Wavelength-resolving and high spatial resolution fluorescence microscopy
A method for wavelength-resolving and high spatial resolution fluorescence microscopy in which fluorescence labels in a sample are repeatedly excited to emit fluorescence radiation and frames including images of isolated labels are produced with a microscope. The positions of the images of the isolated fluorescing labels are localized with a localization precision exceeding the optical resolution of the imaging beam path of the microscope. The imaging beam path of the microscope has a diffractive element which, during the imaging, diffracts the image of the sample comprising the isolated fluorescing labels into a first diffraction order so that each frame contains the first diffraction order images of the isolated fluorescing labels. A parameter of the first diffraction order images of the isolated fluorescing labels is evaluated and an indication of the wavelength of the isolated fluorescing labels is derived from this evaluated parameter.
1. A method for wavelength-resolving and high spatial resolution fluorescence microscopy, comprising:
using an illumination device to repeatedly excite fluorescence labels in a sample to emit fluorescence radiation;
producing a plurality of frames of the sample and a high-resolution image of the sample using a microscope having a detector and an imaging beam path, the imaging beam path having an optical resolution and a spectrally selective diffractive element with known diffraction properties, the frames comprising images of excited and fluorescing labels of the sample imaged onto the detector, wherein the labels are excited such that images of at least some of the fluorescence labels are isolated in each frame and the positions of the images of the isolated fluorescing labels are localized in the frames with a localization precision exceeding the optical resolution, and wherein the diffractive element of the imaging beam path diffracts the image of the sample comprising the isolated fluorescing labels into a first diffraction order such that each frame contains the first diffraction order images of the isolated fluorescing labels;
evaluating at least one parameter of the first diffraction order images of the isolated fluorescing labels in the frames;
deriving an indication of the wavelength of the isolated fluorescing labels from the evaluated parameter; and
wherein the labels comprise emitter ensembles wherein each emitter ensemble is formed by at least one first emitter and a second emitter, wherein in all of the emitter ensembles the second emitter is of the same type and has known spectral emission properties and the first emitter is selected from a group of emitters, with each member of the group of emitters having known spectral emission properties, wherein the members of the group differ in their spectral emission properties and in their capability to mark sample structure, whereby the image of each isolated fluorescing label in the frames consists of a diffraction image of the at least one first emitter and, separated therefrom, a diffraction image of the second emitter, and wherein the positions of the isolated fluorescing labels in the sample are determined from the position of the diffraction images of the second emitters, and the at least one parameter comprises the distance between the diffraction images of the second emitter and the associated at least one first emitter.
2. The method according to claim 1 , wherein the image of each isolated fluorescing label is imaged into a zero diffraction order onto the detector and the position of each isolated fluorescing label is determined from the zero order diffracted image.
3. The method according to claim 1 , wherein the ensembles are pairs.
4. The method according to claim 1 , wherein the diffractive element diffracts the majority of incident radiation into a first diffraction order, and wherein the diffractive element is a blazed grating or a diffractive optical element (DOE).
5. The method according to claim 1 , wherein the diffraction causes a non-rotationally symmetric blurring of the images of the isolated fluorescing labels and a value of the non-rotationally symmetric blurring is the evaluated parameter.
6. The method according to claim 5 , wherein the at least one parameter further comprises a rotational asymmetry of the images of the first emitters.
7. The method according to claim 6 , wherein the ensembles are pairs.
8. The method according to claim 6 , wherein the diffractive element diffracts the majority of incident radiation into a first diffraction order, and wherein the diffractive element is a blazed grating or a diffractive optical element (DOE).
9. The method according to claim 5 , wherein for each image of an isolated fluorescing label, the location indication is corrected for a diffraction-limited offset by using the determined wavelength indication of the label and by taking into account the known diffraction properties of the spectrally selective diffractive element.
10. The method according to claim 1 , wherein the image of each isolated fluorescing label is imaged into a zero diffraction order onto the detector and a distance between the zero diffraction order image and the first diffraction order image of this label is the evaluated parameter.
11. The method according to claim 10 , wherein the image of each isolated fluorescing label is imaged into a zero diffraction order onto the detector and the position of each isolated fluorescing label is determined from the zero order diffracted image.
12. A fluorescence microscope for wavelength-resolving imaging of a sample with a spatial resolution being better than an optical resolution of the microscope, the microscope comprising:
an illumination device configured to repeatedly excite fluorescence labels in the sample to emit fluorescence radiation;
an imaging device, comprising an imaging beam path and a detector, configured to image the sample onto the detector with the optical resolution, so as to produce a plurality of frames in which at least some of the labels excited by the illumination device are isolated in each frame, the imaging beam path including a spectrally selective diffractive element with known diffraction properties configured to diffract the image of the sample comprising the isolated fluorescing labels into a first diffraction order such that each frame contains first diffraction order images of the isolated fluorescing labels; and
a control device configured to control the illumination device and the imaging device, such that the positions of the isolated fluorescing labels are localized with a localization precision exceeding the optical resolution in the frames, and so as to produce a high-resolution image of the sample, and wherein the control device is configured to evaluate, in the frames, the first diffraction order images of the isolated fluorescing labels and derive therefrom an indication of the wavelength of the isolated fluorescing labels; and
wherein the fluorescence labels in the sample comprise emitter ensembles wherein each ensemble is formed by at least one first emitter and a second emitter, wherein in all the emitter ensembles the second emitter is of the same type and has known spectral emission properties and the first emitter is selected from a group of emitters, with each member of the group of emitters having known spectral emission properties, wherein the members of the group differ in their spectral emission properties and in their capability to mark sample structure, whereby the image of each isolated fluorescing label in the frames consists of a diffraction image of the at least one first emitter and, separated therefrom, a diffraction image of the second emitter, and wherein the control device is configured to determine the positions of the isolated fluorescing labels in the sample from the position of the diffraction images of the second emitters, and evaluate the distance between the diffraction images of the second emitter and the associated at least one first emitter to derive therefrom the indication of the wavelength of the isolated fluorescing labels.
13. The fluorescence microscope according to claim 12 , wherein the imaging device is further configured such that the image of each isolated fluorescing label is imaged into a zero diffraction order onto the detector and the control device is configured to determine the position of each isolated fluorescing label from the zero order diffracted image.
14. The fluorescence microscope according to claim 12 , wherein the diffractive element is configured to diffract the majority of incident radiation into a first diffraction order, and wherein the diffractive element is a blazed grating or a diffractive optical element (DOE).
15. The fluorescence microscope according to claim 12 , wherein the diffraction causes a non-rotationally symmetric blurring of the images of the isolated fluorescing labels and the control device is configured to evaluate a value of the non-rotationally symmetric blurring.
16. The fluorescence microscope according to claim 15 , wherein the control device is configured to evaluate a rotational asymmetry of the images of the first emitters to derive therefrom the indication of the wavelength of the isolated fluorescing labels.
17. The fluorescence microscope according to claim 15 , wherein for each image of an isolated fluorescing label, the control device is configured to correct the location indication for a diffraction-limited offset by using the determined wavelength indication of the label and by taking into account the known diffraction properties of the spectrally selective diffractive element.