Method and apparatus for correcting aberrations in fluorescence microscopy
Scanning fluorescence microscopes and related methods are described for microscopically recording images of samples extending in three dimensions. In particular embodiments, the microscopes are configured for recording a first sectional image that is parallel to an optical axis of a microscope objective lens by scanning a sample with a focused excitation light distribution in a sectional area parallel to the optical axis of the microscope objective. The excitation light distribution may be corrected by a correction device according to initial adjustment values for adjustment parameters of an aberration correction function. The first sectional image is then evaluated and new adjustment values for the adjustment parameters are defined. Further image data are then recorded by scanning the sample with the focused excitation light distributions, with the excitation light distribution corrected by the correction device according to the new adjustment values for the adjustment parameters of the aberration correction function.
1 . A scanning fluorescence microscope comprising
an excitation light source configured for providing excitation light having an excitation light distribution,
a microscope objective lens having an optical axis and configured and arranged for focusing the excitation light distribution of the excitation light,
a mounting device configured for mounting a sample,
a scanning device configured and arranged for scanning the sample mounted on the mounting device with the focused excitation light distribution in three spatial dimensions,
a detection device configured and arranged for detecting fluorescence light emitted out of the sample for each scanning position of the scanning device,
a storage device configured for storing sets of image data,
an adjustable correction device configured for correcting aberrations of the excitation light distribution,
a controller configured and connected for adjusting the correction device, and
an evaluation unit configured and connected for evaluating the sets of image data and for defining adjustment values for adjustment parameters of an aberration correction function according to which the adjustable correction device is adjusted by the controller, wherein the aberration correction function is a function of a z-position of the scanning position in a z-direction along the optical axis of the microscope objective lens,
wherein the scanning device is configured for scanning a first sample with the focused excitation light distribution in a sectional area that is parallel to the optical axis of the microscope objective lens such that at least one first sectional image is recorded along the optical axis of the microscope objective lens, wherein the excitation light distribution is corrected by means of the adjustable correction device according to initial adjustment values for the adjustment parameters of the aberration correction function and wherein fluorescence light emitted out of the first sample is detected for each scanning position by means of the detection device,
wherein the evaluation unit is configured for evaluating the at least one first sectional image and for defining new adjustment values for the adjustment parameters of the aberration correction function, and
wherein the scanning device is further configured for scanning the first sample or another sample with the focused excitation light distribution for recording further image data, wherein the excitation light distribution is corrected by means of the adjustable correction device according to the new adjustment values for the adjustment parameters of the aberration correction function, and wherein fluorescence light emitted out of the respective first or other sample is detected for each scanning position by means of the detection device.
2 . The scanning fluorescence microscope of claim 1 , wherein the correction device is arranged in a common path of the excitation light and the fluorescence light.
3 . The scanning fluorescence microscope of claim 1 , comprising a depletion light source configured for providing depletion light, wherein the correction device is arranged in a common path of the excitation light, the fluorescence light and the depletion light.
4 . The scanning fluorescence microscope of claim 3 , wherein an additional correction device configured for correcting aberrations of the depletion light is arranged in a separate path of the depletion light only.
5 . The scanning fluorescence microscope of claim 1 , wherein the correction device comprises a deformable mirror.
6 . A scanning fluorescence microscope comprising
an excitation light source configured for providing excitation light having an excitation light distribution,
a microscope objective lens having an optical axis and configured and arranged for focusing the excitation light distribution of the excitation light,
a mounting device configured for mounting a sample,
a scanning device configured and arranged for scanning the sample mounted on the mounting device with the focused excitation light distribution in three spatial dimensions,
a detection device configured and arranged for detecting fluorescence light emitted out of the sample for each scanning position of the scanning device,
a storage device configured for storing sets of image data,
an adjustable correction device configured for correcting aberrations of the excitation light distribution,
a controller configured and connected for adjusting the correction device,
an input unit configured for receiving user input for defining adjustment values for new adjustment values for adjustment parameters of an aberration correction function according to which the adjustable correction device is adjusted by the controller, wherein the aberration correction function is a function of a z-position of the scanning position in a z-direction along the optical axis of the microscope objective lens, and
a display unit for displaying the sets of image data,
wherein the scanning device is configured for scanning a first sample with the focused excitation light distribution in a sectional area that is parallel to the optical axis of the microscope objective lens such that at least one first sectional image is recorded along the optical axis of the microscope objective lens, wherein the excitation light distribution is corrected by means of the adjustable correction device according to initial adjustment values for the adjustment parameters of the aberration correction function and wherein fluorescence light emitted out of the first sample is detected for each scanning position by means of the detection device,
wherein the display unit is configured for displaying the at least one first sectional image recorded along the optical axis of the microscope objective lens, and
wherein the scanning device is further configured for scanning the first sample or another sample with the focused excitation light distribution for recording further image data, wherein the excitation light distribution is corrected by means of the adjustable correction device according to the new adjustment values for the adjustment parameters of the aberration correction function, and wherein fluorescence light emitted out of the respective first or other sample is detected for each scanning position by means of the detection device.
7 . The scanning fluorescence microscope of claim 6 , wherein the correction device is arranged in a common path of the excitation light and the fluorescence light.
8 . The scanning fluorescence microscope of claim 6 , comprising a depletion light source configured for providing depletion light, wherein the correction device is arranged in a common path of the excitation light, the fluorescence light and the depletion light.
9 . The scanning fluorescence microscope of claim 8 , wherein an additional correction device configured for correcting aberrations of the depletion light is arranged in a separate path of the depletion light only.
10 . The scanning fluorescence microscope of claim 6 , wherein the correction device comprises a deformable mirror.
11 . The scanning fluorescence microscope of claim 1 , wherein the aberration correction function is a linear function of a z-position along the optical axis of the microscope objective lens and wherein the adjustment parameters of the aberration correction function are an aberration correction offset and an aberration correction slope.
12 . The scanning fluorescence microscope of claim 1 , wherein the aberration correction function is a square function of a z-position along the optical axis of the microscope objective lens.
13 . The scanning fluorescence microscope of claim 1 , wherein the aberration correction function is a function of a z-position along the optical axis of the microscope objective lens, that is additively composed of a linear or square function component that is directed to a correction of at least one of a defocus or spherical aberration, and a function component that is directed to a correction of further aberrations.
14 . The scanning fluorescence microscope of claim 1 , wherein the scanning device is configured for scanning the first sample or the other sample with the focused excitation light distribution such that a plurality of first or further sectional images are recorded, wherein the excitation light distribution is corrected by means of the adjustable correction device according to different adjustment values for the adjustment parameters of the aberration correction function.
15 . The scanning fluorescence microscope of claim 1 , wherein the scanning device is configured for scanning the first sample or the other sample with the focused excitation light distribution such that a plurality of first or further sectional images are recorded, wherein the excitation light distribution is corrected by means of the adjustable correction device according to the same adjustment values for the adjustment parameters of the aberration correction function and wherein the sectional areas associated with the plurality of first or further sectional images are arranged at lateral distances or oriented at angles with respect to one another.
16 . The scanning fluorescence microscope of claim 1 , wherein the evaluation unit is configured and connected for separately defining the adjustment values for adjustment parameters of the aberration correction function for different axial partial areas of the sample.
17 . The scanning fluorescence microscope of claim 6 , wherein the aberration correction function is a linear function of a z-position along the optical axis of the microscope objective lens and wherein the adjustment parameters of the aberration correction function are an aberration correction offset and an aberration correction slope.
18 . The scanning fluorescence microscope of claim 6 , wherein the aberration correction function is a square function of a z-position along the optical axis of the microscope objective lens.
19 . The scanning fluorescence microscope of claim 6 , wherein the aberration correction function is a function of a z-position along the optical axis of the microscope objective lens, that is additively composed of a linear or square function component that is directed to a correction of at least one of a defocus or spherical aberration, and a function component that is directed to a correction of further aberrations.
20 . The scanning fluorescence microscope of claim 6 , wherein the scanning device is configured for scanning the first sample or the other sample with the focused excitation light distribution such that a plurality of first or further sectional images are recorded, wherein the excitation light distribution is corrected by means of the adjustable correction device according to different adjustment values for the adjustment parameters of the aberration correction function.
21 . The scanning fluorescence microscope of claim 6 , wherein the scanning device is configured for scanning the first sample or the other sample with the focused excitation light distribution such that a plurality of first or further sectional images are recorded, wherein the excitation light distribution is corrected by means of the adjustable correction device according to the same adjustment values for the adjustment parameters of the aberration correction function and wherein the sectional areas associated with the plurality of first or further sectional images are arranged at lateral distances or oriented at angles with respect to one another.
22 . The scanning fluorescence microscope of claim 6 , wherein the input unit is configured for receiving different axial partial areas of the sample, into which the sample is partitioned on basis of a visual inspection of at least one of the sectional images displayed on the display unit, and wherein the input unit is configured for separately defining the adjustment values for adjustment parameters of the aberration correction function for the different axial partial areas of the sample.
23 . The scanning fluorescence microscope of claim 7 , wherein the scanning device is configured for scanning the first sample or the other sample with the focused excitation light distribution such that a plurality of first or further sectional images are recorded, wherein the excitation light distribution is corrected by means of the adjustable correction device according to different adjustment values for the adjustment parameters of the aberration correction function.
24 . The scanning fluorescence microscope of claim 1 , wherein:
a sectional plane in which the sample is scanned with the focused excitation light distribution is parallel to the optical axis of the microscope lens such that the first sectional image is recorded parallel to the optical axis of the microscope lens.
25 . The scanning fluorescence microscope of claim 24 , wherein:
the aberration correction function is a function of a z-position in the sample that varies along the optical axis of the microscope lens, and
at least when the excitation light distribution is corrected by means of the adjustable correction device according to the new adjustment values for the adjustment parameters of the aberration correction function, the excitation light distribution is not corrected by means of the adjustable correction device for all z-positions in the sample in a same way.