Scanning Microscope and Method for Determining the Point Spread Function (PSF)of a Scanning Microscope

• US 20160267658A1
• Filed: 10/10/2014
• Published: 09/15/2016
• Est. Priority Date: 10/15/2013
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Inventors: KIEWEG, Michael, KLEPPE, Ingo, NOVIKAU, Yauheni, NETZ, Ralf, NIETEN, Christoph
• Granted Patent: US 10,048,481 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: G02B 21/0072 details concerning resolution or correction, including general design of CSOM objectives
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/008 Details of detection or image processing, including general computer control
• CPC Class Codes: G02B 21/06 Means for illuminating specimens
• CPC Class Codes: G02B 27/58 Optics for apodization or superresolution; Optical synthetic aperture systems
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/10064 Fluorescence image
• CPC Class Codes: G06T 2207/10152 Varying illumination
• CPC Class Codes: G06T 5/50 using two or more images, e.g. averaging or subtraction
• CPC Class Codes: G06T 5/73 Deblurring; Sharpening
• CPC Class Codes: G06T 7/97 Determining parameters from multiple pictures depth or shape recovery from multiple images G06T7/55; stereo camera calibration G06T7/85

Abstract

A method for operating a scanning microscope and for determining point spread functions, with which sample images are recorded with the scanning microscope. The method includes scanning a sample with at least one illuminating light beam; recording at least one sample image with a detector device during a scan by the illuminating light beam; and comprising the point spread function, with which a sample image is recorded, from the at least one sample image. A detector device having receiving elements is used, where the distance between the receiving elements is smaller than a diffraction disk that generates a sample point on the detector device. Detector signals, generated by means of the receiving elements, are read out for each of the different positions of the illuminating light beam on the sample, as a result of which the scanning of the sample allows the detector signals, which are read out, to generate a plurality of sample images. In this case the point spread functions with respect to the different detector signals are defined in each instance by means of an illumination point spread function and a detection point spread function. With respect to all of the detector signals, a matching illumination point spread function is assumed that is shifted in accordance with the scanning motion for different detector signals. In addition, with respect to all of the detector signals, a matching detection point spread function is assumed that takes account of a spatial offset between the detector elements. The plurality of sample images are used to compute the illumination point spread function and the detection point spread function, and these functions are used to compute the point spread functions with respect to the different detector signals. In addition, the invention also relates to a corresponding scanning microscope.

Claims

1. A method for operating a scanning microscope and for determining point spread functions, with which sample images are recorded with the scanning microscope, comprising

scanning a sample with at least one illuminating light beam;

recording at least one sample image with a detector device of the scanning microscope during a scan by said at least one illuminating light beam;

computing the point spread function, with which a sample image is recorded by the scanning microscope, from the at least one sample image,

said detector device having receiving elements, where the distance between said receiving elements is smaller than a diffraction disk that generates a sample point on the detector device,

said receiving elements generating detector signals which are read out for each of the different positions of the illuminating light beam on the sample, as a result of which the scanning of the sample allows the read out detector signals to generate a plurality of sample images, wherein the point spread functions with respect to the different detector signals are defined in each instance by means of an illumination point spread function and a detection point spread function,

assuming that a matching illumination point spread function, with respect to all of the detector signals, is shifted in accordance with the scanning motion for different detector signals, and

a matching detection point spread function is assumed with respect to all of the detector signals, that takes account of a spatial offset between the detector elements, and

using the plurality of sample images to compute the illumination point spread function and the detection point spread function, said point spread functions being used to compute the point spread functions with respect to the different detector signals.

2. The method, as claimed in claim 1, wherein the sample images are used, together with the computation of the point spread functions, to compute a complete image.

3. The method according to claim 1, wherein the computed point spread functions are used to compute the size of a measuring range, which determines a pixel of the complete image.

4. The method according to claim 1, wherein the illumination point spread function and the detection point spread function are computed by iteratively adapting a fit function, wherein the fit function describes a correlation of the recorded sample images to at least the illumination point spread function, to the detection point spread function, to the respective offset between the illumination point spread function and the detection point spread function for the various detector signals, and to a sample function, which represents the sample spatially resolved.

5. The method according to claim 4, wherein the fit function comprises an expression, which is to be iteratively adapted and which describes the illumination point spread function, and an expression, which is to be iteratively adapted and which describes the detection point spread function.

6. The method according to claim 5, wherein the two expressions, which are to be iteratively adapted, are formed in each case by Zernike polynomials.

7. The method according to claim 5, further comprising using a theoretical illumination point spread function and a theoretical detection point spread detection, both of which are determined in advance with the given information about the optical imaging means of the scanning microscope, as the starting values for the two expressions that are to be iteratively adapted.

8. The method according to claim 1, further comprising determining a theoretical illumination point spread function and a theoretical detection point spread function by means of given information about the optical imaging means of the scanning microscope, determining variances between the theoretical illumination point spread function and the computed illumination point spread function as well between the theoretical detection point spread function and the computed detection point spread function, and identifying aberrations, based on the variances that are found.

9. The method according to claim 8, further comprising

storing user instructions for handling elements of the scanning microscope, on which the illuminating light or the sample light, emitted by the sample, impinges, in an electronic memory,

storing an allocation of the user instructions to various aberrations,

using electronic evaluation means to select, as a function of the identified aberrations, the associated user instructions, which are outputted to a user.

10. The method according to claim 8, wherein the aberrations of the scanning microscope can be corrected with adaptive optics, and using electronic control and evaluation means as a function of the identified aberrations, to adjust the adaptive optics, in order to reduce aberrations of the scanning microscope.

11. The method according to claim 1, further comprising

recording an additional plurality of samples in order to determine the height dependence of the point spread function, from which at least one additional complete image is computed,

wherein the measurements with respect to various complete images differ in the vertical offset between the illumination point spread function and the detection point spread function, and

using the plurality of complete images to compute the height dependence of the point spread functions.

12. The method according to claim 11, further comprising

recording the sample images for the different complete images without having to adjust the height of the sample, a light source device, which emits the illuminating light, or the detector device, for which purpose

the light source device comprises at least two light source units, which are disposed in planes that are optically conjugate to various sample planes, and/or

the detector device comprises two detector areas, onto which two different planes are mapped.

13. The method according to claim 12, further comprising simultaneously recording the sample images for a first complete image with the sample images for a second complete image.

14. The method according to claim 1, wherein each sample is scanned with illuminating light of different wavelength ranges, and/or that the detector device is used to measure the sample light, coming from the sample and composed of different wavelength ranges;

and the illumination point spread function to be computed and/or the detection point spread function to be computed includes and/or include a parameter to describe the wavelength dependence of the illumination point spread function and/or the detection point spread function.

15. The method according to claim 4, wherein the fit function includes parameters, which are to be adapted and which describe the arrangement of the detector elements relative to each other, in particular, for determining an image magnification, with which a sample plane is mapped onto the detector device, and/or for determining an orientation of the detector device relative to the scanning direction of the illuminating light.

16. A scanning microscope, comprising

a light source device for emitting at least one illuminating light beam, comprising a scanning device for generating a scanning motion of the at least one illuminating light beam across a sample,

a detector device for recording at least one sample image during a scan by the illuminating light beam,

electronic control and evaluation means, for computing a point spread function, with which a sample image is recorded, from the at least one sample image,

said detector device having receiving elements, where the distance between said receiving elements is smaller than a diffraction disk that generates a sample point on the detector device,

said electronic control and evaluation means further being designed to read out the detector signals, generated by means of the receiving elements, for each of the different positions of the illuminating light beam on the sample, as a result of which the scanning of the sample allows the detector signals, which are read out, to generate a plurality of sample images,

wherein the point spread functions with respect to the different detector signals are defined in each instance by means of an illumination point spread function and a detection point spread function, and

wherein said the electronic control and evaluation means are designed to compute an illumination point spread function and an a detection point spread function with the plurality of sample images;

and these two functions are used to compute the point spread functions with respect to the different detector signals,

and wherein with respect to all of the detector signals, there is a matching illumination point spread function that is shifted in accordance with the scanning motion for different detector signals; and

that with respect to all of the detector signals there is a matching detection point spread function that takes account of a spatial offset between the detector elements.