Microscope and method for SPIM microscopy
Method and microscope for SPIM microscopy, wherein, in a first step, with reference to a sample to be examined, a calibration is carried out in that the actual position of the light sheet in different sample planes is detected and stored depending on the position in the sample and, in a second step, the stored position of the light sheet is utilized during observation and/or detection of the sample based on the values stored in the first step to correct the position of the light sheet relative to the focal plane of the detection objective and/or, during the displacement of the sample, an adjustment of the position of the light sheet relative to the focal plane of the detection objective is carried out such that the light sheet executes a relative movement in at least one direction relative to the sample and/or the detection objective.
1. A method for SPIM microscopy with a microscope comprising:
an illumination device comprising:
an illumination light source; and
an illumination beam path for illuminating a sample with a light sheet;
a detection arrangement configured to detect light which is radiated from the sample with a detection objective; and
an adjusting means configured to adjust the illumination beam path at least along the optical axis of the detection objective;
wherein the light sheet is substantially planar in a focus of the detection objective or of a defined plane in a vicinity of a focus of the imaging detection objective;
wherein the detection objective has an optical axis which intersects the plane of the light sheet at a non-zero angle;
wherein the sample and the light sheet are moved relative to each other in direction of the optical axis of the detection objective for acquiring different sample planes; and
wherein the method comprises:
a first step comprising, with reference to a sample to be examined, carrying out a calibration so that the actual position and orientation of the light sheet in different sample planes is detected depending on the position and orientation of the sample and stored; and
a second step comprising utilizing the stored position and orientation of the light sheet during observation and/or detection of the sample based on the values stored in the first step to correct the position and orientation of the light sheet relative to a focal plane of the detection objective.
2. The method according to claim 1 ;
wherein, in the second step, the position and orientation of the light sheet and/or of the detection objective is corrected or adjusted simultaneously with, or subsequent to, the relative movement of the sample.
3. The method according to claim 1 ;
wherein, in the second step, a detection and an evaluation of sample contrast are carried out during the relative movement.
4. The method according to claim 1 ;
wherein, in the second step, the relative movement takes place until a maximum image contrast is obtained or until an image contrast is optimal on the basis of criteria.
5. The method according to claim 1 , further comprising:
moving the light sheet relatively around an adjusted vertical focus position and orientation of the detection objective to obtain a maximum or optimal contrast.
6. The method according to claim 1 ;
wherein the adjusting means is for the detection objective, for the illumination, or for both; and
wherein, in the second step, coupling of the movement of the light sheet and/or of the detection objective relative to the sample is carried out via an actuating unit which is connected to the adjusting means.
7. The method according to claim 1 , further comprising:
tilting the illumination beam path around at least one axis.
8. The method according to claim 7 ;
wherein the at least one axis lies in the optical axis of the detection objective.
9. A microscope, control device, or
combination thereof, configured to implement the method according to claim 1 .
10. A non-transitory computer readable medium comprising:
a computer program configured to implement the method according to claim 1 .
11. A method for SPIM microscopy with a microscope comprising:
an illumination device comprising:
an illumination light source; and
an illumination beam path for illuminating a sample with a light sheet;
a detection arrangement configured to detect light which is radiated from the sample with a detection objective; and
an adjusting means configured to adjust the illumination beam path at least along the optical axis of the detection objective;
wherein the light sheet is substantially planar in a focus of the detection objective or of a defined plane in a vicinity of a focus of the imaging detection objective;
wherein the detection objective has an optical axis which intersects the plane of the light sheet at a non-zero angle;
wherein the sample and the light sheet are moved relative to each other in direction of the optical axis of the detection objective for acquiring different sample planes; and
wherein the method comprises:
adjusting, during the relative movement of the sample, the position and orientation of the light sheet relative to the focal plane of the detection objective so that at least one of the light sheet and the detection objective executes a relative movement in at least one direction relative to the sample.
12. The method according to claim 11 ;
wherein the position and orientation of the light sheet and/or of the detection objective is corrected or adjusted simultaneously with, or subsequent to, the relative movement of the sample.
13. The method according to claim 11 ;
wherein a detection and an evaluation of sample contrast are carried out during the relative movement.
14. The method according to claim 11 ;
wherein the relative movement takes place until a maximum image contrast is obtained or until an image contrast is optimal on the basis of criteria.
15. The method according to claim 11 , further comprising:
moving the light sheet relatively around an adjusted vertical focus position and orientation of the detection objective to obtain a maximum or optimal contrast.
16. The method according to claim 11 ;
wherein the adjusting means is for the detection objective, for the illumination, or for both; and
wherein coupling of the movement of the light sheet and/or of the detection objective relative to the sample is carried out via an actuating unit which is connected to the adjusting means.
17. The method according to claim 11 , further comprising:
tilting the illumination beam path around at least one axis.
18. The method according to claim 17 ;
wherein the at least one axis lies in the optical axis of the detection objective.
19. A microscope, control device, or combination thereof, configured to implement the method according to claim 11 .
20. A non-transitory computer readable medium comprising:
a computer program configured to implement the method according to claim 11 .