METHOD AND DEVICE FOR HIGH-RESOLUTION FLUORESCENCE MICROSCOPY

Abstract

A method for high-resolution fluorescence microscopy, in which a number N of partial images of a specimen marked with fluorophores and excited to emit fluorescence are recorded, wherein the specimen is successively illuminated by N different effective illuminating patterns, a composite image is calculated from the partial images, the composite image having a higher structural resolution than the partial images, and the composite image is subsequently output, wherein each effective illuminating pattern is generated from the superimposition of at least two basic illuminating patterns, with the basic illuminating patterns differing from each other and for each partial image, and wherein the fluorophores contained in the sample are excited to emit fluorescence only where the at least two basic illuminating patterns superimpose to illuminate the sample, with the illumination superimposition of the at least two basic illuminating patterns releasing non-linear excitation and/or emission effects and/or switching effects in the fluorophores.

Claims

1. A method for high-resolution fluorescence microscopy, comprising: recording a number of N partial images of a specimen marked by fluorophores and excited to emit fluorescence, successively illuminating the specimen with N different effective illuminating patterns, and calculating a composite image from the partial images, the composite image having a higher structural resolution than the partial images, and outputting the composite image, wherein each effective illuminating pattern is generated from the superimposition of at least two basic illuminating patterns, with the basic illuminating patterns differing from each other and for each partial image, and wherein the fluorophores contained in the specimen are excited to emit fluorescence only where the at least two basic illuminating patterns superimpose to illuminate the specimen, with the illuminating superimposition of the at least two basic illuminating patterns triggering off non-linear excitation and/or emission effects and/or switching effects in the fluorophores.

2. The method as claimed in claim 1, wherein the basic illuminating patterns are generated statistically as speckle patterns.

3. The method as claimed in claim 2, wherein the speckle patterns change during the recording of a partial image.

4. The method as claimed in claim 1, wherein the at least two basic illuminating patterns are projected onto the specimen and there get superimposed to form the effective illuminating pattern.

5. The method for high-resolution fluorescence microscopy as claimed in claim 1, further comprising: marking the specimen using photoswitchable fluorophores, which with activating light of an activation wavelength are put into a state capable of emitting fluorescence, and which with excitation light of an excitation wavelength that differs from the activation wavelength are excited to emit fluorescence, illuminating the specimen with coherent activating light, wherein the activating light as the first basic illuminating pattern gets a first activation pattern imparted to it, and the first activation pattern is projected onto the specimen, so that this is illuminated by activating light in a structured mode, and simultaneously illuminating the specimen with coherent excitation light, wherein the excitation light as a second basic illuminating pattern gets a first excitation pattern differing from the first activation pattern imparted to it, and the first excitation pattern is projected onto the specimen via a microscope objective, so that the specimen is illuminated by excitation light in a structured mode, for which reason fluorescence signals are emitted by such fluorophores only that are simultaneously illuminated by activation and excitation light, projecting the fluorescence signals onto a flat-panel area detector and recording the projected fluorescence signals as intensity levels, from which a first partial image is generated, interrupting the illumination at least with activating light until the predominating share of the fluorophores has passed into a non-activated ground state, repeating the steps a through d with further activation patterns and further excitation patterns differing from each other, to generate further partial images.

6. The method as claimed in claim 5, wherein, for generating the first activation pattern and the first excitation pattern, in each case the same region of a diffuser disk in the illuminating ray path is illuminated, with the diffuser disk being projected into an entrance pupil of a microscope objective and from there onto the specimen, and that, for generating the further activation and excitation patterns, the diffuser disk is rotated about an optical axis or shifted laterally relative to this axis.

7. A device for high-resolution fluorescence microscopy applied to a specimen marked by fluorophores (1), wherein the fluorophores can, by non-linear processes or switching processes, be excited to emit fluorescence, comprising, an illuminating device, a pattern generator, with which, when they are illuminated by the illuminating device, at least two different basic illuminating patterns can be generated simultaneously, the pattern generator configured to vary the at least two basic illuminating patterns at least between two recordings of partial images, a microscope objective configured to project and superimpose the basic illuminating patterns onto the specimen to form an effective illuminating pattern, a flat-panel area detector, on which fluorescence signals emitted by the specimen are projected by the microscope objective and detected as a partial image, and a computer for computing a composite image from a number of partial images.

8. The device as claimed in claim 7, wherein the pattern generator comprises a speckle generator.

9. The device as claimed in claim 8, wherein the speckle generator comprises LCOSs, DOEs, MEMSs, AOMs, at least one diffuser disk, and/or at least one object having an optically rough surface with a roughness greater than a longest wavelength of the light radiated by the illuminating device.

10. The device as claimed in claim 8, wherein the speckle generator comprises a diffuser disk, which, for varying the basic illuminating patterns, can be rotated about an optical axis or shifted perpendicularly to the optical axis.

11. The device as claimed in claim 7, wherein the illuminating device is adapted to radiate coherent light of two wavelengths, which in interaction with the effective illuminating pattern excite the fluorophores to emit fluorescence where the at least two basic illumination patterns superimpose to effect illumination.

12. The device as claimed in claim 7, wherein the device is configured such that the illumination of the specimen is interrupted for a specified period between the recordings of two partial images.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Below, the invention will be explained in more detail and exemplified with reference to the accompanying drawings, which also show features essential to the invention, among others, and in which

[0033] FIG. 1 the setup of a device for high-resolution fluorescence microscopy,

[0034] FIG. 2A)-C) the mode of operation of photoswitchable fluorophores, and

[0035] FIG. 3 the operating principle of recording high-resolution images.

DETAILED DESCRIPTION OF THE DRAWINGS

[0036] To start with, FIG. 1 shows a device with which a method for high-resolution fluorescence microscopy can be performed. In the exemplary embodiment shown here, a specimen 1 arranged on a specimen slide 2 is first marked by photoswitchable fluorophores. The device comprises means for illumination, viz two coherent light sources, an activation laser 3 and an excitation laser 4. The activation laser 3 radiates activating light of an activation wavelength, with which the fluorophores can be put into a state that enables them to fluoresce. The excitation laser 4 radiates excitation light of an excitation wavelength that differs from the activation wavelength. With this excitation wavelength, the fluorophores, once activated, can be put into a state that enables them to fluoresce. Instead of two different lasers one can use a broadband light source equipped with suitable filters.

[0037] The mode of operation of the photoswitchable fluorophores will now be explained in more detail in connection with FIG. 2, which shows the energy levels of such a fluorophore. In the deactivated ground state shown in FIG. 2A), The energy gap between the ground state S.sub.0 and the excited state S.sub.1 or S.sub.2, respectively, cannot be bridged by illumination with light of the excitation wavelength, i.e., with excitation light; the energy is not absorbed, and no fluorescence is possible. Here, light of the excitation wavelength and the excitation energy are represented by arrows with dashed lines. Upon irradiation of the fluorophore molecules with activating light of the activation wavelength, as shown in FIG. 2B), the molecular structure of the fluorophore and, thus, the energy-level diagram changes. The two excitation levels S.sub.1 and S.sub.2 now have but a closer energetic distance to the ground state.sub.0. Light of the activation wavelength is represented by the arrow with the solid, wavy line; in most cases, the activation wavelength is shorter than the excitation wavelength. Now, when the fluorophore is irradiated by excitation light of the excitation wavelength, as shown in FIG. 2C), light of this wavelength is absorbed. By means of a vibration relaxation between the excited states S.sub.2 and S.sub.1 and the subsequent fluorescence process between the excited state S.sub.1 and the ground state S.sub.0, the fluorescent dye molecule returns to the ground state S.sub.0 once it has emitted light of the fluorescence wavelength, shown here by the dash-and-dot line.

[0038] For creating a high-resolution fluorescence image, a number N of partial images of the specimen marked by the fluorophores and excited to emit fluorescence are recorded. In this process, the specimen is successively illuminated with N different effective illuminating patterns, and a composite image is computed from the partial images, the said composite image having a higher structural resolution than the partial images. The composite image can then be read out. Each of the effective illuminating patterns is generated from the superimposition of at least two basic illuminating patterns, with the basic illuminating patterns being different from each other as well as for each partial image. On this condition, it is, in principle, possible also to choose activation wavelength and excitation wavelength to be equal, provided the fluorophores are selected accordingly.

[0039] In the above example, the specimen 1 is, on the one hand, illuminated by coherent activating light of the activation laser 3, wherein to the activating light, being the first basic illuminating pattern, a first activation pattern is imparted, which is projected onto the specimen, so that the specimen is illuminated in a structured mode by activating light. At the same time, the specimen 1 is illuminated by coherent excitation light of the excitation laser 4, wherein to the excitation light, being a second basic illuminating pattern, a first excitation pattern is imparted that differs from the first activation pattern. For that purpose, in the example shown in FIG. 1, the light of the activation laser 3 and the excitation laser 4 is directed via a dichroic beam splitter 5, an acousto-optical filter 6 and a lens 7 onto a diffuser disk 8, which can be laterally shifted perpendicularly to an optical axis 9 and/or rotated about the said axis. As a fluorescent dye, one can use, e.g., FLIP 565 made by Abberior GmbH; in this case, the wavelengths for activation and excitation are 375 nm and 561 nm, respectively.

[0040] Before a partial image is recorded, the acousto-optical filter 6, triggered by a computer 10, is switched to cause a region of the diffuser disk to be coherently illuminated with light both of the activation and of the excitation wavelength. The region on the diffuser disk should not be as large as to occupy the total diffuser disk, but rather as small as possible in order to keep spatial correlation between different partial images as low as possible. This correlation is the greater, the more regions of the diffuser disk appear in two or more images. Moreover, illumination of the diffuser disk 8 should take place in such a way that the entrance pupil 11 of a microscope objective 12 is illuminated as completely as possible, so that the speckles generated in the specimen plane have minimum extension, i.e. are diffraction-limited.

[0041] On account of the two different wavelengths of the laser light, two different illumination structures that vary statistically in space will form in the plane of the specimen 1, with the said two illumination structures being incoherent relative to each other, even though identical regions of the diffuser disk are illuminated. Because of illumination with the activation pattern, which is generated by the activating light of the activation laser 3, the fluorophores in the specimen 1 are, with spatial and statistical variation, put into a state capable of fluorescein. On account of the excitation pattern that is formed simultaneously by the laser light of the excitation laser 4, the fluorescent fluorophores capable of fluorescein are also excited to emit fluorescence, also with spatial and statistical variation. However, only such fluorophores will be excited to emit fluorescence, and actually do so, which are simultaneously illuminated by activation and excitation light, i.e. where the two basic illuminating patterns superimpose while illuminating the specimen 1. The illuminating patterns are projected into the entrance pupil 11 of the microscope 12 by means of an optical system 13. Fluorescence light emitted by the specimen is projected onto a flat-panel area detector 16, e.g. being a part of an EMCCD camera, by means of the microscope objective 12, another dichroic beam splitter 14, which separates the fluorescence light from the illumination light, and a tube lens 15. The fluorescence light detected by means of the flat-panel area detector 16, which is recorded in terms of intensity levels, is processed into a partial image by means of the computer 10.

[0042] After the recording of the first partial image and after the recording of every further partial image, the acousto-optical filter 6 is switched for no activating light and no excitation light reaching the specimen 1. This state is maintained long enough until a sufficient number, i.e., more than two thirds or, better, more than 80% of the fluorophores have returned to the non-fluorescent ground state. In the meantime, the diffuser disk 8 is moved into another position, e.g., by a stepper motor, as sketched out here by double arrows. Subsequently, the specimen 1 is illuminated as described above. The activation and excitation patterns formed now differ from the patterns used before as well as from each other, because the patterns are generated with different wavelengths, even though the same region is illuminated. Altogether, this leads in the specimen 1 to a fluorescence that varies statistically in space and differs from the fluorescence recorded before. Now another partial image is recorded, and after the recording of a total number of N images, where N may amount, e.g., to between 50 and 300, a high-resolution image of the specimen 1, each time illuminated in a different statistical variation in space, is calculated by an analyzing algorithm as mentioned above as an example, and displayed on a screen of the computer 10.

[0043] Below, the functional principle of pattern generation is explained in yet greater detail with the help of FIG. 3. On account of the structure of the activation pattern of the laser 3 (shown in FIG. 3 by horizontal hatching), a speckle pattern, only sporadic fluorescence molecules are put into a state capable of fluorescein in the specimen. The excitation pattern generated by the light of the excitation laser 4 (shown in FIG. 3 by vertical hatching), a second illuminating speckle pattern, has a different structure that is also varying in a different spatially statistical way, for which reason only part of the fluorescent molecules capable of fluorescein can actually be excited to emit fluorescence. The spatial region in the specimen 1, from which, after projection onto the flat-panel area detector 16 by the microscope objective 12, fluorescence light can be detected, results from the overlap region (shown in FIG. 3 by horizontal and vertical hatching); in the example shown in FIG. 3 there is only one single fluorophore, which is marked by a white circle. It is only in that overlap region that the fluorophores are actually activated and simultaneously excited to emit fluorescence. The fluorescence region, i.e., the overlap region, is smaller, as a rule, than the size of the speckle patterns used for illumination. This leads to a smaller effective illumination structure in the specimen 1 than would be possible with a conventional diffraction-limited structured illumination.

LIST OF REFERENCE NUMBERS

[0044] 1 specimen [0045] 2 specimen slide [0046] 3 activation laser [0047] 4 excitation laser [0048] 5 dichroic beam splitter [0049] 6 acousto-optical filter [0050] 7 lens [0051] 8 diffuser disk [0052] 9 optical axis [0053] 10 computer [0054] 11 entrance pupil [0055] 12 microscope objective [0056] 13 optical system [0057] 14 dichroic beam splitter [0058] 15 tube lens [0059] 16 flat-panel area detector