Microinjection apparatus and automatic focal point adjustment method
A microinjection apparatus, which injects an object into a cell with a needle, acquires a first image that is an image of the cell at a first focal position and a second image that is an image of the cell at a second focal position, and decides a state of the cell based on a difference image obtained from the first image and the second image. Execution of such sequence of processing makes it possible to measure a state of a cell without involving human work and confirm the state of the cell in performing a microinjection, without a necessity of experienced skill, efficiently, and simply.
1. A microinjection apparatus that injects an object into a cell on a base surface with a needle, the microinjection apparatus comprising:
an image acquiring unit that acquires a plurality of images of the cell at different focal positions;
a state deciding unit that decides a state of the cell based on a difference image obtained from a first image and a second image included in the plurality of images;
a first focal position detecting unit that detects a first focal position of the image acquiring unit at which a differential aggregate distribution according to focal positions of a first focal point interval assumes a maximum value as calculated based on difference images obtained from the first image and images of the cell acquired by the image acquiring unit at the focal positions of the first focal point interval, at an observation position on the base surface at which the state of the cell is measured by the state deciding unit; and
a second focal position detecting unit that detects a second focal position of the image acquiring unit at which a differential aggregate distribution according to focal positions of a second focal point interval assumes a minimum value as calculated based on difference images obtained from the first image and images of the cell acquired by the image acquiring unit at the focal positions of the second focal point interval narrower than the first focal point interval, at the observation position, each of the focal positions of the second focal point interval being within a predetermined range from the first focal position detected by the first focal position detecting unit.
2. The microinjection apparatus according to claim 1 , wherein the state deciding unit decides the state of the cell based on a brightness of the difference image.
3. The microinjection apparatus according to claim 2 , wherein the state deciding unit decides the state of the cell based on a correlation of areas in the difference image having brightness lower than a threshold and higher than the threshold.
4. The microinjection apparatus according to claim 2 , wherein the state of the cell includes at least one of presence or absence of the cell, life or death state of the cell, and adherent state of the cell.
5. The microinjection apparatus according to claim 1 , further comprising an inclination calculating unit that calculates an inclination of the base surface based on the second focal position and the first focal position at least at three observation positions on the base surface.
6. The microinjection apparatus according to claim 5 , further comprising a distance keeping unit that keeps a constant distance between the base surface and a leading edge of the needle, irrespective of the observation positions, according to calculated inclination of the base surface.
7. A method of automatically adjusting a focal point of a lens relative to a cell for injecting an object into the cell with a needle, the method comprising:
acquiring with an image acquiring unit a plurality of images of the cell at different focal positions;
deciding a state of the cell based on a difference image obtained from a first image and a second image included in the plurality of images;
first detecting including detecting a first focal position of the image acquiring unit at which a differential aggregate distribution according to focal positions of a first focal point interval assumes a maximum value as calculated based on difference images obtained from the first image and images of the cell acquired by the image acquiring unit at the focal positions of the first focal point interval, at an observation position on the base surface at which the state of the cell is decided at the deciding; and
second detecting including detecting a second focal position of the image acquiring unit at which a differential aggregate distribution according to focal positions of a second focal point interval assumes a minimum value as calculated based on difference images obtained from the first image and images of the cell acquired by the image acquiring unit at the focal positions of the second focal point interval narrower than the first focal point interval, at the observation position, each of the focal positions of the second focal point interval being within a predetermined range from the first focal position detected by the first detecting.
8. The method according to claim 7 , wherein the deciding includes deciding the state of the cell based on a brightness of the difference image.
9. The method according to claim 8 , wherein the deciding includes deciding the state of the cell based on a correlation of areas in the difference image having brightness lower than a threshold and higher than the threshold.
10. The method according to claim 8 , wherein the state of the cell includes at least one of presence or absence of the cell, life or death state of the cell, and adherent state of the cell.
11. The method according to claim 7 , further comprising calculating an inclination of the base surface based on the second focal position and the first focal position at least at three observation positions on the base surface.
12. The method according to claim 11 , further comprising keeping a constant distance between the base surface and a leading edge of the needle, irrespective of the observation positions, according to calculated inclination of the base surface.