Live Review: Tile Light Sheet Microscopy and Application of Tissue Clearing Technology

In the interactive session of the report, teachers raised many representative questions about the current subject direction.

Q: What is tiled light sheet technology? And what is the principle of the control of this technology?

A: Tile light sheet technology is a new type of selective plane illumination microscopy (SPIM) three-dimensional imaging technology, which increases the FOV of SPIM without increasing the thickness of the light sheet and without reducing the confinement ability of the excitation light sheet

The control principle of tiled light sheet technology:

When imaging, whether by moving the objective lens or moving the sample, it will affect the imaging quality and cause artifacts

Figure 1.

For detailed principles of tiled light sheet technology, please refer to the following documents:

1.

2.

Q: When using lectin to label blood vessels, should the tail vein be injected and then the organ should be taken out and then transparentized, or should the lectin be labeled with a staining instrument first?

Q: Which clearing method is better combined with immunostaining?

In general, fluorophores are also better preserved in aqueous environments

In addition, large tissue immunostaining is also related to sample size

Q: What clearing methods can be used for matrigel extracellular matrix glue?

Q: The collected images have a large amount of information, how to process the data? A large number of optical sections need to be spliced, how to choose the software? Also, what software is used to achieve cell segmentation and single cell tracking? Can third-party software be used for quantitative analysis? What format is the image output in?

A: The amount of large sample imaging data collected by the tiled light sheet microscope can reach hundreds of GB or TB level.

Figure 2.

At present, there are many third-party software that can realize 3D large image stitching, such as Amira and Imaris, all of which can process the original data in .

The above-mentioned third-party professional image processing software all have corresponding image analysis modules, which can realize functions such as segmentation, single-cell tracking and quantitative analysis

Q: Does tissue clearing cause chemical damage to the tissue itself? Will it destroy fluorescent substances (such as GFP) in the tissue?

A: Tissue clearing methods are divided into two categories: active clearing methods and passive clearing methods.
The clearing of tissues is accomplished by introducing external force or passive diffusion of biochemical reagents.
Connections and subtle features
.
Different tissue clearing methods have different effects on tissue morphology, transparency, transparency efficiency, lipid retention, endogenous fluorescent signals, nucleic acid substances, and immunostaining due to their different mechanisms
.
For example, the organic solvent clearing method in the passive clearing method, by first removing the moisture of the tissue, and then using a high refractive index organic solution for refractive index matching, although it achieves a good transparency effect, it will quench the signal of the fluorescent protein.
kill effect
.
The hydrophilic groups in fluorescent proteins make the hydrophilic solvent clearing method more conducive to the preservation of fluorescence
.
The passive hydrophilic solvent clearing method gradually replaces the low-refractive index tissue fluid, cell fluid or high-refractive index lipid by immersion to achieve the same refractive index.
Although this method is more conducive to fluorescence preservation, it takes a long time
.
The active clearing method based on CLARITY/SHIELD technology starts from protecting the conformation of fluorescent proteins, not only can retain endogenous proteins and nucleic acids, but also accelerate the removal of lipids by electrophoresis, which improves the efficiency of clearing
.

references:

Feng Yi.
Three-dimensional imaging of medical tissue transparency [M].
Fudan University Press, 2020: 3-21.

Chung et al.
, Structural and molecular interrogation of intact biological systems.
Nature, 2013.

Park et al.
, Protection of tissue physicochemical properties using polyfunctional crosslinkers.
Nature Biotechnology, 2019.
 
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The development of tissue clearing technology and light sheet fluorescence microscopy enables researchers to observe and functionally analyze the internal structure, physiological and pathological characteristics of biological tissues from macro to micro
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