Light microscope and method for recording images with a light microscope

Patent metadata

Field	Value
Publication number	US-10976534-B2
Application number	US-201715788918-A
Country	US
Kind code	B2
Filing date	Oct 20, 2017
Priority date	Mar 28, 2012
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

Abstract

The invention relates to a method for recording images with a light microscope, wherein a specimen container with a specimen is arranged on a specimen holder of the light microscope, and wherein illuminating light is guided onto the specimen. The illuminating light can hereby be cut in a cross-section transversely to an optical axis of the light microscope through a wall of the specimen container to a limited cross-sectional region. First and second diaphragm settings are determined and set, for the limited cross-sectional region of the illuminating light defined by the wall of the specimen container, in which the diaphragm covers equal sized portions of the limited cross-sectional region. In addition the invention relates to a light microscope which is adapted in particular to carry out the method.

First claim

The invention claimed is:

1. A method for recording images with a light microscope, comprising:
arranging a specimen container with a specimen on a specimen holder of the light microscope,
guiding illuminating light onto the specimen,
bringing a diaphragm for restricting the illuminating light into an optical path of the illuminating light,
calculating a phase contrast image based on a difference between brightness values of image pixels of an illuminated specimen region of a first image and an illuminated specimen region of a second image, wherein the illuminated specimen region of the first image is identical to the illuminated specimen region of the second region, and wherein the illuminated specimen region of the first image and the illuminated specimen region of the second region are illuminated with different diaphragm settings in a pupil plane, for a limited cross-sectional region of the illuminating light which is defined by a wall of the specimen container, those diaphragm settings are determined and set as diaphragm settings, in which the diaphragm covers equal sized portions of the limited cross-sectional region,
wherein the illuminating light is restricted to the limited cross-sectional region by the wall of the specimen container, in a cross-section transverse to an optical axis of the light microscope.

2. The method of claim 1 ,
wherein
the diaphragm is brought into the optical path between the specimen and image recording means.

3. The method of claim 1 ,
wherein
the diaphragm is brought into the optical path between an illuminating light source and the specimen.

4. The method of claim 1 ,
wherein
a mid-point of the limited cross-sectional region of the illuminating light is determined,
as a straight line of symmetry a straight line extending through the mid-point is determined, and
as a first and second diaphragm setting those diaphragm settings are set, in which the area covered by the diaphragm in the first diaphragm setting and the area covered by the diaphragm in the second diaphragm setting lie symmetrical to the straight line of symmetry.

5. The method of claim 4 ,
wherein
the diaphragm settings differ in a rotation angle of a diaphragm edge of the diaphragm around a rotation axis, and
as a straight line of symmetry a straight line is fixed which cuts the mid-point and the rotation axis.

6. The method of claim 5 ,
wherein
a position of the rotation axis is determined in a calibration measurement.

7. The method of claim 6 ,
wherein
at least two pupil images are recorded in the calibration measurement, wherein the diaphragm edge is brought into different rotation angles around the rotation axis,
the position of the rotation axis is determined from the orientations of the diaphragm edge in the different pupil images.

8. The method of claim 1 ,
wherein
as a straight line of symmetry, a connecting straight line from the optical axis or from the rotation axis to a closest lying wall region of the specimen container is determined,
wherein the closest lying wall region is that part of the wall of the specimen container which is closest to the optical axis or to the rotation axis, and
as first and second diaphragm settings, those settings are applied, in which the area covered by the diaphragm in the first diaphragm position and the area covered by the diaphragm in the second diaphragm position lie symmetrical to the straight line of symmetry.

9. The method of claim 4 ,
wherein
an overview image of the specimen is recorded for the purpose of determination of the first and second diaphragm setting in an overview measurement,
the position of a wall of the specimen container is determined in the overview image by means of image processing means, and
a closest lying wall region to the optical axis is determined from the determined position of the wall; or the mid-point of the limited cross-sectional region is determined with the determined position of the wall.

10. The method of claim 1 ,
wherein
for the purpose of determination of the first and second diaphragm settings the diaphragm is brought into the optical path of the illuminating light, the diaphragm is rotated and in different rotation positions of the diaphragm test images of the specimen are recorded, a respective pair of test images, for which associated rotation positions of the diaphragm are rotated by 180° relative to each other, are set against each other having regard to an evaluation criterion, one of the respective pair of test images is selected with the evaluation criterion and associated two diaphragm settings are set as the first and second diaphragm settings.

11. The method of claim 1 ,
wherein
the diaphragm is rotated continuously,
a camera sensor of the light microscope is exposed via multiple rotations of the diaphragm for a first image recording,
a light pulse as illuminating light is sent for the first image recording whenever the diaphragm is at a first rotation position as a first diaphragm setting, and otherwise no illuminating light is emitted,
the camera sensor of the light microscope is exposed via multiple rotations of the diaphragm for a second image recording,
a light pulse as illuminating light is sent for the second image recording whenever the diaphragm is at a second rotation position as a second diaphragm setting, and otherwise no illuminating light is emitted.

12. The method of claim 1 ,
wherein
the diaphragm has a circle segment form, of which the circle segment opening has a tip which contacts the optical axis of the light microscope,
the diaphragm is rotated continuously,
as a first diaphragm setting the diaphragm moves over a first rotation region and as a second diaphragm setting the diaphragm moves over a second rotation region.

13. The method of claim 11 ,
wherein
a test image is recorded with at least one of: the first and the second diaphragm setting,
an image brightness of the recorded test image or the recorded test images is determined,
an exposure time of a camera chip of the light microscope is fixed with the determined image brightness so that a desired image brightness is achieved with the first and second diaphragm settings,
and
a rotation speed of the diaphragm is selected so a time which the diaphragm requires to move over the first and the second rotation region is respectively equal to the fixed exposure time.

14. A light microscope, comprising:
a specimen holder for holding a specimen container with a specimen,
optical means for guiding illuminating light onto the specimen,
a diaphragm which can be arranged for restricting the illuminating light in an optical path of the illuminating light,
wherein
a phase contrast image is calculated using a controller based on a difference between brightness values of image pixels of an illuminated specimen region of a first image and an illuminated specimen region of a second image, wherein the illuminated specimen region of the first image is identical to the illuminated specimen region of the second region, and wherein the illuminated specimen region of the first image and the illuminated specimen region of the second region are illuminated with different diaphragm settings in a pupil plane,
the controller is adapted, for a limited cross-sectional region of the illuminating light which is defined through a wall of the specimen container, to determine and set as diaphragm settings those diaphragm settings, in which the diaphragm covers equal sized portions of the limited cross-section region,
wherein restricting the illuminating light to the limited cross-sectional region in a cross-section

Assignees

• Zeiss Carl Microscopy Gmbh

Inventors

• Steinmeyer Ralf
• Schoen Peter
• Langhorst Matthias
• Bendlin Cornelia

Classifications

• G02B21/14 Primary

  affording illumination for phase-contrast observation · CPC title

• G02B21/34

  Microscope slides, e.g. mounting specimens on microscope slides · CPC title

• G02B21/361 Primary

  Optical details, e.g. image relay to the camera or image sensor (G02B21/364 takes precedence; illumination details G02B21/06 and subgroups) · CPC title