METHOD AND AN APPARATUS FOR LOCALIZATION OF SINGLE DYE MOLECULES IN THE FLUORESCENT MICROSCOPY

• US 20090237501A1
• Filed: 03/16/2009
• Published: 09/24/2009
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: Ruprecht-Karls-Universitã¤T Heidelberg
• Original Assignee: Universitat Heidelberg
• Inventors: Baddeley, David, Lemmer, Paul, Cremer, Christoph, Eipel, Heinz
• Granted Patent: US 8,212,866 B2
• Time in Patent Office: Days
• US Class Current: 348/79
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• 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 and apparatus are provided for obtaining a sub-resolution spatial information of a sample labeled with at least one type fluorescent label. The sub-resolution spatial information has localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction. The method acquires localization image data by employing fluorescence localization microscopy. The acquired localization image data is processed to obtain the localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction. The step of processing includes determining in each of the detected images of the series the positions of the barycenters of the detected fluorescence emission distributions from the single fluorescent molecules of the one or more fluorescent labels in at least one spatial direction.

Claims

1. A method for obtaining a sub-resolution spatial information of a sample labeled with at least one type fluorescent label, said sub-resolution spatial information comprising localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction, comprising the steps:

acquiring localization image data by employing fluorescence localization microscopy, wherein said localization image data comprises a series of images obtained by

illuminating a region of interest of the sample with illumination light having intensity in the range of approximately 1 kW/cm² to 1 MW/cm²,

detecting by an information acquiring sensor of at least a portion of the fluorescent light emitted by at least a portion of the fluorescent molecules of the at least one type fluorescent label upon illumination, thereby obtaining an image of the region of interest;

repeating the steps of illuminating and detecting of the emitted fluorescent light a plurality of times, thereby obtaining the series of images, each image being taken at a different time step;

processing the acquired localization image data to thereby obtain said localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction, wherein the step of processing comprises determining in each of the detected images of the series the positions of the barycenters of the detected fluorescence emission distributions from the single fluorescent molecules of the one or more fluorescent labels in at least one spatial direction.

2. The method according to claim 1, wherein upon illumination, at least a portion of the fluorescent molecules are transferred from a first fluorescent state to a second, reversibly bleached state.

3. The method according to claim 2, wherein upon illumination, at least a portion of the fluorescent molecules of the at least one type fluorescent label are transferred from the first state to the second state and after recovery to the first state to a third, inactive state.

4. The method according to claim 3, wherein the third state is an irreversibly bleached state.

5. The method according to claim 1, wherein the one or more fluorescent labels comprise fluorescent proteins and/or their derivatives and/or modifications.

6. The method according to claim 1, wherein the one or more fluorescent labels comprise non-protein based fluorescent labels and/or their derivatives.

7. The method according to claim 1, wherein the one or more fluorescent labels comprise green fluorescent protein (GFP) and/or its derivatives and/or modifications, for example, green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), OFP, enhanced green fluorescent protein (eGFP), emGFP, enhanced yellow fluorescent protein (eYFP).

8. The method according to claim 1, wherein the one or more fluorescent labels comprise monomeric red fluorescent protein (mRFP) and its derivatives and modifications, for example mCherry.

9. The method according to claim 1, wherein the one or more fluorescent labels comprise rhodamin derivatives, for example Alexa- and/or attodyes for non-proteins.

10. The method according to claim 1, wherein the one or more fluorescent labels comprise xanthen derivatives, for example fluorescein.

11. The method according to claim 1, wherein the one or more fluorescent labels comprise rhodamin derivatives, for example Alexa- and/or attodyes.

12. The method according to claim 1, wherein the one or more fluorescent labels comprise coumarin derivatives.

13. The method according to claim 1, wherein the one or more fluorescent labels comprise cyanin derivatives.

14. The method according to claim 1, wherein the employed fluorescence localization microscope has a confocal set-up of the optical illumination path, a wide field set-up of the optical illumination path, a 4Pi set-up, a STED set-up or STED-4Pi set-up.

15. The method according to claim 1, wherein the used illumination light is monochromatic.

16. The method according to claim 1, wherein a plurality of different types of fluorescent labels are employed and the step of acquiring localization image data of one or more objects in a region of interest by employing fluorescence localization microscopy is carried out separately for each fluorescent label using illumination light with an optimal intensity selected from within the range of approximately 1 kW/cm² to 1 MW/cm².

17. The method according to claim 1, wherein the step of processing the acquired localization image data comprises fitting of a model function f(x,y) to the acquired the acquired fluorescent emission distributions from the single fluorescent molecules in each of the two dimensional images of the time series:

$f<br><br>\left(x,y\right)=A<br><br><br><br>\exp \left(-\frac{{\left(x_0-x\right)}^2+{\left(y_0-y\right)}^2}{2<br><br>{\mathrm{\sigma <br><br>}}^2}\right)+B_0+B_1<br><br>\left(x_0-x\right)+B_2<br><br>\left(y_0-y\right),$

wherein x and y are Cartesian coordinates in an object plane, perpendicular to the optical axis of the microscope;

x₀ and y₀ are the starting parameters for the position, which are determined as the center of the segmented signal;

A is the amplitude of the distribution, and B₀, B₁, B₂ are parameters describing linear background.

18. The method according to claim 1, wherein in the step of acquiring localization image data the one or more objects in the region of interest are illuminated by a structured illumination light.

19. The method according to claim 1, further comprising acquiring additional wide field image data comprising a series of wide field images of the region of interest by employing a wide field fluorescence microscopy using illumination light, which is spatially structured, respectively modulated along an optical axis of the microscope, said acquiring additional wide field image data being obtained by:

illuminating the one or more objects in the region of interest with the structured illumination light;

detecting a wide field image of the fluorescent light emitted from the fluorescent molecules of the one or more fluorescent labels;

moving the object and/or the structured illumination light in discrete steps along the optical axis and detecting at each step a wide field image of the fluorescent light emitted from the fluorescent molecules, thereby obtaining said series of wide field images of the region of interest,

wherein said step of acquiring additional wide field image data of the one or more objects is carried out before the step of acquiring localization image data.

20. The method of claim 19 further comprising:

processing the acquired additional wide field image data to obtain additional spatial information comprising information about the spatial extension along the optical axis of at least one fluorescently labeled object in the sample and/or additional spatial information of the positions of the barycenters of the detected fluorescence emission distribution of the single fluorescent molecules in the direction of the optical axis; and

combining the localization information obtained by the localization microscopy with the additional spatial information obtained by the wide field fluorescence microscopy using structured illumination light.

21. The method of claim 19, further comprising:

generating a common theoretical three dimensional model function for all detected signals within the one or more objects, wherein said three dimensional model function is divided in plurality of two dimensional layers along the optical axis, and

performing a lateral cross correlation of the acquired three dimensional image data and said three dimensional model function, wherein the maxima of the correlation function represent both one object identification and one three-dimensional localization.

22. The method of claim 21, further comprising applying a threshold method to the obtained correlation maxima.

23. Method of claim 19 further comprising a step of carrying out a spatial calibration of the structured illumination, wherein the spatial calibration is carried out with the help of at least one fluorescent reference point.

24. Method of claim 19, wherein

during acquiring additional image data of said one or more objects with wide field fluorescence microscopy using structured illumination light, the one or more objects are illuminated with the structured illumination such, that at least a portion of the fluorescence molecules of the at least one fluorescent label is transferred in an active state and is used for the corresponding far-field observation, whereas a second portion of the fluorescence molecules remains in an inactive state;

and

during the step of acquiring localization image data by employing fluorescence localization microscopy a second portion of the fluorescence molecules is activated by changing of the illumination light intensity of the optical radiation to the one lying within the range of approximately 1 kW/cm² to 1 MW/cm², wherein said step of acquiring localization image data by employing fluorescence localization microscopy is carried out on the basis of the second part of the fluorescent molecules.

25. A fluorescence localization microscope for obtaining a sub-resolution spatial information of a sample labeled with at least one type fluorescent label, said sub-resolution spatial information comprising localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction, said microscope comprising:

an illumination optics defining an optical illumination path, configured to illuminate the one or more objects in a region of interest;

at least one additional optical element positioned within the optical illumination path of the localization microscope, the at least one additional optical element configured to enable a switching of the intensity of the illumination light to an intensity lying within the range of 1 kW/cm² to 1 MW/cm² and/or an adjustment, respectively regulation of the intensity of the illumination light within the range of 1 kW/cm² to 1 MW/cm²;

at least one information acquiring sensor positioned in an optical detection path, configured to detect at least a portion of the emitted fluorescent light, thereby obtaining an image of the illuminated region of interest.

26. The microscope of claim 25, wherein the localization microscope has a confocal set-up of the optical illumination path, a wide field set-up of the optical illumination path, a 4Pi set-up, a STED set-up or STED-4Pi set-up.

27. The microscope of claim 25, wherein the at least one additional optical element comprises one or more of

at least one lens or a lens system;

at least one gray filter or a gray filter set;

at least one polarization filter;

at least one acoustooptical modulator; and

/or

at least one electrooptical modulator.

28. The microscope of claim 25, further comprising at least one additional sensor, which is used in at least one additional optical detection path, said additional sensor and said additional optical detection path configured to register at least a portion of the fluorescent light, which does not reach the information acquiring sensor.

29. The microscope of claim 28, further comprising at least one second additional optical element positioned in the least one additional optical detection path.

30. The microscope according to claim 25 comprising furthermore a processing unit, configured to process the acquired localization image data to obtain sub-resolution spatial information of a sample labeled with at least one type fluorescent label, said sub-resolution spatial information comprising localization information about the positions of fluorescent molecules of the at least one type fluorescent label in at least one spatial direction.