Microscope for determining properties of moving objects in a biological sample
A microscope for determining properties of moving objects in a biological sample can include a sample space for arranging a biological sample and an objective for capturing a detection radiation coming from a confocal volume created in the sample. The microscope can have a detection beam path along which a captured detection radiation is steered to a detector in the form of a detector array having a plurality of detector elements, with the detector arranged in a plane conjugate to a back-side image plane of the objective. Measurement values from the detector elements can each be read and analyzed on an individual basis. A light source can provide an excitation radiation. In an illumination beam path, a beam-shaping optical unit can shape the excitation radiation to form a confocal volume in the sample space and/or, in the detection beam path, a beam-shaping optical unit can shape the detection radiation.
1 . A microscope for determining properties of moving objects in a biological sample, comprising:
a sample space for arranging a biological sample;
an objective for capturing a detection radiation coming from a confocal volume created in the sample;
a detection beam path along which a captured detection radiation is steered to a detector in the form of a detector array having a plurality of detector elements, wherein the detector is arranged in a plane conjugate to a back-side image plane of the objective, and measurement values from the detector elements can each be read and analyzed on an individual basis;
a light source for providing an excitation radiation and there being, in an illumination beam path, a beam-shaping optical unit for shaping the excitation radiation to form a confocal volume in the sample space and/or, in the detection beam path, a beam-shaping optical unit for shaping the detection radiation, and
an analysis and control unit configured to use the detector to capture, at a plurality of times over a measurement duration, a detection radiation from a confocal volume created in the sample and to be able to in each case analyze the measurement values from the detector elements on an individual basis; and configured to select at least one pair of detector elements, wherein each partner of a pair is formed by at least one detector element;
in a step B configured to create correlations of the acquired measurement values from the detector elements of at least one pair among themselves, wherein
in a step B1 correlations are calculated starting from one of the selected detector elements in a first direction to the further selected detector element; and optionally
in a step B2 correlations are additionally calculated starting from the further selected detector element in a second direction (back direction) to the first selected detector element; and
in a step C configured to analyze the correlations in respect of the occurrence of a change in the intensity of the measurement values from selected detector elements.
2 . The microscope according to claim 1 , wherein the beam-shaping optical unit for shaping the excitation radiation and/or the beam-shaping optical unit for shaping the detection radiation, which is present, comprises a slit stop, a beam expander, or a pinhole.
3 . The microscope according to claim 1 , wherein the beam-shaping optical unit for shaping the excitation radiation and/or the beam-shaping optical unit for shaping the detection radiation, which is present, comprises a spatial light modulator.
4 . The microscope according to claim 1 , wherein
in B2 correlations are additionally calculated starting from the further selected detector element in a second direction (back direction) to the first selected detector element.
5 . The microscope according to claim 4 , wherein the correlations of B1 and/or B2 are cross correlations.
6 . The microscope according to claim 5 , wherein correlations of a plurality of pairs are calculated, and wherein all pairs have at least one detector element as a common partner.
7 . The microscope according to claim 6 , wherein the respective further partners of the pairs are arranged at an identical distance from the common partner.
8 . The microscope according to claim 1 , wherein a plurality of detector elements are combined to form a respective one of the partners of an extended pair.
9 . The microscope according to claim 1 , wherein a group of a plurality of pairs or plurality of extended pairs of detector elements is defined, wherein the imaginary connecting lines of the center points of the pairs or extended pairs run parallel to one another.
10 . The microscope according to claim 9 , wherein a mean value of the cross correlations in a first direction and/or a second direction, created from the measurement values of the pairs or extended pairs, is formed.
11 . The microscope according to claim 9 , characterized by a determination of a plurality of groups of detector elements, wherein the respective imaginary connecting lines between the groups are aligned differently from one another.
12 . The microscope according to claim 9 , wherein cross correlations of measurement values of the pairs or extended pairs of at least two different groups are formed.
13 . The microscope according to claim 12 , wherein a speed and/or a movement direction of an object is determined on the basis of the cross correlations by virtue of a plurality of cross correlations.
14 . The microscope according to claim 8 , wherein the partners of a pair are spatially separated in each case by at least one detector element not belonging to the relevant pair.
15 . The microscope according to claim 13 , wherein the speed and/or the movement direction of an object is calculated over pairs or extended pairs with different alignments and all curves obtained thus are fitted with the directions and/or a speed as a parameter.
16 . The microscope according to claim 1 , comprising the beam-shaping optical unit for shaping the excitation radiation.
17 . The microscope according to claim 1 , comprising the beam-shaping optical unit for shaping the detection radiation.
18 . The microscope according to claim 16 , comprising the beam-shaping optical unit for shaping the detection radiation.
19 . A microscope for determining properties of moving objects in a biological sample, comprising:
a sample space for arranging a biological sample;
an objective for capturing a detection radiation coming from a confocal volume created in the sample;
a detection beam path along which a captured detection radiation is steered to a detector in the form of a detector array having a plurality of detector elements, wherein the detector is arranged in a plane conjugate to a back-side image plane of the objective, and measurement values from the detector elements can each be read and analyzed on an individual basis; and
a light source for providing an excitation radiation and there being, in an illumination beam path, a beam-shaping optical unit for shaping the excitation radiation to form a confocal volume in the sample space and/or, in the detection beam path, a beam-shaping optical unit for shaping the detection radiation,
wherein a group of a plurality of pairs or plurality of extended pairs of detector elements is defined, wherein the imaginary connecting lines of the center points of the pairs or extended pairs run parallel to one another,
wherein cross correlations of measurement values of the pairs or extended pairs of at least two different groups are formed, and
wherein a speed and/or the movement direction of an object is calculated over pairs or extended pairs with different alignments and all curves obtained thus are fitted with the directions and/or a speed as a parameter.