Time lapse observation method, and time lapse observation apparatus and multiphoton microscope used therefor
View Patent ↗Time lapse observation method includes: before a process to obtain a first time lapse image, capturing an image of a reference area on a sample being a partial area of a target area or an area in a vicinity of the target area being a smaller area than the target area to obtain a reference image; storing a position of a capturing area in capturing the reference image as a reference position; before a process to obtain the time lapse image performed, setting a position of the capturing area sequentially at different positions in the optical axis direction of an objective including the reference position and capturing an image at each of the positions to obtain comparison target images; and matching the capturing area with the target area, based on a comparison result of the reference image and the comparison target images.
1. A time lapse observation method matching the image capturing area with an image capturing target area on a sample set in advance, capturing an image of the image capturing target area at certain time intervals, to obtain a time lapse image, comprising:
before a process to obtain the time lapse image for a first time,
capturing an image of a reference area on the sample being a partial area of the image capturing target area or an area in a vicinity of the image capturing target area being a smaller area than the image capturing target area to obtain a reference image;
storing a position and a size of the image capturing area in capturing the reference image as a reference position, reference size respectively; and
before a process to obtain the time lapse image performed at certain time intervals,
setting a position of the image capturing area sequentially at a plurality of different positions in the optical axis direction of an objective including the reference position and capturing an image at each of the plurality of positions to obtain a plurality of comparison target images; and
matching the image capturing area in obtaining the time lapse image with the image capturing target area, based on a comparison result of the reference image and the plurality of comparison target images.
2. The time lapse observation method according to claim 1 , wherein:
the comparison target image with a highest correlation with the reference image is identified by a pattern matching process of the reference image and each of the plurality of comparison target images; and
a position of the image capturing area in the optical axis direction in obtaining the time lapse image and a position of the image capturing target in the optical axis direction are matched, based on a positional relation of the comparison target image with the highest correlation and the reference image in the optical axis direction.
3. The time lapse observation method according to claim 2 , wherein
a position of the image capturing area in an XY direction orthogonal to the optical axis direction in obtaining the time lapse image and a position of the image capturing target area in the XY direction are matched, based on a comparison result of the reference image and the comparison target image with the highest correlation.
4. The time lapse observation method according to claim 2 , wherein
a position of the image capturing area in an XY direction orthogonal to the optical axis direction in obtaining the time lapse image and a position of the image capturing target area in the XY direction are matched, based on a comparison result of the reference image and the comparison target image captured at the reference position.
5. The time lapse observation method according to claim 2 , wherein
the plurality of comparison target images are obtained by irradiation with a smaller light amount per unit area compared with a light amount per unit area to irradiate the image capturing target area in obtaining the time lapse image.
6. In the time lapse observation method according to claim 5 , wherein
the plurality of comparison target images are obtained by irradiation for a shorter illumination time per unit area compared with a irradiation time per unit area to irradiate the image capturing target area with light in obtaining the time lapse image.
7. The time lapse observation method according to claim 6 , wherein
the plurality of comparison target images are obtained by a setting of a higher sensitivity of a detector compared with a setting of a sensitivity of the detector in obtaining the time lapse image.
8. The time lapse observation method according to claim 5 , wherein
the plurality of comparison target images are obtained by irradiation with light of a weaker intensity compared with an intensity of light to irradiate the image capturing target area in obtaining the time lapse image.
9. The time lapse observation method according to claim 2 , further comprising:
every time when the time lapse image is obtained, updating the reference image, and updating the reference position by a position of the image capturing area at a time when the updated reference image was captured.
10. The time lapse observation method according to claim 9 , wherein
every time when the time lapse image is obtained, the reference image is updated by the comparison target image having the highest correlation identified by the pattern matching process from the plurality of comparison target images obtained before obtaining the last time lapse image, and the reference position is updated by a position of the image capturing area at a time when the updated reference image was captured.
11. The time lapse observation method according to claim 2 , wherein
by a movement of the objective in the optical axis direction, a position of the image capturing area in the optical axis direction in capturing the time lapse image and a position of the image capturing target area in the optical axis direction area matched.
12. The time lapse observation method according to claim 2 , wherein
by a movement of a stage on which the sample is placed in the optical axis direction, a position of the image capturing area in the optical axis direction in capturing the time lapse image and a position of the image capturing target area in the optical axis direction are matched.
13. The time lapse observation method according to claim 2 , wherein
by changing a scanning area of a scanning unit, a position of the image capturing area in an XY direction orthogonal to the optical axis direction in capturing the time lapse image and a position of the image capturing target area in the XY direction are matched.
14. The time lapse observation method according to claim 2 , wherein
by the movement of a stage on which the sample is placed in the XY direction orthogonal to the optical axis direction, a position of the image capturing area in the XY direction in capturing the time lapse image and a position of the image capturing target area in the XY direction are matched.
15. The time lapse observation method according to claim 2 , wherein
before a process to obtain the time lapse image for a first time,
a plurality of reference areas are captured to obtain a plurality of reference images;
a plurality of reference positions area stored; and
before a process to obtain the time lapse image performed at certain time intervals,
a plurality of comparison target images corresponding to each of the plurality of reference images are obtained; and
the image capturing area in obtaining the time lapse image is matched with the image capturing target area, based on a comparison result of the plurality of reference images and the plurality of corresponding comparison target images.
16. The time lapse observation method according to claim 2 , wherein
each of the time lapse image and the comparison target image is a fluorescent image.
17. The time lapse observation method according to claim 2 , wherein
each of the time lapse image and the comparison target image is an SHG image.
18. A time lapse observation apparatus used for the method according to claim 2 .
19. A multiphoton microscope used for the method according to claim 2 .