US-12619057-B2 - Light-field microscopy image capturing method and light-field microscope

Abstract

The invention relates to a light-field microscope and a light-field microscopy image capturing method, including the steps of providing an illumination radiation from at least one light source; modulating the illumination radiation by virtue of adjusting the intensity distribution thereof; generating a dynamic light sheet in a sample space, in which a sample to be imaged can be arranged; scanning a region of the sample to be imaged using the light sheet; collecting a detection radiation and detecting image signals using light-field technology. The illumination radiation is modulated by virtue of the light source being controlled and an intensity of the illumination radiation provided by the light source being altered, especially in a controlled fashion over time. Image recordings of the sample are triggered for different positions of the light sheet.

Inventors

• Tiemo Anhut
• Daniel Schwedt

Assignees

• CARL ZEISS MICROSCOPY GMBH

Dates

Publication Date

20260505

Application Date

20230404

Priority Date

20220411

Claims (8)

1. 1 . Light-field microscopy image capturing method, comprising: providing an illumination radiation from at least one light source; modulating the illumination radiation by virtue of adjusting the intensity distribution thereof; generating a dynamic light sheet in a sample space, in which a sample to be imaged can be arranged, by virtue of the modulated illumination radiation being radiated into an entrance location in an entrance pupil of a common illumination and detection objective and a first angle of incidence of the modulated illumination radiation being altered multiple times; wherein a layer of the sample is illuminated with said dynamic light sheet only thinly at a time, avoiding bleaching outside the light sheet; scanning a region of the sample to be imaged using the dynamic light sheet, the dynamic light sheet being brought into a number of different positions; using the common objective to collect a detection radiation coming from the sample space; guiding the collected detection radiation along a detection beam path and generating a plurality of partial image representations in a detection plane by means of a microlens array; detecting the partial image representations as respective image signals of an image recording and evaluating the captured image signals on the basis of their associated location information and angle information; modulating the illumination radiation by virtue of the light source being controlled directly and an intensity of the illumination radiation provided by the light source being altered in controlled fashion over time, and recording an image whenever the dynamic light sheet is brought into a position; or recording an image while the dynamic light sheet adopts different positions.
2. 2 . Image capturing method according to claim 1 , wherein the illumination radiation is modulated while the latter is scanned along a scanning path transversely to its propagation direction in relation to the sample space, with the modulation and the change of the first angle of incidence of the illumination radiation being matched to one another.
3. 3 . Image capturing method according to claim 2 , wherein a modulation function is determined on the basis of an intensity distribution, known in advance, of the illumination radiation at rest and said modulation function is retrievably stored, with locations along at least one section of the scanning path being assigned modulation factors by way of the modulation function, with a desired intensity profile being implemented over the relevant section of the scanning path in the case of the control-technical implementation of said modulation factors in respect of location-related intensities of the scanned illumination radiation.
4. 4 . Image capturing method according to claim 2 wherein the intensity of the illumination radiation is time-modulated during a scan transversely to the propagation direction such that a dynamic light sheet that is structured over its width is generated.
5. 5 . Image capturing method according to claim 4 , wherein unwanted background signals are reduced by means of a combination by calculation of at least two image recordings.
6. 6 . Image capturing method according to claim 1 wherein the modulated illumination radiation is shaped into a line-shaped light distribution and the line-shaped light distribution is radiated into the entrance location and a first angle of incidence of the line-shaped light distribution is altered multiple times.
7. 7 . Image capturing method according to claim 1 wherein the dynamic light sheet is directed into the sample space at an inclination angle greater than zero degrees with respect to the optical axis of the common objective by virtue of the entrance location in the entrance pupil being chosen away from the optical axis.
8. 8 . Image capturing method according claim 6 , wherein the optical axis passes through the chosen entrance location and the structured light sheet is generated in at least one position parallel to the optical axis.

Description

RELATED APPLICATION The present application is a U.S. National Stage application of German Application No. DE 10 2022 203 632.8 filed on Apr. 11, 2022, the contents of which are herein incorporated by reference in their entirety. FIELD OF THE INVENTION The invention relates to a light-field microscopy image capturing method and a light-field microscope according to the preambles of the independent claims. BACKGROUND OF THE INVENTION Light field microscopy can be used as a method for simultaneous three-dimensional imaging of even relatively large objects such as the brains of small mammals and living samples such as fish embryos or fruit fly larvae. To enable imaging, the objects to be imaged are specifically provided with markers to this end, the markers emitting a detection radiation as a consequence of being excited with a suitable wavelength. Usually, fluorescence markers or fluorophores serve marking purposes in this context. As a result of illumination with radiation at an excitation wavelength, a fluorescence radiation, as detection radiation, can be excited, captured and used for imaging. By way of example, if thick biological samples are illuminated using a spectral lamp, fluorescence radiation is likewise excited, albeit quite unspecifically, in the entire illuminated sample as a result of the effect of the illumination radiation. Light-field microscopy is a far-field method, by means of which a three-dimensional image data record can be generated from the recorded parallactic views of the sample. However, it proves not possible to discriminate emission light generated away from the focus. As a result, what is known as optical cutting, as known from laser scanning microscopes, for example, is not possible. To improve the contrast of the desired signal vis-à-vis background signals, it is possible to combine the light-field detection with appropriate illumination modalities. An example to this end has been disclosed in DE 10 2014 102 215 A1. It proposes the illumination of a sample using a light sheet radiated into the sample at an angle. The thin light sheet is moved (scanned) over the region of the sample to be imaged. The illumination by means of a light sheet and the capture of image data can be implemented in various ways. In the case of a continuous light sheet scan, the entire sample field to be imaged is scanned during the camera recording period. However, on account of the oblique incoming radiation of the light sheet, the excitation light leaves the sample field after a certain propagation length, with the result that relatively deep depths in the sample reached in that case no longer contribute to the detection radiation captured by the camera. Moreover, backscattering of excitation light is not implemented over the entire numerical aperture (NA) of the detection objective, with the result that the collection efficiency is significantly reduced in this respect. In comparison with the illumination by a spectral lamp (NA=0), this method brings about a slight improvement in the image contrast with a hardly reduced volume rate. A further option consists in a triggered light-sheet scan. In this case, the camera is triggered at each position of the light sheet and an image recording is captured. This prevents the out-of-focus background generated at other positions by the departing light sheet from adding to the desired signal on the camera. Thus, unwanted background signals can initially be removed by calculation before a three-dimensional view of the sample is generated. The image contrast is significantly increased in this method, but to the detriment of the achievable volume rate. This method is particularly suitable if the volumes of interest, that is to say the regions of the sample to be image, take up a relatively small field of view. In a third procedure, structured illumination is generated by virtue of the light intensity of the light sheet being modulated at different positions. Moreover, the entire field of view is scanned during one camera recording period. The structured illumination can be used to eliminate out-of-focus background light by calculation. Only a few—generally three—image recordings are required to this end. Hence, the loss of volume rate is still manageable and an improvement in the contrast is obtained vis-à-vis the continuous light-sheet scan. OBJECTIVES OF THE INVENTION The invention is based on the object of proposing an option for image capture by means of light field detection that is improved over the prior art. The object is achieved by means of the subjects of the independent claims. The dependent claims relate to advantageous developments of the invention. This object is achieved by an image capturing method from the field of light-field microscopy. As one step, the method includes the provision of an illumination radiation from at least one light source. The illumination radiation is modulable by virtue of the intensity distribution thereof being adjusted