], which provides a highly multiplexed in situ detection scheme for different types of RNA by color sequence coding multiple imaging, which is not limited by the number of spectral and microscopic detection channels. Although the ISS method achieved in situ sequencing of the transcripts for the first time, only the known sequences of the target sites can be determined. Fluorescent in situ sequencing (FISSEQ) technology [16] uses random primers to generate complementary DNA (cDNA) and then circularizes the cDNA by single-stranded DNA cyclase. After magnifying the rolling circle amplification (RCA) signal, the transcript itself is sequenced to obtain the transcript sequence information, and the RNA expression at the transcriptome level can be located and analyzed simultaneously in a non-targeting manner. Image-based spatial transcriptomics technology theoretically has high spatial resolution, but due to factors such as optical crowding, there is only a low gene number detection throughput.

With the rapid development of in situ capture technology, high-throughput single-cell multi-omics sequencing technology, and increasingly sophisticated microfluidic manipulation technology, spatial omics sequencing methods have dramatically developed. It has the advantages of high throughput, high sensitivity, a large tissue area for analysis, and the ability for multimodal analysis, making it an important technique for single-cell spatial resolution analysis.

The application of spatial profiling technology is of particular concern. In theory, any tissue containing active mRNA or intact, well-fixed mRNA is suitable for spatial sequencing technology. However, due to the defects of existing methods, the application of spatial omics is limited. When choosing the tissues, try not to choose the degraded FFPE tissues or the fragile fresh frozen tissues. When placed on a slide or chip for analysis, the tissue section is easy to tear and deform. Therefore, rare and valuable tissue samples or tissues that need serial section sequencing may have technical operation problems. At the same time, low-resolution spatial barcoded chips will cause important information to be missing, especially when applying some porous tissue sections.

It can be seen that to expand the spatial sequencing application range, resolution and gene detection number are key points. To improve this, two directions can be taken: spatial barcoded array design and last-step single-cell sequencing. Barcoded oligo sequences in liquid have diffusion issues, which will hinder the tight arrangement of the barcoded array, thereby further reducing resolution. The use of solid-phase microspheres or microarray sample applicators may alleviate this problem. The high-sensitivity sequencing method is also another focus to improve the quality of spatial sequencing technology. By simultaneously improving the fabrication of the upstream spatial array and the downstream sequencing sensitivity, the application range will become wider and wider.

Lastly, spatial omics technology is widely desired; however, many researchers have a relatively high threshold to operate and obtain spatial sequencing information because of the complexity of the operation, leading to a huge demand for commercial spatial omics sequencing platforms. At present, mature commercial platforms are developed using early spatial sequencing technology with limited performance. Therefore, the commercialization of high-resolution advanced spatial sequencing technology is also one of the development directions for spatial sequencing technology.