Biological sample preparation method applicable to ultra-thin section and fluorescence imaging

Abstract

The invention relates to a biological sample preparation method applicable to ultra-thin section and fluorescence imaging and belongs to the technical field of biological engineering. The method comprises the following steps of: fixing biological tissue which is marked by fluorescent protein by using 4 percent paraformaldehyde; after a sample is rinsed in a phosphate buffer solution (PBS) with concentration of 0.01 mol / L, performing gradient dehydration by using an ethanol solution, wherein dehydration concentration does not exceed 95 percent; and permeating and embedding the sample by using improved glycidyl methacrylate (GMA). An embedded biological sample which is prepared by the method has hardness for continuous ultra-thin section and can keep a fluorescence signal. The method is simple, low in cost and suitable for popularization in common laboratories.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a biological sample preparation method suitable for ultrathin section and fluorescence imaging. Background technique [0002] Since the discovery of fluorescent protein (GFP) and its derivatives (XFPs) in the 1990s and their application as a new type of marker in zoology, botany, biology, and pharmacy, life science has developed by leaps and bounds. Using modern molecular genetic technology, XFPs can be connected to any protein of interest, and then by observing its fluorescence, the spatial position, movement and interaction of the marked protein can be observed. Not only that, with the help of XFPs labeling technology, it can also specifically label various cells, such as the three-dimensional spatial distribution and connection relationship of neurons in the brain, which plays an important role in studying the structure and function of organisms. [0003] In early stud...

AI Technical Summary

Problems solved by technology

However, the fluorescence sample preparation methods provided in the above-mentioned documents still have the following limitations: (1) expensive equipment such as fast high-pressure freezers and freezer substitution instruments are used, which cannot be popularized in ordinary laboratories; (2) fast high-pressure freezers only use It is suitable for processing tissues with a thickness of less than 200 microns, so the sample preparation method based on this equipment can only be used to process biological samples with a size of less than 200 microns; It is very toxic, and it will also reduce the fluorescence of XFPs; (4) the resulting biological samples still have a certain amount of fluorescence loss, some even as high as more than 40%; (5) all operations need to be carried out at -90 ° C to -20 ° C It is carried out in an ultra-low temperature environment, and the operation is cumbersome, and only professionally trained personnel can master it.

[0005] In view of the fact that new three-dimensional imaging methods such as micro-optical tomography system (MOST) can perform continuous ultra-thin sectioning and imaging of centimeter-sized biological samples, and real-time imaging of moving slices during the slicing process, so the fluorescence of the sample is required The signal is relatively high, and the existing fluorescent sample preparation methods cannot meet the requirements of preparing large-sized biological samples and completely maintaining fluorescence

Images