Traditional histological examination involves physical sectioning of frozen or paraffin-embedded tissue samples. Thin tissue sections several microns thick allow scientists to label and observe individual cells, but this processing method cannot analyze the three-dimensional characteristics of biological samples, and only a small part of the complete sample is analyzed, while most parts of the sample are not obtained. The tissue clearing and volumetric fluorescence imaging technology enables scientists to directly analyze complete samples, thereby exploring the spatial relationships and structural features of biological samples that cannot be detected by traditional histological examinations.

Figure 1．Imaging of tumor tissue samples

CD BioSciences has a series of advanced imaging equipment and a service support team with rich experience in the imaging field. Through tissue clearing and volumetric fluorescence imaging, we can provide you with high-quality tissue imaging services to realize the visual analysis of complete organs and living bodies. If you have any needs, please feel free to contact us.

Tissue Clearing and Volumetric Fluorescence Imaging

Although there are many types of tissue clearing and volumetric fluorescence imaging technologies, their basic principles and steps are similar. The main difference lies in the reagents, instruments, and experimental time used in each step adjusted according to different experimental purposes. In addition, the order of the steps may be slightly different. The technique mainly includes four steps:

Figure 2．General steps for tissue permeabilization, clearing and 3D imaging (Almagro J, et al. 2021).

In recent years, with the improvement and dissemination of tissue clearing and volumetric fluorescence imaging technology, it has been applied in developmental biology, immunology and oncology research. The details of the reagent combination and imaging system for tissue clearing and volumetric fluorescence imaging technology are as follows:

Core Reagent Set

Tissue clearing and volumetric fluorescence imaging technology first uses a series of reagents to construct cocktail formulas of different components and concentrations for fixation, dehydration, permeabilization and refractive index matching. The transparent fluorescence three-dimensional imaging technology for different tissues mainly selects reagents based on factors such as epitope stability and expression, tissue texture, and marker compatibility.

Imaging System Selection

The volume of the specimen and the resolution and magnification required for the experiment determine which imaging system to use. Taking the mouse mammary gland as an example, the fluorescence microscope observed the mammary gland slice sample, and the smallest unit was the mammary gland structure; Light slice microscope observed intact breast tissue, even intact mice, and the smallest unit was cells; Confocal microscope can observe the intact breast tissue, the smallest unit that can be observed is the subcellular structure.

Figure 3．Imaging approaches for transparent tissues (Almagro J, et al. 2021).

Comparison of Imaging Systems

Imaging Systems	Resolution	Signal-to-noise ratio	Shooting speed	Sample volume	Sample	Data	Fluorescence quenching rate
Fluorescence stereo microscope	Low	Low	Fast	Small	Tissue	2D/3D	Fast
Light sheet microscope	Middle	High	Fast	Big	Cell	3D	Slow
Confocal microscope	High	High	Slow	Small	Subcellular	2D/3D	Fast

Application of Tissue Clearing and Volumetric Fluorescence Imaging Technology in Different Tissues

Intestine and respiratory tract: There is a single layer of epithelial structure, that is, a single layer of cells in direct contact with the basement membrane and connective tissue.

Mammary gland epithelium: It includes double-layer ducts and underlying basal cells, which together constitute the ductal tree of the mammary glands.

Skin: It is composed of complex multilayer epithelium, hard outer epidermis, different layers of extracellular matrix and a thick layer of basal adipose tissue.

Pancreas: In addition to the number and three-dimensional arrangement of the epithelial layer, there is also a large amount of connective tissue that wraps the pancreatic islets.

Organs and organisms: tissue clearing fluorescence three-dimensional imaging technology is also suitable for imaging organoids, such as various mouse and human liver cells, airways, intestines or breast organoids.

1. Almagro J, Messal H A, Zaw Thin M, et al. Tissue clearing to examine tumour complexity in three dimensions[J]. Nature Reviews Cancer, 2021: 1-13.

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