MICROSCOPE AND METHOD FOR HIGH-RESOLUTION 3D FLUORESCENCE MICROSCOPY

• US 20150160446A1
• Filed: 12/13/2012
• Published: 06/11/2015
• Est. Priority Date: 01/24/2012
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Inventors: Kalkbrenner, Thomas, Wolleschensky, Ralf
• Granted Patent: US 9,885,860 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 2201/12 Circuits of general importance; Signal processing
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/361 Optical details, e.g. image relay to the camera or image sensor G02B21/364 takes precedence; illumination details G02B21/06 and subgroups
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: G02B 27/58 Optics for apodization or superresolution; Optical synthetic aperture systems

Abstract

A method for high-resolution 3D fluorescence microscopy, wherein fluorescence emitters in a sample are repeatedly excited to emit fluorescence, and still images are produced of the sample by means of a microscope. The microscope has an imaging beam path with an optical resolution and a focal plane, wherein the fluorescence emitters are excited to emit fluorescence in such a manner that at least a subset of the fluorescence emitters is isolated in each still image so that the images of these fluorescence emitters can be separated in the still images within the optical resolution. The positions of the fluorescence emitters are localized in the generated still images, from the images of the isolated fluorescence emitters, with a location accuracy exceeding the optical resolution, and a high-resolution composite image is generated therefrom.

Claims

1. A method for high-resolution 3D fluorescence microscopy, comprising

repeatedly exciting fluorescence emitters in a sample to emit fluorescence, and producing still images of the sample by means of a microscope having an imaging beam path with an optical resolution and a focal plane, said fluorescence emitters being stimulated to emit fluorescence in such a manner that at least a subset of the fluorescence emitters is isolated in each still image and so that the images of these fluorescence emitters can be separated in the still images within the optical resolution,

localizing, in the resulting still images, the positions of said fluorescence emitters from the images of the isolated fluorescence emitters, with a location accuracy exceeding the optical resolution, and a high-resolution composite image being generated therefrom,

dividing each still image into a first and a second partial image by means of a dividing element, said first partial image images a first focal plane in the sample, and said second partial image images a second focal plane in the sample,

both said first and second partial images being imaged separately,

using an adaptive mirror as said dividing element arranged substantially in a pupil of the imaging beam path,

adjusting said adaptive mirror in such a manner that it images said first and second partial images separately in time or in space, and

further adjusting said adaptive mirror is adjusted in such a manner that it implements two different focal lengths for the two partial images.

2. The method according to claim 1, wherein said adaptive mirror images the two partial images along diverging optical axes, in adjacent image capture regions.

3. The method according to claim 2, further comprising adjusting an intensity ratio of the partial images by mirror surface fractions of the adaptive mirror, which provide the imaging of the first partial image, being reduced in size, wherein mirror surface fractions of the adaptive mirror which provide the imaging of the second partial image are enlarged by the same degree.

4. The method according to claim 1, further comprising changing a difference in the focal lengths by means of the adjustment of the adaptive mirror to implement the adjustment of a depth resolution or a detected depth region.

5. A The method according to claim 1, further comprising detecting radiation reflected by the mirror by means of a wave front sensor, and the mirror is adjusted for the purpose of correcting imaging errors of the imaging beam path.

6. The method according to claim 1, further comprising adjusting the two focal lengths synchronously for a depth position adjustment.

7. A The method according to claim 1, wherein prior to the depth-resolving microscopy process, the partial images are first generated with the same focal length, and positionally aligned in this state, in an alignment step of the partial images.

8. A The method according to claim 1, wherein two image capture regions are on one camera, and prior to the depth-resolving microscopy process, in an alignment step, the two partial images are first imaged superimposed on the camera and positionally aligned in this state, and then the mirror is adjusted in such a manner that the partial images come to lie next to each other in the image capture regions.

9. A fluorescence microscope for the purpose of three-dimensional imaging of a sample with a location accuracy exceeding the optical resolution, comprising:

an illumination device which is designed for the purpose of repeatedly exciting fluorescence emitters in the sample to emit fluorescence,

an imaging device having an imaging beam path with the optical resolution, designed for the purpose of producing still images of the sample at the optical resolution,

a control device which is designed for the purpose of controlling the illumination device and the imaging device in such a manner that multiple still images of the sample are produced, wherein the fluorescence emitters are excited to emit fluorescence in such a manner that at least a subset of the fluorescence emitters in each still image is isolated in such a manner that the images of these fluorescence emitters can be separated in the still images within the optical resolution, wherein

the control device being designed for the purpose of localizing the positions of the isolated fluorescing fluorescence emitters in the generated still images with a location accuracy exceeding the optical resolution, and of generating a high-resolution composite image therefrom,

the imaging beam path having a dividing element which divides each still image into a first and a second partial image, wherein the first partial image images a first focal plane in the sample and the second partial image images a second focal plane in the sample,

the imaging device having at least one camera on which the two partial images are imaged next to each other,

the dividing element being designed as an adaptive mirror which is arranged substantially in a pupil of the imaging beam path,

the control device being designed for the purpose of adjusting the adaptive mirror in such a manner that it images the two partial images separately in time or in space, and

the control device being designed for the purpose of adjusting the adaptive mirror in such a manner that it implements two different focal lengths for the two partial images.

10. The fluorescence microscope according to claim 9, wherein the control device is designed for the purpose of controlling the adaptive mirror in such a manner that it images the two partial images along diverging optical axes, in adjacent image capture regions of the camera.

11. The fluorescence microscope according to claim 10, wherein said control device is designed for the purpose of adjusting the adaptive mirror, for the purpose of adjusting an intensity ratio of the partial images, in such a manner that the size of mirror surface fractions of the adaptive mirror which provide the imaging of the first partial image is reduced, and the size of mirror surface fractions of the adaptive mirror which provide the imaging of the second partial image is enlarged by the same degree.

12. The fluorescence microscope according to claim 9, wherein the control device is designed for the purpose of adjusting the adaptive mirror in such a manner that a difference in the focal lengths is changed, for the purpose of implementing the adjustment of a depth resolution or a detected depth region.

13. The fluorescence microscope according to claim 9, further comprising a wave front sensor which detects radiation reflected by the mirror in order to adjust the mirror for the purpose of correcting imaging errors of the imaging beam path.

14. The fluorescence microscope according to claim 9, wherein said control device is designed for the purpose of adjusting the two focal lengths synchronously, for a depth position adjustment.

15. The fluorescence microscope according to claim 9, wherein said control device is designed for the purpose of controlling the mirror in such a manner that the partial images are first generated with the same focal length, and a position alignment signal is generated in this state, for the purpose of alignment prior to the depth-resolving imaging process.

16. The fluorescence microscope according to claim 9, wherein said image capture regions are provided by a camera, and the control device is designed for the purpose of controlling the mirror in such a manner that the partial images are depicted superimposed on the camera, a position alignment signal is generated in this state, and then the mirror is adjusted in such a manner that the partial images are imaged next to each other in the image capture regions, for the purpose of alignment prior to the depth-resolving measurement process.