Fluorescece obsevation method and fluorescence obsevation apparatus

Abstract

A fluorescence observation method in which it is possible to observe plural types of fluorescence emitted from plural kinds of fluorescent molecules simultaneously without the necessity of plural kinds of exciting wavelengths and with a simple optical structure, an observed object for which a fluorescent molecule is used as a fluorescent label suffers little damage, and it is possible to use glass that is in common use as an optical material used for fluorescence observation, and a fluorescence observation apparatus are offered. A fluorescence observation method of the present invention for detecting plural types of fluorescence emitted from two or more kinds of fluorescent molecules includes: subjecting each of the two or more kinds of fluorescent molecules to multi-photon excitation by exciting light having an exciting wavelength equal to or shorter than 700nm in a visible region, to generate fluorescence upon making use of an absorption wavelength band in a deep ultraviolet region of each of the two or more kinds of fluorescent molecules; and simultaneously detecting plural types of fluorescence generated on a shorter-wavelength side or on both of the shorter-wavelength side and a longer-wavelength side of the exciting wavelength of the exciting light.

Description

technical field [0001] The present invention relates to a fluorescence observation method of observing fluorescence emitted from fluorescent molecules such as various fluorescent proteins, and to a fluorescence observation apparatus. Background technique [0002] Using fluorescent molecules to observe biomolecules is a standard observation method in the field of medicine or life sciences. Various fluorescent molecules having different fluorescence wavelengths have been developed, and various biomolecules can be observed in combination with each other using these fluorescent molecules. [0003] Among conventional methods of observing fluorescence emitted by fluorescent molecules, for example, there is a method in which fluorescent molecules are excited by single-photon excitation using light in the visible region and fluorescence generated on the longer wavelength side of the wavelength of the excitation light is detected (Stokes shift); and a method in which fluorescent mol...

AI Technical Summary

Problems solved by technology

[0005] (Problem 1) In the case of simultaneous observation of multiple types of fluorescent molecules, in order to simultaneously irradiate the sample with multiple types of excitation lights having different wavelengths from each other, an optical system for mixing the multiple types of excitation lights in the same path is required so that Complex optical structure;

[0006] (Problem 2) When fluorescence observation is performed using a plurality of excitation lights having different wavelengths from each other, the plurality of excitation lights to be collected on the sample surface are distorted due to chromatic aberration that occurs when they pass through optical systems located on their paths. cause their focal position to vary with their wavelength so that spatial coincidence is broken

As a result, this problem can lead to artifacts in the analysis of overlap between various biomolecules or the localization of biomolecules; and

[0007] (Problem 3) Although fluorescence observation is performed one by one for each fluorescent molecule while sequentially changing a plurality of excitation lights having different wavelengths, the process does not allow simultaneous observation of the fluorescent light emitted from the individual fluorescent molecules

[0011] (Problem 4) In order to collect and guide light in the deep ultraviolet region to fluorescent molecules, there are few optical members available, and there are many restrictions in the configuration of the optical system, and therefore, it is difficult to manufacture an optical system that satisfies desired specifications

[0012] (Question 5) Compared with visible light or infrared light, light in the deep ultraviolet region has greater light energy and causes large photodamage to living bodies

As a result, in the case where the object to be observed is a living sample such as a living cell, light in the deep ultraviolet region is not suitable as excitation light for observing fluorescence emitted from fluorescent molecules labeled on the sample

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