HIGH RESOLUTION MICROSCOPY BY MEANS OF STRUCTURED ILLUMINATION AT LARGE WORKING DISTANCES

• US 20150260978A1
• Filed: 09/26/2013
• Published: 09/17/2015
• Est. Priority Date: 09/28/2012
• Status: Abandoned Application

• Current Assignee: Friedrich-Schiller-Universitat Jena, Universitat Heidelberg
• Original Assignee: Universitat Heidelberg
• Inventors: Cremer, Christoph, Heintzmann, Rainer, Best, Gerrit, Amberger, Roman
• Application Number:
  US14/431,956
• Publication Number:
  US 20150260978A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: G02B 21/0004 specially adapted for specific applications
• CPC Class Codes: G02B 21/0012 Surgical microscopes counterbalanced structures for surgical microscopes G02B7/001
• CPC Class Codes: G02B 21/06 Means for illuminating specimens
• 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 obtaining a sub-resolution image of a specimen using a microscope is provided. The method includes projecting an illumination pattern of illumination light onto the specimen, thereby illuminating the specimen, at least one of detecting at least a portion of fluorescent light emitted from the specimen and detecting at least a portion of illumination light reflected from the specimen, thereby capturing a series of images of the specimen at a plurality of different relative positions of the specimen with respect to the illumination pattern projected onto the specimen, wherein between the capturing of at least two images of the series the relative position of the specimen with respect to the illumination pattern projected onto the specimen is shifted in a non-controlled manner, and processing the captured images to extract a sub-resolution image of the specimen.

Claims

1. A method for obtaining a sub-resolution image of a specimen (52) using a microscope (100), the method comprising:

projecting an illumination pattern of illumination light onto the specimen (52), thereby illuminating the specimen (52);

at least one of detecting at least a portion of fluorescent light emitted from the specimen (52) and detecting at least a portion of illumination light reflected from the specimen (52), thereby capturing a series of images of the specimen (52) at a plurality of different relative positions of the specimen (52) with respect to the illumination pattern projected onto the specimen (52), wherein between the capturing of at least two images of the series the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) is shifted in a non-controlled manner; and

processing the captured images to extract a sub-resolution image of the specimen (52).

2. The method according to claim 1, wherein the relative position of the specimen (52) with respect to the illumination pattern is shifted in a non-controlled manner by utilizing at least one of stochastic movements of the specimen (52), stochastically shifting the specimen (52), stochastic stage scanning, and stochastic movements of a focusing lens system (42, 44) of a microscope objective (40).

3. The method according to claim 1, wherein processing the captured images comprises:

for each image of the series, determining the non-controlled shift of the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52);

a-posteriori shifting of each image of the series to reverse the determined shift of the relative position of the specimen (52) with respect to the projected illumination pattern, thereby obtaining a corresponding shifted image; and

processing the shifted images to extract a sub-resolution image of the specimen (52).

4. The method according to claim 3, wherein determining the non-controlled shift of the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) comprises:

applying an analytic deconvolution of each of a second and subsequent images of the series by a first image of the series to obtain a corresponding deconvolved image;

determining the position of a maximum (P) of the analytic deconvolution of each of the deconvolved images; and

determining the non-controlled shift of the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) for each of the second and subsequent images of the series from the determined position of the maximum (P) of the analytic deconvolution.

5. The method according to claim 3, wherein determining of the non-controlled shift of the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) comprises:

Fourier-transforming of each of the images of the series;

dividing each of the Fourier-transformed second and each subsequent images by the Fourier-transformed first image to obtain a corresponding divided image;

inverse Fourier-transforming each of the divided images;

determining the position of a maximum (P) of intensity of each of the inverse Fourier-transformed divided images; and

determining the non-controlled shift of the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) for each of the second and subsequent images of the series from the determined position of the maximum (P) of intensity of the inverse Fourier-transformed divided image.

6. The method according to claim 1, wherein the illumination pattern is projected onto the specimen (52) through at least one objective (40) of the microscope (100) having a numerical aperture of less than 0.5.

7. The method according to claim 1, wherein the specimen (52) is illuminated only when images are captured.

8. The method according to claim 7, wherein the duration of on-times during which the specimen (52) is illuminated is shorter than the duration of off-times during which the illumination of the specimen (52) is interrupted.

9. The method according to claim 1, further comprising:

rotating the specimen (52) along an axis perpendicular to the optical axis of the microscope (100) to a plurality of rotation angles;

at each rotation angle obtaining a set of images including a plurality of images captured at a plurality of different relative positions of the specimen (52) with respect to the illumination pattern projected onto the specimen (52); and

generating a three-dimensional image based on the obtained sets of images at the plurality of rotation angles.

10. The method according to claim 1, further comprising:

focusing the illumination pattern of illumination light in a plurality of different focal planes;

for each focal plane obtaining a set of images including a plurality of images captured at a plurality of different relative positions of the specimen (52) with respect to the illumination pattern projected onto the specimen (52); and

generating a three-dimensional image based on the obtained sets of images for the plurality of different focal planes.

11. The method according to claim 1, further comprising sorting-out of one or more of the captured images, wherein a particular image is sorted out if it is determined that the stochastic variation of the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) during the capturing of the image exceeds a predetermined threshold.

12. A large distance microscope (100) for fluorescence and/or reflection illumination observations of a specimen (52), the microscope (100) comprising:

a light source (10;

11;

12) configured to emit illumination light;

a pattern generation system (20;

22;

24) arranged in the optical path of the illumination light, said pattern generation system (20;

22;

24) configured to generate an illumination pattern of the illumination light;

at least one objective (40) arranged and configured to illuminate the specimen (52) by projecting the illumination pattern onto the specimen (52); and

an image capturing system (70) configured to detect at least one of at least a portion of fluorescent light emitted from the specimen (52) and to detect at least a portion of illumination light reflected from the specimen (52), thereby capturing a series of images of the specimen (52) at a plurality of different relative positions of the specimen (52) with respect to the illumination pattern projected onto the specimen (52), wherein between the capturing of at least two images of the series the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) is shifted in a non-controlled manner.

13. The large distance microscope (100) according to claim 12, wherein the at least one objective (40) has a numerical aperture of less than 0.5.

14. The large distance microscope (100) according to claim 12, further comprising a data processing system configured to process the series of captured images, thereby producing a sub-resolution image of the specimen (52).

15. The large distance microscope (100) according to claim 12, further comprising a pattern shifting component configured to stochastically shift the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) and wherein the pattern shifting component is configured to at least one of stochastically shift the specimen (52), perform a stochastic stage scanning, and stochastically move the focusing lens system (42, 44) of the at least one objective (40).

16. The large distance microscope (100) according to claim 12, wherein the light source (10;

11;

12) is configured to emit stroboscopic illumination light having on- and off-times, wherein the specimen (52) is illuminated only during the on-times, and wherein the on-times are respectively shorter than the off-times during which the illumination of the specimen (52) is interrupted.

17. A computer implemented method for generating sub-resolution images of a specimen (52) based on a series of images of the specimen (52) obtained by a microscope (100), wherein the series of images is obtained by projecting an illumination pattern of illumination light onto the specimen (52), thereby illuminating the specimen (52), and at least one of detecting at least a portion of fluorescent light emitted from the specimen (52) and detecting at least a portion of illumination light reflected from the specimen (52), thereby capturing a series of images at a plurality of different relative positions of the specimen (52) with respect to the illumination pattern projected onto the specimen (52), wherein between the capturing of at least two images of the series the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52) is shifted in a non-controlled manner, and wherein said method comprises the steps of:

receiving the series of images of the specimen (52);

for each received image determining the non-controlled shift of the relative position of the specimen (52) with respect to the illumination pattern projected onto the specimen (52);

a-posteriori shifting of each received image to reverse the corresponding non-controlled shift of the relative position of the specimen (52) with respect to the projected illumination pattern, thereby obtaining a corresponding shifted image; and

processing the shifted images to extract a sub-resolution image.

18. A computer program product, which, when loaded into the memory of a computer and executed by the computer performs a computer implemented method according to claim 17.