Fluorescence microscope apparatus
View Patent ↗To observe and analyze intermolecular interactions such as diffusion and/or binding behaviors of molecules in a sample in a reacting state against optical stimulation, while applying the optical stimulation to a desired region in the sample by irradiating stimulus light. There is provided a fluorescence microscope apparatus comprising: a fluorescence image-capturing optical system; a stimulus light-irradiation optical system which includes a scanner for; a control unit which acquires temporal observation data by repeatedly capturing images using said image-capturing optical system while applying optical stimulation using the stimulus light-irradiation optical system; an analysis unit; and a display unit.
1. A fluorescence microscope apparatus comprising:
an irradiation optical system which generates fluorescence by irradiating an image capture region of a sample with excitation light as a spotlight;
a fluorescence image-capturing optical system for capturing fluorescence images of image capture region;
a stimulus light-irradiation optical system which includes a scanner for applying only optical stimulation to an optional region in the sample by irradiating stimulus light, the optional region differing from the image capture region in size and/or shape;
a control unit which acquires temporal observation data by repeatedly capturing images using the image-capturing optical system while applying optical stimulation using the stimulus light-irradiation optical system;
an analysis unit which analyzes two-dimensional distribution of diffusion and/or binding behaviors of molecules through analysis of changes in fluorescence intensity caused by molecular fluctuations within a confocal volume of the sample by executing raster image correlation spectroscopy with use of the temporal observation data of the image capture region; and
a display unit which displays an analysis result from the analysis unit, wherein
said irradiation optical system includes an excitation scanner that two-dimensionally scans the irradiated excitation light on the image capture region of the sample, and
said fluorescence image-capturing optical system has a confocal aperture for a confocal detection of fluorescence light under the conditions where one pixel size is smaller than the spotlight size of the excitation light, the fluorescence light emitted from a position where the excitation light is irradiated.
2. A fluorescence microscope apparatus according to claim 1 , wherein said irradiation optical system generates fluorescence through multiphoton absorption.
3. A fluorescence microscope apparatus according to claim 1 , comprising:
a region specifying unit which specifies a part of the image capture region in which the temporal observation data is acquired as a desired region with respect to the temporal observation data, wherein
said analysis unit carries out analysis of diffusion and/or binding behaviors of molecules caused by the optical stimulation, with use of the fluorescence intensity data within the specified region.
4. A fluorescence microscope apparatus according to claim 1 , wherein the pixel size is half the size or less than a diameter of the spotlight size of the excitation light.