US-12626475-B2 - Sample observation device and sample observation method

US12626475B2US 12626475 B2US12626475 B2US 12626475B2US-12626475-B2

Abstract

In a sample observation device, an image acquisition unit 6 acquires a plurality of pieces of image data of a sample in a Y-axis direction, and an image generation unit generates luminance image data on luminance of the sample on the basis of the plurality of pieces of image data, binarizes luminance values of each of the plurality of pieces of image data to generate a plurality of pieces of binarized image data, and generates area image data on an existing area of the sample on the basis of the plurality of pieces of binarized image data.

Inventors

Satoshi Yamamoto
Masanori Matsubara

Assignees

HAMAMATSU PHOTONICS K.K.

Dates

Publication Date: 20260512
Application Date: 20210614
Priority Date: 20200616

Claims (20)

1 . A sample observation device comprising: an irradiation optical system comprising an optical shaping element, configured to irradiate a sample with planar light on an XZ surface; a scanning unit comprising a moving stage, configured to scan the sample in a Y-axis direction to pass through an irradiation surface of the planar light; an imaging optical system comprising a lens, having an observation axis inclined with respect to the irradiation surface and configured to image an observation light generated in the sample by irradiation with the planar light; an image acquisition unit comprising an image sensor, configured to acquire a plurality of pieces of XZ image data corresponding to an optical image of the observation light imaged by the imaging optical system; an image generation unit comprising a processor, configured to generate observation image data of the sample on the basis of the plurality of pieces of XZ image data acquired by the image acquisition unit; and an analysis unit comprising a processor, configured to extract information on the sample on the basis of the observation image data and to execute analysis on the sample, wherein the image acquisition unit acquires a plurality of pieces of the XZ image data in the Y-axis direction, and wherein the image generation unit generates luminance image data on luminance of the sample on the basis of the plurality of pieces of XZ image data, binarizes luminance values of each of the plurality of pieces of XZ image data to generate a plurality of pieces of binarized XZ image data, generates area image data on an existing area of the sample on the basis of the plurality of pieces of binarized XZ image data, and generates Z-axis position X image data from the area image data, a pixel value of the generated Z-axis position X image data is a coordinate value indicating a position in a Z-axis direction.
2 . The sample observation device according to claim 1 , wherein the analysis unit integrates values of each of the plurality of pieces of binarized XZ image data constituting the area image data in the Z-axis direction to generate thickness X image data and couples pieces of the thickness X image data in the Y-axis direction to generate thickness XY image data on a thickness of the sample.
3 . The sample observation device according to claim 1 , wherein the analysis unit extracts a top pixel position in the Z-axis direction from each of the plurality of pieces of binarized XZ image data constituting the area image data to generate top position X image data and couples pieces of the top position X image data in the Y-axis direction to generate top position XY image data on a top position of the sample.
4 . The sample observation device according to claim 3 , wherein the analysis unit generates top luminance XY image data indicating a luminance value at the top position of the sample on the basis of the top position XY image data and the luminance image data.
5 . The sample observation device according to claim 1 , wherein the analysis unit extracts a bottom pixel position in the Z-axis direction from each of the plurality of pieces of binarized XZ image data constituting the area image data to generate bottom position X image data and couples pieces of the bottom position X image data in the Y-axis direction to generate bottom position XY image data on a bottom position of the sample.
6 . The sample observation device according to claim 5 , wherein the analysis unit generates bottom luminance XY image data indicating a luminance value at the bottom position of the sample on the basis of the bottom position XY image data and the luminance image data.
7 . The sample observation device according to claim 1 , wherein the analysis unit extracts a specific pixel position in the Z-axis direction from each of the plurality of pieces of binarized XZ image data constituting the area image data to generate specific position X image data and couples pieces of the specific position X image data in the Y-axis direction to generate specific position XY image data on a specific position of the sample.
8 . The sample observation device according to claim 7 , wherein the analysis unit generates specific luminance XY image data indicating a luminance value at the specific position of the sample on the basis of the specific position XY image data and the luminance image data.
9 . The sample observation device according to claim 1 , wherein the analysis unit integrates luminance values of each of the plurality of pieces of XZ image data constituting the luminance image data in the Z-axis direction to generate integrated luminance X image data and couples pieces of the integrated luminance X image data in the Y-axis direction to generate integrated luminance XY image data on integrated luminance of the sample.
10 . A sample observation device comprising: an irradiation optical system comprising an optical shaping element, configured to irradiate a sample with planar light on an XZ surface; a scanning unit comprising a moving stage, configured to scan the sample in a Y-axis direction to pass through an irradiation surface of the planar light; an imaging optical system comprising a lens, having an observation axis inclined with respect to the irradiation surface and configured to image an observation light generated in the sample by irradiation with the planar light; an image acquisition unit comprising an image sensor, configured to acquire a plurality of pieces of XZ image data corresponding to an optical image of the observation light imaged by the imaging optical system; an image generation unit comprising a processor, configured to generate observation image data of the sample on the basis of the plurality of pieces of XZ image data acquired by the image acquisition unit; and an analysis unit comprising a processor, configured to extract information on the sample on the basis of the observation image data and to execute analysis on the sample, wherein the image acquisition unit acquires a plurality of pieces of the XZ image data in the Y-axis direction, wherein the image generation unit generates luminance image data on luminance of the sample on the basis of the plurality of pieces of XZ image data, binarizes luminance values of each of the plurality of pieces of XZ image data to generate a plurality of pieces of binarized XZ image data, and generates area image data on an existing area of the sample on the basis of the plurality of pieces of binarized XZ image data, and wherein the analysis unit extracts a maximum luminance value in a Z-axis direction from each of the plurality of pieces of XZ image data constituting the luminance image data to generate maximum luminance X image data and couples pieces of the maximum luminance X image data in the Y-axis direction to generate maximum luminance XY image data on a maximum luminance value of the sample.
11 . A sample observation device comprising: an irradiation optical system comprising an optical shaping element, configured to irradiate a sample with planar light on an XZ surface; a scanning unit comprising a moving stage, configured to scan the sample in a Y-axis direction to pass through an irradiation surface of the planar light; an imaging optical system comprising a lens, having an observation axis inclined with respect to the irradiation surface and configured to image an observation light generated in the sample by irradiation with the planar light; an image acquisition unit comprising an image sensor, configured to acquire a plurality of pieces of XZ image data corresponding to an optical image of the observation light imaged by the imaging optical system; an image generation unit comprising a processor, configured to generate observation image data of the sample on the basis of the plurality of pieces of XZ image data acquired by the image acquisition unit; and an analysis unit comprising a processor, configured to extract information on the sample on the basis of the observation image data and to execute analysis on the sample, wherein the image acquisition unit acquires a plurality of pieces of the XZ image data in the Y-axis direction, wherein the image generation unit generates luminance image data on luminance of the sample on the basis of the plurality of pieces of XZ image data, binarizes luminance values of each of the plurality of pieces of XZ image data to generate a plurality of pieces of binarized XZ image data, and generates area image data on an existing area of the sample on the basis of the plurality of pieces of binarized XZ image data, and wherein the analysis unit extracts a minimum luminance value in a Z-axis direction from each of the plurality of pieces of XZ image data constituting the luminance image data to generate minimum luminance X image data and couples pieces of the minimum luminance X image data in the Y-axis direction to generate minimum luminance XY image data on a minimum luminance value of the sample.
12 . The sample observation device according to claim 1 , wherein the image acquisition unit acquires a plurality of pieces of the XZ image data corresponding to an optical image of the observation light having a plurality of wavelengths, and wherein the image generation unit generates luminance image data on luminance of the sample on the basis of the plurality of pieces of XZ image data corresponding to an optical image of the observation light having one wavelength of the plurality of wavelengths, binarizes luminance values of each of the plurality of pieces of XZ image data corresponding to an optical image of the observation light having another wavelength of the plurality of wavelengths to generate a plurality of pieces of binarized XZ image data, and generates area image data on an existing area of the sample on the basis of the plurality of pieces of binarized XZ image data.
13 . The sample observation device according to claim 1 , wherein the image acquisition unit acquires a plurality of pieces of the XZ image data corresponding to an optical image of the observation light having a plurality of wavelengths, and wherein the image generation unit binarizes luminance values of each of the plurality of pieces of XZ image data corresponding to an optical image of the observation light having one wavelength of the plurality of wavelengths to generate a plurality of pieces of first binarized XZ image data, generates luminance image data on luminance of the sample on the basis of the first binarized XZ image data, binarizes luminance values of each of the plurality of pieces of XZ image data corresponding to an optical image of the observation light having another wavelength of the plurality of wavelengths to generate a plurality of pieces of second binarized XZ image data, and generates area image data on an existing area of the sample on the basis of the plurality of pieces of second binarized XZ image data.
14 . A sample observation method comprising: an irradiation step of irradiating a sample with planar light on an XZ surface; a scanning step of scanning the sample in a Y-axis direction to pass through an irradiation surface of the planar light; an imaging step of imaging of an observation light generated in the sample by irradiation with the planar light using an imaging optical system having an observation axis inclined with respect to the irradiation surface; an image acquisition step of acquiring a plurality of pieces of XZ image data corresponding to an optical image of the observation light imaged by the imaging optical system; an image generation step of generating observation image data of the sample on the basis of the plurality of pieces of XZ image data; and an analysis step of extracting information on the sample on the basis of the observation image data and executing analysis on the sample, wherein, in the image acquisition step, a plurality of pieces of the XZ image data are acquired in the Y-axis direction, and wherein, in the image generation step, luminance image data on luminance of the sample is generated on the basis of the plurality of pieces of XZ image data, luminance values of each of the plurality of pieces of XZ image data are binarized to generate a plurality of pieces of binarized XZ image data, area image data on an existing area of the sample is generated on the basis of the plurality of pieces of binarized XZ image data, and Z-axis position X image data is generated from the area image data, a pixel value of the generated Z-axis position X image data is a coordinate value indicating a position in a Z-axis direction.
15 . The sample observation method according to claim 14 , wherein, in the analysis step, values of each of the plurality of pieces of binarized XZ image data constituting the area image data are integrated in the Z-axis direction to generate thickness X image data, and pieces of the thickness X image data are coupled in the Y-axis direction to generate thickness XY image data on a thickness of the sample.
16 . The sample observation method according to claim 14 , wherein, in the analysis step, a top pixel position in the Z-axis direction from each of the plurality of pieces of binarized XZ image data constituting the area image data is extracted to generate top position X image data, and pieces of the top position X image data are coupled in the Y-axis direction to generate top position XY image data on a top position of the sample.
17 . The sample observation method according to claim 16 , wherein, in the analysis step, top luminance XY image data indicating a luminance value at the top position of the sample is generated on the basis of the top position XY image data and the luminance image data.
18 . The sample observation method according to claim 14 , wherein, in the analysis step, a bottom pixel position in the Z-axis direction from each of the plurality of pieces of binarized XZ image data constituting the area image data is extracted to generate bottom position X image data, and pieces of the bottom position X image data are coupled in the Y-axis direction to generate bottom position XY image data on a bottom position of the sample.
19 . The sample observation method according to claim 18 , wherein, in the analysis step, bottom luminance XY image data indicating a luminance value at the bottom position of the sample is generated on the basis of the bottom position XY image data and the luminance image data.
20 . The sample observation method according to claim 14 , wherein, in the analysis step, a specific pixel position in the Z-axis direction from each of the plurality of pieces of binarized XZ image data constituting the area image data is extracted to generate specific position X image data, and pieces of the specific position X image data are coupled in the Y-axis direction to generate specific position XY image data on a specific position of the sample.

Description

TECHNICAL FIELD The present disclosure relates to a sample observation device and a sample observation method. BACKGROUND ART Selective plane illumination microscopy (SPIM) is known as one of methods for observing the inside of a sample having a three-dimensional structure such as a cell. As a technique related to such a method, for example, there is a sample observation device described in Patent Literature 1. The sample observation device of Patent literature 1 includes an irradiation optical system that irradiates a sample with planar light on an XZ surface, a scanning unit that scans the sample in a Y-axis direction with respect to an irradiation surface of the planar light, and an imaging optical system that has an observation axis inclined with respect to the irradiation surface and images an observation light generated in the sample by irradiation with the planar light. In this sample observation device, a plurality of pieces of XZ image data of the sample are acquired in the Y-axis direction, and pieces of X image data generated by integrating luminance values of an analysis area in the XZ image data in the Z direction are coupled in the Y-axis direction to generate XY image data of the sample. CITATION LIST Patent Literature [Patent Literature 1] Japanese Unexamined Patent Publication No. 2019-184401 SUMMARY OF INVENTION Technical Problem In the sample observation device as described above, it is possible to acquire three-dimensional information on the sample in a state where the influence of variation in a luminance value of background light is sufficiently reduced. Although various feature amounts of the sample can be analyzed from the three-dimensional information, it is desirable that the sample observation device obtain an analysis result of the sample almost at the same time as the end of a measurement operation. Therefore, it is necessary to speed up the processing required from the acquisition of the XZ image data of the sample to the analysis of the sample based on the data. The present disclosure has been made to solve the above problem, and an object of the present disclosure is to provide a sample observation device and a sample observation method capable of speeding up the processing required for sample analysis. Solution to Problem According to an aspect of the present disclosure, there is provided a sample observation device including: an irradiation optical system configured to irradiate a sample with planar light on an XZ surface; a scanning unit configured to scan the sample in a Y-axis direction to pass through an irradiation surface of the planar light; an imaging optical system having an observation axis inclined with respect to the irradiation surface and configured to image an observation light generated in the sample by irradiation with the planar light; an image acquisition unit configured to acquire a plurality of pieces of XZ image data corresponding to an optical image of the observation light imaged by the imaging optical system; an image generation unit configured to generate observation image data of the sample on the basis of the plurality of pieces of XZ image data acquired by the image acquisition unit; and an analysis unit configured to extract information on the sample on the basis of the observation image data and to execute analysis on the sample, wherein the image acquisition unit acquires a plurality of pieces of the XZ image data in the Y-axis direction, and wherein the image generation unit generates luminance image data on luminance of the sample on the basis of the plurality of pieces of XZ image data, binarizes luminance values of each of the plurality of pieces of XZ image data to generate a plurality of pieces of binarized XZ image data, and generates area image data on an existing area of the sample on the basis of the plurality of pieces of binarized XZ image data. In this sample observation device, the planar light is applied on the XZ surface while the sample is scanned on the Y axis, and the plurality of pieces of XZ image data corresponding to the optical image of the observation light generated in the sample are acquired. Then, the observation image data is generated on the basis of the plurality of pieces of XZ image data, the information on the sample is extracted on the basis of the observation image data, and the analysis regarding the sample is executed. Here, in the sample observation device, when the observation image data is generated, the luminance image data on the luminance of the sample is generated on the basis of the plurality of pieces of XZ image data, the luminance values of each of the plurality of pieces of XZ image data are binarized to generate the plurality of pieces of binarized XZ image data, and the area image data on the existing area of the sample is generated on the basis of the plurality of pieces of binarized XZ image data. By performing the analysis using the necessary image data among the generated luminance image