INSIHGT: a multimodal 3D spatial biology platform for 3D histology
This Learning Wednesday paper note highlights “INSIHGT: an accessible multi-scale, multimodal 3D spatial biology platform,” a study introducing a 3D histochemistry method designed for deep, homogeneous tissue staining. The paper is relevant to 3D spatial biology because it connects volumetric imaging, multimodal analysis, and downstream compatibility with traditional pathology workflows.
Selected notes from the paper
“The complexity of biological systems mandates high-dimensional measurements to obtain an integrative understanding”
“Spatially resolved transcriptomics and highly multiplexed immunohistochemistry have proven to be powerful approaches to extract spatial molecular insights from tissue slices, but the two-dimensional (2D) readout limits the representativeness of the information extracted. Meanwhile, three-dimensional (3D) multiplexed visualization of tissue structural and molecular features can reveal previously unknown organization principles.”
“Here, we report the development of In situ Host-Guest Chemistry for Three-dimensional Histology (INSIHGT). INSIHGT is a user friendly 3D histochemistry method”.
“INSIHGT was designed to be a minimally perturbative, deeply and homogeneously penetrating staining method for 3D histology. […] INSIHGT involves simply incubating the conventional formaldehyde-fixed tissues in [B12H12]]2-/ PBS with antibodies, then in 2HP.CD/PBS. both at room temperature with no specialized equipment.”
“We found that INSIHGT achieved the deepest immunolabeling penetration with the best signal homogeneity throughout the penetration depth.”
“We also performed RNA integrity number (RIN) and whole genome DNA extraction analyses on INSIHGT-treated samples. We found each step of the INSIHGT protocol did not result in a significant decrease in RNA integrity number (RIN).”
“With RNA sequencing whole transcriptomic comparing an INSIHGT-treated sample and a paired control sample, the results showed essentially no differentially expressed genes profiles.”
“Hence, unsurprisingly, we found single molecule fluorescent in situ hybridization (FISH) is also applicable for co-detection of protein antigens and RNAs with INSIHGT.”
“In addition to 3D imaging of human samples, we found that INSIHGT processed samples can be retrieved and processed as naïve tissues for traditional 2D histology via paraffin wax embedding and sectioning.”
“We recognize that INSIHGT still has room for further improvements. Immunostaining penetration homogeneities for larger tissues and denser antigens can be further enhanced.”
“In addition to not interfering with downstream clinical processes, the preserved quality of special staining allows for multi-modal cross-validation of 3D fluorescent imaging findings, making INSIHGT the ideal platform choice for next generation histopathology.”
Alpenglow Perspective
This paper reinforces a central point for 3D spatial biology: tissue context matters. When molecular and structural features are measured across depth, researchers can begin to understand organization patterns that may be missed in thin sections alone. Methods that preserve tissue integrity while supporting downstream analysis are especially valuable for building confidence in the integration of 3D imaging with established pathology approaches.
