Fluorescence control method of light-controlled fluorescent protein marker biological tissue embedding sample

Abstract

The invention discloses a fluorescence control method of a light-controlled fluorescent protein marker biological tissue embedding sample. Activation light is used for activating the light-controlled fluorescent protein on the surface layer of the sample, and exciting light is used for exciting the light-controlled fluorescent protein on the surface layer to emit fluorescence. The method comprises the following steps: selecting appropriate wavelength ranges of the activation light and the exciting light, and controlling an appropriate included angle between the activation direction and the upper surface of the sample at the same time, thereby realizing the precise control of the surface layer fluorescence of the light-controlled fluorescent protein marker biological tissue embedding sample, wherein the activation is convenient, and the thickness of the activated surface layer is thin. Only the fluorescence on the surface layer of the biological tissue embedding sample is activated when the fluorescence control method disclosed by the invention is used for activating; the exciting light excites the activated surface layer to perform fluorescence imaging when the method is applied to the fluorescent tomography of the biological tissue embedding sample, the thickness of the activated surface layer of the sample is the axial resolution of the fluorescence imaging, and the axial resolution is high.

Description

technical field [0001] The invention belongs to the field of fluorescence microscopic imaging, and in particular relates to a fluorescence control method for embedding samples of biological tissues marked with light-controlled fluorescent proteins. Background technique [0002] The photophysical properties of some fluorescent proteins can be precisely controlled by light, and these fluorescent proteins are called light-controlled fluorescent proteins. It includes at least the following two categories, photoactivatable fluorescent proteins and photoconversion fluorescent proteins. Light-activated fluorescent proteins do not fluoresce in the initial state, and can be excited to fluoresce after being activated by light at a specific wavelength, such as PAGFP. Photoconvertible fluorescent proteins can fluoresce in the initial state, but after being activated by light, they can be excited to fluoresce in another color, such as mEos3.1, mEos3.2, etc. The biological tissue labele...

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Problems solved by technology

[0003] Aiming at the above defects or improvement needs of the prior art, the present invention provides a fluorescence control method for labeling biological tissue-embedded samples with light-controlled fluorescent proteins, the purpose of which is to label biological tissue-embedded samples with light-sensitive fluorescent proteins Use specific wavelengths of activating light to activate the photoluminescent protein on the surface of the sample, and then excite the fluorescent protein on the surface of the sample with the corresponding wavelength of excitation light to realize the control of the surface fluorescence of the photoluminescent protein-labeled biological tissue-embedded sample, and by optimizing the fluorescence control condition to make its surface activation thickness thinner, and apply it to fluorescence tomography. Due to the lack of effective fluorescence control methods, due to the poor tomographic ability and low axial resolution during fast wide-field fluorescence imaging, the high-resolution point scanning imaging method is too slow for large-volume biological tissue-embedded samples.

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