Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems

Abstract

SUMMARY Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (C e 3D)

“…Recently described tissue clearing techniques have been valuable for visualization of macroscale 3D tissue architecture, suggesting that such approaches may also allow examination of the fine-grained relationships between various cell populations and their microanatomical surroundings. However, consistent with reports by others (2,3,8,12), in our hands all major clearing methodologies (CUBIC, CLARITY, PACT, SeeDB, ClearT, ClearT2, Scale, AbScale, SWITCH, DISCO-based methods, BABB) failed to simultaneously yield optimal tissue transparency, bright reporter protein fluorescence, strong immunolabeling via directly conjugated antibodies, as well as minimally perturbed tissue and cellular morphology (SI Appendix, Figs. S1 and S2 A and B and Table S1).…”

“…Hydrogel-embedding methods, which remove tissue lipids, can result in extensive protein cross-linking and poor epitope-based immunolabeling (6,7). A recent glutaraldehydebased method successfully demonstrated volumetric multiplexed imaging, but uses suboptimal fixation reagents and extreme pH treatments that can compromise antibody-based labeling and increase tissue autofluorescence, while also requiring multiple timeintensive incubation steps and complex 3D image registration algorithms (8). The C e 3D technology reported here is largely devoid of these limitations, inducing rapid optical transparency in most tissues using simple and inexpensive reagents and permitting highquality, multiplexed, large volumetric microscopy of cells with varied morphology and phenotypic complexity in structurally intricate organs.…”

Large Scale Imaging by Fine Spatial Alignment of Multi-Scanning Data with Gel Cube Device

“…It frequently happens that undesired noise deteriorates the laser illumination imaging, and many studies of measurement systems [16,17], image algorithms [18,19], and reagent for sample preparation [20,21] have been improved to enhance image quality. However, achieving large-scale (millimeters) high-resolution 3D imaging remains difficult, due to the trade-off between the viewing range and NA of an objective lens.…”

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

“…On the other hand, some antibodies, such as Tuj1 or Map2, often labeled only the surface of the tissues. This might be improved by other methods, such as SWITCH 15 . Another potential limitation is that it might be difficult to preserve fine protein structures in ACT-processed tissue because of the tissue swelling and shrinkage during the tissue-clearing step.…”