METHOD AND MICROSCOPE FOR THREE-DIMENSIONAL RESOLUTION-ENHANCED MICROSCOPY

• US 20120224034A1
• Filed: 08/26/2010
• Published: 09/06/2012
• Est. Priority Date: 09/30/2009
• Status: Active Grant

• Current Assignee: Carl Zeiss Microscopy GmbH (Carl-Zeiss-Stiftung)
• Original Assignee: CARL Zeiss Microimaging GmbH (Carl-Zeiss-Stiftung)
• Inventors: Kalkbrenner, Thomas, Wolleschensky, Ralf
• Granted Patent: US 8,970,688 B2
• Time in Patent Office: Days
• US Class Current: 348/49
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• 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

The invention relates to a high resolution microscope for three-dimensionally determining the position of objects, in particular individual fluorophores, and preferably for the high spatial resolution luminescence microscopy of a sample, which is marked with marker molecules that can be activated or switched using a signal such that they can be induced to emit certain luminescent radiation only in the activated state. The object is represented by means of an imaging system, preferably the microscope lens, on a surface detector consisting of individual detector elements. At least one microlens array is located in front of the detector elements, and different, preferably adjacent, detector elements receive light from microlenses having different focal lengths and from different object planes, or wherein by means of at least once microlens array, located in part in front of the detector elements, a different object plane is represented on the detector elements in the direction of the light behind the microlenses than on detector elements having no microlenses in front of the latter.

Claims

1. A method for high-resolution microscopy for three-dimensional determination of the position of objects, in particular individual fluorophores, for spatial high-resolution luminescence microscopy of a sample labeled with marker molecules which can be activated or switched by means of a signal in such a way that they can be excited to emit certain luminescent radiation only in the activated state, wherein the method comprises the following steps:

a) introduction of a signal onto the sample in such a way that only a subquantity of the marker molecules present in the sample are activated, wherein the sample contains subquantities in which the distance between the closest neighboring activated marker molecules is at least greater than or equal to a length resulting from a predefined optical resolution,

b) excitation of the activated molecules to emit luminescent radiation,

c) detection of the luminescent radiation with the predefined optical resolution, and

d) generation of an individual picture from the luminescent radiation captured in step c), wherein the geometrical locations of the marker molecules emitting luminescent radiation are identified by means of a spatial resolution increased above the predetermined optical resolution, wherein the steps are repeated several times and the plurality of individual pictures obtained in this fashion are combined into a complete image, and wherein

an object is imaged onto a surface detector consisting of individual detector elements by means of an imaging system, preferably the microscope lens, wherein a different object plane is imaged onto the detector element in the direction of light behind the microlenses than onto the detector element in front of which no microlenses are located through at least one microlens array which is partly located in front of the detector elements.

2. The high-resolution microscopy method according to claim 1, further comprising symmetrical distribution of the microlenses.

3. The high-resolution microscopy method according to claim 1, wherein

a different object plane is imaged onto the detector elements in the direction of light behind the microlenses than onto the detector elements in front of which no microlenses are located.

4. The high-resolution microscopy method according to claim 1, wherein the microlenses are arranged in front of the detector and/or in or in the vicinity of an intermediate image plane.

12. The microscopic method, according to claim 1 for the generation of a three-dimensionally resolved image, by further comprising imaging objects onto a surface detector consisting of individual detector elements by means of an imaging system, and where a different object plane is imaged onto the detector elements in the direction of the light behind the microlenses than onto detector elements in front of which no microlenses or microlenses with a different focal width are located through at least one microlens array which is located at least partly in front of the detector elements.

13. The microscopic method, according to claim 1, wherein one or a plurality of detector elements of a surface detector arranged around a central detector element are jointly evaluated in such a way that a two- or three-dimensional position recognition and/or object recognition is achieved by mathematically linking the signals of the detectors arranged around a central detector element and by compiling the location and/or movement information based on the mathematical result and/or its sign.

14. The method according to claim 1 further comprising tracking one or a plurality of particle pathways.

15. The method according to claim 1 wherein said method is used in a pair of or as optical tweezers.

5. A method of high-resolution microscopy for the three-dimensional determination of the position of objects, in particular individual fluorophores, for spatial high-resolution luminescent microscopy of a sample labeled with marker molecules which can be activated or switched by means of a signal in such a way that they can only be excited to emit certain luminescent radiation in the activated state, wherein the method comprises the following steps:

a) introduction of the signal onto the sample in such a way that only a subquantity of the marker molecules present in the sample are activated, wherein the sample contains subquantities in which the distance between the closest neighboring activated marker molecules is at least greater than or equal to a length resulting from a predefined optical resolution,

b) excitation of the activated molecules to emit luminescent radiation,

c) detection of the luminescent radiation with the predefined optical resolution and

d) generation of an individual picture from the luminescent radiation captured in step c), wherein the geometrical locations of the marker molecules emitting luminescent radiation are identified by means of a spatial resolution increased above the predetermined optical resolution, wherein the steps are repeated several times and the plurality of individual pictures obtained in this fashion are combined into a complete image, and wherein an object is imaged onto a surface detector consisting of individual detector elements by means of an imaging system, preferably the microscope lens, wherein at least one microlens array is located in front of the detector elements and different, preferably neighboring detector elements obtain light from microlenses with different focal lengths and from different object planes.

6. The high-resolution microscopy method according to claim 5, wherein the microlenses are arranged in front of the detector and/or in or in the vicinity of an intermediate image plane.

7. A method for high-resolution microscopy for three-dimensional determination of the position of objects, in particular individual fluorophores, for the spatial high-resolution luminescent microscopy of a sample labeled with marker molecules which can be activated or switched by means of a signal in such a way that they can be excited to emit certain luminescent radiation only in the activated state, wherein the method comprises the following steps:

a) introduction of the signal onto the sample in such a way that only a subquantity of the marker molecules present in the sample are activated, wherein the sample contains subquantities in which the distance between the closest neighboring activated marker molecules is at least greater than or equal to a length resulting from a predefined optical resolution,

b) excitation of the activated molecules to emit luminescent radiation,

c) detection of the luminescent radiation with the predefined optical resolution and

d) generation of an individual picture from the luminescent radiation captured in step c), wherein the geometrical locations of the marker molecules emitting luminescent radiation are identified by means of a spatial resolution increased above the predetermined optical resolution, wherein the steps are repeated several times and the plurality of individual pictures obtained in this fashion are combined into a complete image, and wherein

an object is imaged onto a surface detector consisting of individual detector elements by means of an imaging system, preferably the microscope lens, and one or a plurality of detector elements arranged around a central detector element are jointly evaluated in such a way that a two- or three-dimensional position recognition and/or object recognition is achieved by mathematically linking the signals of the detectors arranged around a central detector element and by compiling location- and/or movement-related information based on the mathematical result and/or its sign.

8. A microscope for carrying out the method of claim 7, wherein detectors are arranged in a horizontal plane (X, Y) and/or a vertical plane (X,Z;

Y,Z).

9. The microscope according to claim 7, wherein the imaging onto at least one detector element is performed by means of an anamorphotic imaging element for the determination of a Z-position of an object.

10. The microscope according to claim 9, where the spatial location in Z is determined based on the distortion of the detected image.

11. The microscope according to claim 9, wherein the anamorphotic element is a combination of microlenses and cylinder lenses.