DE102022112384B4 - METHOD, LIGHT MICROSCOPE AND COMPUTER PROGRAM FOR SETTING A TIME DELAY BETWEEN LIGHT PULSES - Google Patents

Abstract

Method for setting a time delay (td) between light pulses (P1, P2), wherein a sample (2) is illuminated with light from first light pulses (P1) and second light pulses (P2) by means of at least one light source (3, 4) of a light microscope (1). is, wherein a light signal (L) is detected by means of a detector (10, 10a, 10b) of the light microscope (1), which is generated by a combination of the first light pulses (P1) and the second light pulses (P2), characterized in that the light signal (L) is detected for different time delays (td) between the first light pulses (P1) and the second light pulses (P2), the time delay that results in a maximum light signal (L) being set as a reference value, and starting from the reference value, the time delay (td) is set to a desired value so that imaging parameters of a microscopic image of the sample (2) are optimized.

Description

Technical field of the invention

The invention relates to a method, a light microscope and a computer program for setting a time delay between light pulses.

State of the art

In light microscopy, especially in fluorescence microscopy, pulsed illumination of the sample is used for various purposes, e.g. B. for the analysis of the fluorescence lifetime, for the so-called pulse-interleaved microscopy, for confocal microscopy and for STED (stimulated emission depletion) or RESOLFT microscopy (reversible saturable optical linear fluorescence transitions).

In STED and RESOLFT microscopy, an excitation light focus is overlaid with a light distribution of fluorescence avoidance light or switching light with a local intensity minimum that depopulates the excited state of the fluorophores down to a narrow central region at the focus, resulting in an increase in resolution below the diffraction limit leads.

Light pulses can e.g. B. with internally or externally pulsed lasers or z. B. can be generated with CW lasers in combination with a pulse picker.

For certain applications the light pulses need to be synchronized with other events. For example, in so-called pulse-interleaved microscopy, the time delay between the pulses of different laser sources must be adjusted in order to generate a desired pulse sequence.

Such as in the patent application DE 10 2019 110 160 A1 described, the resolution of pulsed STED microscopy also depends heavily on the time delay between the excitation pulses and the STED pulses. These must hit the sample later than the excitation pulses to ensure that the excited state of the fluorophores is sufficiently populated to achieve effective depletion of the excited state. However, if the STED pulse occurs too late, a significant portion of the fluorophores spontaneously decay, including in areas around the central intensity minimum of the STED donut, leading to suboptimal spatial resolution of the resulting image.

The excitation of fluorophores with two photons of different wavelengths is also referred to as two-color two-photon excitation and is, for example, in the publications Lakowicz JR, Gryczynski I, Malak H, Gryczynski Z. Two-color two-photon excitation of fluorescence. Photochem Photobiol. 10/1996; 64(4):632-5 . doi: 10.1111/j.1751-1097.1996.tb03116.x and Quentmeier S, Denicke S, Ehlers JE, Niesner RA, Gericke KH. Two-color two-photon excitation using femtosecond laser pulses. J Phys Chem B. May 8, 2008; 112(18):5768-73 . doi: 10.1021/jp7113994. Quentmeier S, Denicke S, Gericke KH. Two-color two-photon fluorescence laser scanning microscopy. J Fluoresc. 11/2009; 19(6):1037-43 . doi: 10.1007/s10895-009-0503-x further describes an application of this technique to laser scanning microscopy.

The publication of C. Polzer, S. Ness, M. Mohseni, T. Kellerer, M. Hilleringmann, J. Rädler and T. Hellerer, “Correlative two-color two-photon (2C2P) excitation STED microscopy,” Biomed. Opt Express 10, 4516-4530 (2019), describes a combination of two-color two-photon excitation and two-photon STED microscopy by combining a femtosecond fiber laser with a diode laser that can optionally generate pulses with pulse lengths of 37 ps and 538 ps. In a first imaging mode, a first fluorophore is excited by a two-photon process triggered by illumination with a combination of two laser pulses of different colors. For this purpose, the diode laser is operated in short-pulse mode and the pulses of the two lasers are synchronized to the same time. In a second imaging mode, the same laser sources are used for two-photon STED microscopy, with the femtosecond fiber laser exciting a second fluorophore through a two-photon process and the diode laser generating time-delayed STED pulses in the long-pulse operating mode To reduce fluorescence of the second fluorophore in an area around the central minimum. The light from the diode laser is phase modulated in this imaging mode to form a donut-shaped STED light distribution at the focus. This setup enables the acquisition of super-resolution images of a structure along with confocal microscopy images of a counterstain of the sample that are inherently aligned with the super-resolution images. Furthermore, the publication mentioned describes a method in which a light signal is generated by the combined laser pulses by means of sum frequency generation in a substrate made of iron (II) iodate. Here, the diode laser is operated in short-pulse mode (as in two-color two-photon microscopy). The light signal is measured in a control experiment at different pulse delays in order to determine the pulse shape of the diode laser from the cross-correlation of the pulses and to verify that the two-photo nen excitation takes place effectively despite the different pulse widths.

The DE 10 2005 027 896 A1 describes a method for increasing the photon yield in a STED or two-photon microscopy method. A sample is illuminated with pulsed light and the repetition rate of the light pulses (or alternatively the scanning speed across the sample) is adjusted so that the distances between adjacent pulses are greater than the lifetime of a triplet state of fluorophores in the sample, around the triplet -to depopulate the state.

In the DE 10 2019 110 160 A1 discloses a STED microscope and a STED method for imaging a sample with pulsed illumination of the sample, in which a delay of the STED pulses relative to the excitation pulses is adjusted to a value optimized for STED efficiency by determining a fluorescence decay curve and the pulse delay is adjusted until the decay curve corresponds to a target curve.

From the DE 10 2019 110 157 A1 a fluorescence scanning microscope and a method for imaging a sample using a STED microscopy method are known, in which fluorescence lifetimes are evaluated in order to determine a spatial offset between the STED beam and the excitation beam and to spatially adjust the STED beam to the excitation beam.

In the DE 20 2015 001 565 U1 A confocal fluorescence microscope is described, which illuminates a sample with pulsed excitation light and pulsed manipulation light (e.g. STED light). The microscope has a pulse pattern generator and a detector that is synchronized with the pulse pattern generator and detects in a time-resolved manner. Among other things, an application of the microscope is described in which the sample is subjected to special pulse patterns, so that the effect of the excitation light alone, the manipulation light alone and the combination of the excitation light and the manipulation light in the sample can be evaluated separately by evaluating the fluorescence in different time windows can be.

The US 2002/0027202 A1 describes a method of multi-photon microscopy in which the pulse repetition rate of the excitation light, i.e. the pulse spacing of similar light pulses, is adjusted to the fluorescence lifetime of the fluorophore in order to optimize the photon yield.

The publication DE 694 24 271 T2 describes a method for the generation and detection of long-lived fluorescence and phosphorescence by two-photon excitation while optimizing the pulse repetition frequency to the properties of the fluorophore. Furthermore, a test arrangement with a europium-activated yttrium oxide sulfide crystal as a test substrate is described.

As already mentioned above, the pulse delay between light pulses, for example between the excitation pulse and the STED pulse in STED microscopy, can have a significant impact on the image quality achieved. However, the actual pulse delay depends on environmental parameters and can vary over the course of an experiment or between different experiments, even with constant settings. This has a negative impact on the reproducibility of the microscopy data obtained.

Objective problem

The present invention is therefore based on the object of providing a light microscopy method with pulsed illumination in which the pulse delay between different light pulses can be set in a reproducible manner.

Solution

This goal is achieved by the subject matter of independent claims 1 (method), 11 (light microscope) and 12 (computer program). Advantageous embodiments of the invention are specified in subclaims 2 to 10 and described below.

Description of the invention

A first aspect of the invention relates to a method for setting a time delay between light pulses, wherein a sample is illuminated with light from first light pulses and second light pulses by means of at least one light source of a light microscope, a light signal being detected by means of a detector of the light microscope, which is determined by a combination of the first light pulses and the second light pulses are generated. According to the invention, the light signal is detected one after the other for different time delays between the first light pulses and the second light pulses, with the time delay that results in a maximum light signal being set as a reference value. Starting from the reference value, the time delay is set to a desired value (which is not zero) so that imaging parameters of a microscopic image of the sample are optimized.

The first light pulses and the second light pulses can have any pulse length suitable for the microscopic experiment, in particular in the femtosecond, picosecond or nanosecond range. The light pulses can be generated in particular by a triggerable or non-triggerable pulse laser or by a CW laser using a pulse selector or pulse picker.

The first and second light pulses are in particular each generated by a first and second light source. Alternatively, it is also possible to generate the first and second light pulses with a single light source, for example by means of a white light laser, the light of which is split into two partial beams via a beam splitter. A respective spectral range can then be selected from the light of the partial beams, e.g. using optical filters or acousto-optically tunable filters (AOTF).

The first light pulses and the second light pulses in particular have different wavelengths. For example, the first and second light pulses can have wavelengths with which different light-emitting units (e.g. fluorophores) in the sample can be excited.

In the context of this application, the time delay between the light pulses is to be understood as the time difference between the times of the peak intensity of the light pulses.

The reference value in question is in particular a time delay of zero. In this case, the light pulses are first synchronized in time. Based on this, a desired time delay value other than zero can then be set.

By determining the reference value, the subsequent setting of the time delay becomes independent of environmental fluctuations and equipment influences. In this way, the reproducibility of microscopy results can be increased. In particular, the method according to the invention makes it easier to compare the results of experiments that are carried out at different times with the same light microscope and of experiments that are carried out with different light microscopes.

The term “microscopic image” is to be understood in the context of the present application in the sense that an image of structures of the sample is created using a light microscope. However, this does not necessarily require an optical image of the sample in the actual sense. Rather, the creation of a localization map of individual light-emitting units in the sample also falls under the term “microscopic imaging”. Such localization maps can be created, for example, using so-called PALM/STORM microscopy or MINFLUX microscopy. With these methods, positions of individual fluorophores in the sample are determined and a localization map is then calculated from a large number of position data.

If optical imaging actually occurs, this can be done by wide-field exposure of the sample with excitation light and detection of the emitted light (e.g. fluorescent light) with an area detector such as a camera. Alternatively, as e.g. B. is common in confocal microscopy and STED microscopy, the sample can be scanned with focused excitation light (e.g. with a galvoscanner) and the emitted light can be detected with a point detector, the point detector being arranged in particular confocally to the focal plane in the sample . A confocal pinhole can be arranged in front of the detector. The detected emission light can also pass through the scanner (so-called descanned configuration) or the detection beam path can be divided in front of the scanner as seen from the sample (so-called non-descanned configuration).

According to one embodiment, light-emitting units (in particular luminophores, more particularly fluorophores) in the sample are excited by means of the first light pulses, with a light distribution of emission prevention light with a local minimum being formed in the sample by means of the second light pulses in order to reduce the light emission of the light-emitting units in one To prevent the area around the local minimum and to image the sample microscopically with a resolution below a diffraction limit. The imaging parameters of the said microscopic image with the resolution below the diffraction limit are optimized by setting the time delay according to the invention.

In this specification, “resolution” means the minimum distance that two structures must have in order to be perceived as separate structures in a microscopic image. A “resolution below the diffraction limit” is, in particular, a resolution below the so-called Abbe limit. The Abbe limit can be expressed for the lateral resolution by the quotient of the wavelength of the detected light and twice the numerical aperture of the objective used.

The term “light-emitting units” used here refers to molecules, particles or molecular complexes that emit light in response to illuminating light (here in particular the light of the first light pulses). The emitted light can be generated, for example, by reflection or scattering of the illuminating light, or, if the illuminating light is excitation light that can excite the light-emitting units, the emitted light can be luminescent light, in particular fluorescent light or phosphorescent light. The light-emitting units can be individual molecules, complexes or aggregates of molecules or particles. The light-emitting entity may itself emit light or may contain one or more molecules that emit light in a complex, aggregate, or particle. In fluorescence microscopy, the light-emitting units can be, for example, fluorophores (small molecules or fluorescent macromolecules such as fluorescent proteins) or single molecules labeled with one or more fluorophores (e.g. proteins formed by a chemical bond between amino acid side chains and fluorophores). or multiple fluorophores bound, or proteins bound to an antibody or similar moiety, the antibody being labeled with a fluorophore). An example of a light-reflecting particle is a gold nanoparticle. Fluorescent particles can e.g. B. be so-called quantum dots.

The emission prevention light can in particular be STED light, which transfers the light-emitting units from the excited state through stimulated emission depletion into the ground state or into an oscillation state that is energetically arranged above the ground state, from which the light-emitting unit spontaneously falls back to the ground state. In this case, the microscopic imaging method is STED microscopy. Alternatively, the emission prevention light can also be a switching light, which converts the light-emitting units, in particular from the excited state, into a dark state, for example a triplet state. This effect is particularly exploited in so-called RESOLFT (reversible saturable optical linear fluorescence transitions) microscopy.

Using these methods, a resolution below the diffraction limit of light can be achieved (so-called superresolution microscopy) by narrowing the effective detection point spread function through the additional STED or switching light in the area around the local minimum of the light distribution.

The light distribution with the local minimum can be achieved in particular by phase modulation of the corresponding light beam. For example, the light beam can be modulated by a phase pattern generated using a phase plate or a spatial light modulator. A modulation with a vortex-shaped phase pattern in a plane conjugate to the objective pupil, for example, leads to a so-called donut light distribution in the focal plane. By means of an annular phase jump in the said plane conjugate to the pupil, a so-called bottle-beam distribution can be created at the focus, which is sometimes also referred to as a 3D donut.

According to a further embodiment, the light signal is generated by sum frequency generation, difference frequency generation or frequency mixing of the first light pulses and the second light pulses. For this purpose, a substrate with a suitable second-order electrical susceptibility can be provided in the sample or at another suitable location separate from the sample. This embodiment has the advantage that the intensity of the light signal depends heavily on the temporal coordination of the first and second light pulses. Therefore, the reference value of the time delay can be determined very precisely using the effects mentioned. In addition, there is the advantage that detected light of a unique wavelength can be generated, which can differ in particular from the illuminating light and the light emitted by the light-emitting unit. This light can therefore be separated from the remaining light, for example using appropriate filters or dichroic beam splitters.

According to a further embodiment, the light signal is generated by a two-photon excitation, in particular by a two-color, two-photon excitation (in the event that the wavelengths of the first and second light pulses differ from each other), in particular from light-emitting units in the sample or through the substrate or through a material of an active surface of a detector, generated by the first light pulses and the second light pulses. For example, emitters (e.g. fluorophores) that are suitable for two-photon microscopy at the wavelengths of the first and second light pulses can be introduced into the sample. The maximum of the emitted light is detected when the first light pulses and the second light pulses are precisely timed to one another. The light emitting units may be the same units used for microscopic imaging of the sample or, as described further below, different types of light emitting units may be used for the light signal and imaging. The advantage of using the two-photon effect is that: Light intensity of the light signal depends heavily on the temporal synchronization of the light pulses and the reference value can therefore be determined very precisely.

According to a further embodiment, first light-emitting units in the sample are excited by the first light pulses, wherein the second light pulses prevent the light emission of the first light-emitting units in the area around the local minimum, and wherein the light signal is generated by the two-photon excitation of second light-emitting units in the sample, which are different from the first light-emitting units, is generated by the first light pulses and the second light pulses.

According to a further embodiment, first light-emitting units in a first sample (e.g. a measurement sample) are excited by the first light pulses, the second light pulses preventing the light emission of the first light-emitting units in the area around the local minimum, and the light signal passing through the two-photon excitation of second light-emitting units in a second sample (e.g. a calibration sample), which are different from the first light-emitting units, is generated by the first light pulses and the second light pulses.

According to a further embodiment, the light signal is generated by illuminating a substrate with the light of the first light pulses and the second light pulses. This substrate can, for example, comprise a material with a suitable second-order electrical susceptibility, so that the light signal can be generated, for example, by sum frequency generation, difference frequency generation or frequency mixing. The substrate may be part of the sample, associated with the sample, or contained in the sample. Alternatively, the substrate can also be separated from the sample and, in particular, arranged at a different location in the beam path of the light microscope.

According to a further embodiment, the light signal is detected by means of a first detector, light emanating from light-emitting units in the sample excited by the first light pulses being detected by means of a second detector, the second detector being different from the first detector. When using two separate detectors, the detectors can be selected in an ideal manner for the respective measurement requirements. For example, the detectors can be designed for the detection of light in different wavelength ranges. Alternatively, the light signal and the light emitted by the light-emitting units excited by the first light pulses can also be detected with the same detector.

According to a further embodiment, the light signal arises on an active surface of the first detector, in particular wherein the active surface has a semiconductor material that can be excited by means of two-photon excitation from a combination of the first light pulses and the second light pulses. The semiconductor material can be, for example, gallium arsenide phosphide or a photophysically similar material. This embodiment has the advantage that no separate substrate is necessary to generate the light signal. This leads to a particularly simple and user-friendly implementation of the method. In particular, a separate second detector is provided for detecting the light emitted by the light-emitting units in the sample.

According to a further embodiment, the reference value is determined based on a cross-correlation between the first light pulses and the second light pulses. In particular, a cross-correlation function between the first light pulses and the second light pulses is calculated. In particular, the reference value is the time delay between the first light pulses and the second light pulses for which a maximum of the cross-correlation function results.

According to a further embodiment, the imaging parameters optimized by setting the time delay include a resolution and/or a signal-to-background ratio. In other words, by appropriately selecting the time delay, the resolution and/or the signal-to-background ratio of the microscopic image of the sample is optimized. Particularly in STED microscopy, the delay between the excitation pulse and the STED pulse affects the resolution. This is particularly the case because, depending on when the STED pulse reaches the sample, different proportions of the fluorophores are in the excited state and in the ground state. In particular, this can also have an influence on the signal-to-background ratio, especially since the extent of undesirable direct excitation of the fluorophores by the STED light depends on the number of fluorophores in the ground state.

A second aspect of the invention relates to a light microscope comprising a device for setting a time delay between light pulses, in particular according to the method according to the first aspect. The device comprises at least one light source which is designed to provide a sample with light from first light pulses and second light to illuminate pulses. The device further has at least one detector which is designed to detect a light signal which can be generated by a combination of the first light pulses and the second light pulses. In addition, the device comprises a control device which is designed to set different time delays between the first light pulses and the second light pulses, wherein the at least one detector of the light microscope is designed to detect the light signal for the different time delays. Finally, the device has a data processing device which is designed to determine the time delay that results in a maximum light signal and to set it as a reference value, the control device being designed to set the time delay to a desired value based on the reference value so that Imaging parameters of a microscopic image of the sample can be optimized.

In particular, light-emitting units in the sample can be excited by means of the first light pulses, with a light distribution of emission prevention light with a local minimum being able to be generated in the sample by means of the second light pulses in order to prevent the light emission of the light-emitting units in an area around the local minimum and to image the sample microscopically with a resolution below a diffraction limit.

According to a further embodiment, the device comprises a first light source which is designed to generate the first light pulses and/or the device has a second light source which is designed to generate the second light pulses. The device furthermore has, in particular, at least one beam combiner (e.g. one or more dichroic mirrors), which is designed to combine the light from the first light pulses and the second light pulses in a common beam path.

According to a further embodiment, the device has a light modulator which is designed to generate the light distribution of the light of the second light pulses with the local minimum at the focus in the sample.

According to a further embodiment, the device has a control device which is connected to the first light source and the second light source, the control device being designed to adjust the time delay between the first light pulses and the second light pulses.

According to one embodiment, the light microscope has a first detector which is designed to detect the light signal which can be generated by a combination of the first light pulses and the second light pulses. According to a further embodiment, the light microscope has a second detector that is different from the first detector and is designed to detect light emitted by light-emitting units in the sample that were excited by the first light pulses.

A third aspect of the invention relates to a computer program comprising program code designed to cause the light microscope according to the second aspect to carry out the method according to the first aspect.

All features of the method according to the first aspect also apply analogously to the light microscope according to the second aspect and the computer program according to the third aspect.

Advantageous developments of the invention result from the patent claims, the description and the drawings and the associated explanations for the drawings. The described advantages of features and/or combinations of features of the invention are merely examples and can have an alternative or cumulative effect.

With regard to the disclosure content (but not the scope of protection) of the original application documents and the patent, the following applies: Further features can be found in the drawings - in particular the relative arrangements and active connections shown. The combination of features of different embodiments of the invention or of features of different patent claims is also possible, deviating from the selected relationships of the patent claims, and is hereby encouraged. This also applies to features that are shown in separate drawings or are mentioned in their description. These features can also be combined with features of different patent claims. Likewise, features listed in the patent claims may be omitted for further embodiments of the invention, but this does not apply to the independent patent claims of the granted patent.

The reference symbols contained in the patent claims do not represent a limitation on the scope of the subject matter protected by the patent claims. They merely serve the purpose of making the patent claims easier to understand.

Brief description of the drawings

• 1 shows a first embodiment of the light microscope according to the invention;
• 2 shows schematically the setting of a time delay between first light pulses and second light pulses by maximizing a light signal obtained by two-photon excitation according to an embodiment of the method according to the invention;
• 3 shows a second embodiment of the light microscope according to the invention;
• 4 shows a third embodiment of the light microscope according to the invention.

Description of the drawings

1 shows a light microscope 1 for the microscopic analysis of a sample 2, for example by imaging structures in the sample 2, in particular structures that are marked with light-emitting units (e.g. fluorophores), or by localizing individual light-emitting units in the sample 2. The in 1 The light microscope 1 shown is in particular a STED or RESOLFT microscope and has a device for setting a time delay between first light pulses P1 and second light pulses P2.

The light microscope 1 comprises a first light source 3, which is designed to generate first light pulses P1 and a second light source 4, which is designed to generate second light pulses P2 to illuminate the sample 2. The first light source 3 and the second light source 4 can each be pulsed lasers. The first light pulses P1 and the second light pulses P2 are combined in a main beam path of the light microscope 1 via a first dichroic mirror 5a. The combined first and second light pulses P1+P2 are transmitted by a second dichroic mirror 5b, pass through a scanner 6 with one or more (typically at least two) scanning mirrors 61 and a scanning lens 62 as well as a tube lens 7 and are passed through an objective 8 focused into sample 2.

Light-emitting units in the sample 2 are excited by the light from the first light pulses P1 and then emit light (e.g. fluorescent light). The second light pulses P2 include prevention light that depletes the excited state of the light-emitting units in the sample 2 (e.g. by stimulated emission depletion, STED), or switching light that depletes the light-emitting units into a non-emitting dark state (e.g. a triplet condition) (as known, for example, from RESOLFT microscopy).

A light modulator 11 is arranged between the second light source 4 and the first dichroic mirror 5a, which forms the light of the second light pulses P2 in the focal plane in the sample 2 into a hollow, in particular donut- or bottle-beam-shaped light distribution with a local intensity minimum . As a result, the light emission of the light-emitting units only occurs in a small area around the geometric focus, which leads to a resolution below the diffraction limit. The light modulator 11 can z. B. a programmable spatial light modulator with pixels (a so-called spatial light modulator, e.g. operated in diffraction or reflection mode or in transmission mode) or a phase plate which has a delay pattern for phase modulation of the light beam formed by the second light pulses P2. So can e.g. For example, a so-called vortex phase pattern can be displayed to form a donut-shaped focus, and a ring-shaped phase pattern with a phase jump by the value π can be displayed to form a bottle-beam-shaped focus.

The light emitted by the light-emitting units in the sample 2 travels back from the sample 2 through the objective 8 and the tube lens 7, is descanned by the scanner 6 and, based on its wavelength, is reflected by the second dichroic mirror 5b.

The reflected emission light passes through a confocal pinhole 9 and is recorded by a detector 10, in particular a point detector, such as an avalanche photodiode, a photomultiplier or a hybrid detector.

The first light source 3 and the second light source 4 are coupled to a control device 13, which is designed to adjust the time delay t _d between the first light pulses P1 and the second light pulses P2.

innerhalb des Anregungsspektrums des Fluorophors liegt, wobei λ₁ die Wellenlänge des Lichts der ersten Lichtpulse P1 ist, und wobei λ₂ die Wellenlänge des Lichts der zweiten Lichtpulse P2 ist. Hierdurch wird gemäß der in 1 gezeigten Ausführungsform das Lichtsignal L erzeugt. Dies erfolgt insbesondere in einem Kalibrierungsschritt, um die Zeitverzögerung t_d zwischen den ersten Lichtpulsen P1 und den zweiten Lichtpulsen P2 auf den Wert Null einzustellen und damit einen Referenzwert zu erhalten.If the time delay is set to the value zero, suitable light-emitting units, namely certain fluorophores, in the sample 2 can be excited by a two-color two-photon excitation by the combination of the first light pulses P1 and the second light pulses P2 and then fluorescent light emit. This requires that the effective excitation wavelength ${\lambda }_e=\frac{{\mathrm{<figure-callout\; id="1"\; label="\lambda "\; filenames="DE102022112384B4\_0002.png,DE102022112384B4\_0003.png"\; state="\{\{state\}\}">\lambda </figure-callout>}}_1\cdot {\mathrm{<figure-callout\; id="2"\; label="\lambda "\; filenames="DE102022112384B4\_0002.png,DE102022112384B4\_0003.png"\; state="\{\{state\}\}">\lambda </figure-callout>}}_2}{{\mathrm{<figure-callout\; id="1"\; label="\lambda "\; filenames="DE102022112384B4\_0002.png,DE102022112384B4\_0003.png"\; state="\{\{state\}\}">\lambda </figure-callout>}}_1+{\mathrm{<figure-callout\; id="2"\; label="\lambda "\; filenames="DE102022112384B4\_0002.png,DE102022112384B4\_0003.png"\; state="\{\{state\}\}">\lambda </figure-callout>}}_2}$

lies within the excitation spectrum of the fluorophore, where λ ₁ is the wavelength of the light of the first light pulses P1, and where λ ₂ is the wavelength of the light of the second light pulses P2. This means that according to in 1 Embodiment shown generates the light signal L. This is done in particular in a calibration step to determine the time delay t _d between the first light pulses P1 and the second light pulses P2 to the value zero and thus obtain a reference value.

The light signal L is according to the in 1 shown embodiment detected by the detector 10. A data processing device 12, which evaluates the detected light signal L, is coupled to the detector 10. According to one embodiment, the data processing device 12 sends control signals to the control device 13 in the calibration step in order to adjust the time delay t _d until the detected light signal L reaches a maximum. Then the set time delay t _d corresponds to the reference value. The time delay t _d can then be set to a desired value greater than zero, starting from the reference value, for example in order to optimize the resolution when imaging sample 2 using STED microscopy.

Finally, the sample 2 is microscopically imaged in a measuring step by scanning via the scanner 6 with the combined excitation light and STED light of the first light pulses P1 and second light pulses P2 with a resolution below the diffraction limit. In this case, light-emitting units in the sample 2, in particular light-emitting units other than those that were excited in the calibration step by means of two-color two-photon excitation, are excited by means of the light of the first light pulses P1 and in an area around the central local minimum the light distribution caused by the second light pulses P2 at the focus is de-excited by stimulated emission. The emitted fluorescent light from the area of the local minimum reaches the detector 10 via the objective 8, the tube lens 7, the scanner 6, the dichroic mirror 5b and the confocal pinhole 9. An image of the sample 2 can then be calculated using the data processing device 12.

The detector 10 is therefore according to the in 1 Embodiment shown is used both for the detection of the light signal L during the calibration step and for the microscopic imaging of the sample 2 during the measurement step.

The setting of the time delay t _d between the first light pulses P1 and the second light pulses P2 according to an embodiment of the method according to the invention is in 2 shown. The time delay t _d between the first light pulses P1 and the second light pulses P2 is set by the control device 13 and the light signal L generated by the two-color two-photon process described above is measured by the detector 10. In the graphic above 2 In the situation shown, a weak light signal L is detected due to the lack of time overlap between the first light pulses P1 and the second light pulses P2. However, if the time delay t _d is adjusted (middle diagram in 2 ), the light signal L increases and reaches a maximum at a time delay t _d equal to zero, at which the first light pulses P1 and the second light pulses P2 occur essentially at the same time. The bottom diagram in 2 shows an example of an intensity curve of the light signal L depending on the time delay t _d .

After the time delay t _d has been set to zero, which corresponds to the reference value, the time delay t _d can be adjusted from the reference value to a desired value greater than zero, in particular in order to optimize emission prevention efficiency and thus the spatial resolution of STED microscopy. Advantageously, through the inventive prior calibration of the first light pulses P1 and the second light pulses P2, the optimized time delay t _d can be set very precisely and reproducibly from experiment to experiment, which leads to accurate and reproducible imaging results.

In 3 and 4 Two further exemplary embodiments of a light microscope 1, in particular a STED or RESOLFT microscope, are shown with a device according to the invention for setting a time delay t _d between light pulses P1, P2. Some components of the light microscope 1 are identical to those of the in 1 shown light microscope 1. These are in 3 and 4 with to 1 marked with identical reference numbers. Regarding the arrangement and function of these components, see the description 1 referred.

In contrast to the embodiment according to 1 have the embodiments according to 3 and 4 two separate detectors 10a, 10b, namely a first detector 10a for detecting the light signal L for determining the reference value of the time delay t _d between the first light pulses P1 and the second light pulses P2 and a second detector 10b for detecting light emitted by the light emitting Units in sample 2 are emitted after excitation by the light of the first light pulses P1.

At the in 3 In the embodiment shown, the excitation light of the first light pulses P1 generated by the first light source 3 is reflected into a main beam path via a dichroic mirror 5a and the STED light or switching light generated by the second light source 4 is phase-modulated by a light modulator 11 and then by means of a second dichroic mirror 5b also coupled into the main beam path. The combined light of the first and second light pulses P1+P2 then reaches a beam splitter 14, which directs a certain portion of the light from both light pulses P1, P2 to the first detector 10a. Here, the combined light of the first and second light pulses P1+P2 generates the light signal L, which is used to adjust the time delay t _d between the first light pulses P1 and the second light pulses P2, directly on an active surface of the first detector 10a, which is, for example May contain semiconductor material such as gallium arsenide phosphide. The light signal L can arise, for example, through two-photon excitation, in particular two-color two-photon excitation, of the semiconductor material.

Another portion of the combined light from the first and second light pulses P1+P2 is transmitted by the beam splitter 14 and reaches the objective 8 via the scanner 6 and the tube lens 7, which focuses the light into the sample 2. There, light-emitting units, e.g. fluorophores, are excited by the light from the first light pulses P1. The light of the second light pulses P2, phase-modulated by the light modulator 11, forms a light distribution with a local minimum, for example a donut-shaped light distribution, at the focus in the sample 2. The light-emitting units are de-excited by the light of the second light pulses P2 in an area around the local minimum, so that only the light-emitting units positioned close to the local minimum emit light, e.g. fluorescent light. The emitted light passes through the objective 8, the tube lens 7 and the scanner 6 back into the main beam path, is transmitted by the second dichroic mirror 5b and the first dichroic mirror 5a due to its longer wavelength compared to the excitation light and the prevention light and is reached by the confocal pinhole 9 through the second detector 10b. A scan image of the sample 2 with a resolution below the diffraction limit is created from the light intensities measured for a large number of scan positions with the second detector 10b.

As already above for the in 1 As described in the embodiment shown, in particular, in a calibration step with the control device 13, which is connected to the first light source 3 and the second light source 4, the time delay t _d between the first light pulses P1 and the second light pulses P2 is adjusted and the light intensity of the light signal L is measured by the first detector 10a. Based on the measured light intensity, the data processing device 12 calculates the time delay t _d for which a maximum light intensity is measured and sets this as a reference value. In particular, this reference value is zero. Using the reference value, the control device 13 adjusts the time delay t _d to a value greater than zero in order to optimize the resolution of the microscopic image of the sample 2 using STED or RESOLFT microscopy.

A scan image of the sample 2 is then recorded with the optimized time delay t _d as described above using the second detector 10b.

In the 4 The illustrated embodiment of the light microscope 1 is largely identical to the light microscope 1 according to the beam path 3 . However, the beam splitter 14 is missing and the first detector 10a is instead arranged on the side of the sample 2 facing away from the objective 8. In this case, the sample 2 has a substrate 15 on the side facing away from the lens 8, which can be present, for example, as a coating on a sample carrier such as a microscope slide. When the substrate 15 is irradiated with the light of the combined first and second light pulses P1+P2 irradiating the sample 2, the substrate 15 generates a light signal L, which is detected by the first detector 10a. In the substrate 15, the light signal L can arise, for example, through sum frequency generation, difference frequency generation or frequency mixing. The substrate 15 can, for. B. contain iron (II) iodate or similar substances and can be made available in the form of a crystalline powder. Since the light emanating from the substrate 15 in the in 4 configuration shown does not pass through the scanner 6, it may be advantageous to coat the slide with the substrate 15 over a larger area so that the light signal L is generated independently of the scanning position.

In this case, the calibration step can possibly even be carried out simultaneously with the measuring step or in parallel to the measuring step. The light signal L and the light generated by the light-emitting units in the sample 2 can therefore be detected in parallel with the first detector 10a and the second detector 10b. This can be used, for example, for an ongoing determination of the reference value and thus an ongoing calibration of the time delay between the first light pulses P1 and the second light pulses P2.

To ensure that only the desired light signal L reaches the first detector 10a, suitable filters can be placed between the substrate 15 and the first detector 10a (not shown). Possible interference signals to avoid include SHG (second harmonic generation) signals. It may also be necessary to enter the beam path of the light microscope 1 at a suitable point between the objective 8 and the two th detector 10b to provide filters to ensure that only the emission light generated by the light-emitting units in the sample 2, but not the light signal L generated by the substrate 15, is detected by the second detector 10b.

Alternatively, the coating of the slide with the substrate 15 can also be carried out only at certain points, in particular in an area of the slide that is not covered by structures of interest in the sample 2. In this way, the substrate 15 can be specifically controlled with the scanner 6 in the calibration step, with the light signal L, which may interfere with the imaging, not being present during the measuring step.

List of reference symbols

1

light microscope

2

sample

3

First light source

4

Second light source

5a

First dichroic mirror

5b

Second dichroic mirror

6

scanner

7

tube lens

8th

lens

9

Pinhole

10

detector

10a

First detector

10b

Time detector

11

Light modulator

12

Data processing device

13

Control device

14

Beam splitter

15

Substrate

61

Scan mirror

62

Scanning lens

P1

First pulse of light

P2

Second light pulse

t

Time

td

Time Delay

I

intensity

Imax

Maximum intensity

L

Light signal

Claims (12)

Method for setting a time delay (t _d ) between light pulses (P1, P2), wherein a sample (2) is exposed to light from first light pulses (P1) and second light pulses (P2) by means of at least one light source (3, 4) of a light microscope (1). is illuminated, a light signal (L) being detected by means of a detector (10, 10a, 10b) of the light microscope (1), which is generated by a combination of the first light pulses (P1) and the second light pulses (P2), characterized in that that the light signal (L) is detected for different time delays (t _d ) between the first light pulses (P1) and the second light pulses (P2), the time delay that results in a maximum light signal (L) being set as a reference value, and where Starting from the reference value, the time delay (t _d ) is set to a desired value so that imaging parameters of a microscopic image of the sample (2) are optimized. Procedure according to Claim 1 , characterized in that light-emitting units in the sample (2) are excited by means of the first light pulses (P1), and a light distribution of emission prevention light with a local minimum is formed in the sample (2) by means of the second light pulses (P2). to prevent the light emission of the light-emitting units in an area around the local minimum and to image the sample (2) microscopically with a resolution below a diffraction limit. Procedure according to Claim 1 or 2 , characterized in that the light signal (L) is generated by sum frequency generation, difference frequency generation or frequency mixing of the first light pulses (P1) and the second light pulses (P2). Procedure according to Claim 1 or 2 , characterized in that the light signal (L) is generated by a two-photon excitation, in particular a two-color two-photon excitation, in particular of light-emitting units in the sample (2), by the first light pulses (P1) and the second light pulse (P2) is generated. Procedure according to Claim 4 , characterized in that first light-emitting units in the sample (2) are excited by the first light pulses (P1), the second light pulses (P2) preventing the light emission of the first light-emitting units in the area around the local minimum, and wherein the light signal (L) is generated by the two-photon excitation of second light-emitting units in the sample (2) from the first light-emitting units Units are different, generated by the first light pulses (P1) and the second light pulses (P2). Method according to one of the preceding claims, characterized in that the light signal (L) is generated by illuminating a substrate (15) separate from the sample (2) with the light of the first light pulses (P1) and the second light pulses (P2). Method according to one of the preceding claims, characterized in that the light signal (L) is detected by means of a first detector (10a), light emanating from light-emitting units in the sample (2) excited by the first light pulses (P1) being detected by means of a second detector (10b) is detected, the second detector (10b) being different from the first detector (10a). Procedure according to Claim 7 , characterized in that the light signal (L) arises on an active surface of the first detector (10a). Method according to one of the preceding claims, characterized in that the reference value is determined on the basis of a cross-correlation between the first light pulses (P1) and the second light pulses (P2). Method according to one of the preceding claims, characterized in that the imaging parameters include a resolution and/or a signal-to-background ratio. Light microscope (1) comprising a device for setting a time delay between light pulses (P1, P2), in particular according to the method according to one of Claims 1 until 10 , comprising - at least one light source (3, 4), which is designed to illuminate a sample (2) with light from first light pulses (P1) and second light pulses (P2), - a detector (10, 10a, 10b), which is designed to detect a light signal (L), which can be generated by a combination of the first light pulses (P1) and the second light pulses (P2), and - a control device (13) which is designed to set different time delays (t _d ) between the first light pulses (P1) and the second light pulses (P2), the detector (10) being designed to detect the light signal (L) for the different time delays (t _d ), - a data processing device (12), which is designed to set the time delay that results in a maximum light signal (L) as a reference value, the control device (13) being designed to set the time delay (t _d ) to a desired value based on the reference value so that imaging parameters a microscopic image of the sample (2) can be optimized. Computer program comprising program code which is designed to use the light microscope (1) according to Claim 11 to cause the procedure to be carried out in accordance with one of the Claims 1 until 10 to carry out.

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