Method and microscope with correction device for correcting aberration-induced imaging errors

Abstract

To correct aberration-induced imaging errors in an optical system having an objective lens (14) and adaptive optics (18), the light (5) and sample (20) are selected such that, when acting on the sample (20), the light (5) reduces a measurement signal (28) from the sample (20), the relative change in the measurement signal (28) being dependent on the intensity of the light (5). The measurement signal (28) is detected from a focal region of the optical system within the sample (20) over first and subsequent second time periods (38, 37) to determine first and second measurement values. The light (5) is focused by the optical system onto the focal region over a third time period (39) overlapping the first and / or second time periods. From the first and second measurements, a degree index for the relative change in the measurement signal (28) is determined and used as a metric to be optimized when controlling the adaptive optics (18).

Description

Technical Field

[0001] The present invention relates to a method for correcting aberration-induced imaging errors in an optical system having an objective lens and an adaptive optical system in the optical path passing through the objective lens. Further, the present invention relates to a laser scanning microscope having a first light source for excitation light, a second light beam for stimulation light, an objective lens, and a correction device for correcting aberration-induced imaging errors, the correction device including an adaptive optical system in the optical path passing through the objective lens.

Background Art

[0002] Aberration-induced imaging errors may, on the one hand, be caused by the optical system itself if, for example, the optical system is not sufficiently achromatic. On the other hand, regions of a sample that are in front of the focal region of the optical system may also cause fundamental aberrations.

[0003] The aberration-induced imaging errors to be corrected in the optical system affect both the projection of the light intensity distribution onto the respective samples by the objective lens and the imaging of the samples by the objective lens.

[0004] From Non-Patent Literature 1, the correction of aberration-induced imaging errors is known, and these imaging errors are due to the non-uniformity of the optical properties of the sample, especially thick samples. Here, a method called COAT (Coherent Optical Adaptive Technique) is applied, in which the phase distortion due to each sample is measured and compensated for. A Spatial Light Modulator (SLM) is used for both the measurement and compensation of phase distortion, and the SLM further modulates the phase wavefront of the stimulating light so that the stimulating light focused on the sample has a donut-shaped intensity distribution. The phase distortion due to the sample is measured by imprinting a phase pattern on the stimulating light ray by the SLM, and in this phase pattern, the phase shift is modulated by different frequencies in different regions. Here, the intensity of the spot where the stimulating light ray is subsequently focused is recorded in time resolution and analyzed by Fourier transform. In practice, a phase shift that compensates for aberration-induced imaging errors is obtained in each region of the phase pattern imprinted by the SLM. Specifically, the spot intensity is detected by scanning gold nanoparticles with the spot and recording the stimulating light scattered from the gold nanoparticles. Correspondingly, each STED microscope must be driven in scattered light mode, and gold nanoparticles must be positioned where aberration-induced imaging errors of each sample need to be compensated for. In practice, gold nanoparticles were placed only on the upper and lower sides of the sample to demonstrate the functionality of this method.

[0005] From Patent Document 1, a method for correcting aberrations in a STED microscope using an adaptive optical system is known, in which a metric combining image sharpness and image brightness is used. This metric is maximized or minimized by controlling an optical modulator, which is an SLM or a deformable mirror. Image sharpness can only be determined after the sample has been imaged. Image brightness depends on the concentration and density of the fluorescent markers that mark the target structure in the sample. Therefore, the respective maximums and minimums of the combined metric of image brightness and image sharpness have only local expressive power and cannot be compared to other maximums or minimums in other areas of the sample.

[0006] Non-patent document 2 describes a known method for aligning a donut-shaped stimulation light focus with a spot-shaped excitation light focus, in which the discrepancy between the STED image and the lifetime image of the fluorescent nanobeads is determined and eliminated. Specifically, to achieve this, the discrepancy between the centroid of the normalized spatial intensity distribution of the fluorescence light from the nanobeads and the centroid of the normalized spatial lifetime intensity distribution of the fluorescence light from the nanobeads is determined and adjusted.

[0007] From Patent Document 2, it is known that a precise adjustment of a STED microscope is performed so that the maximum intensity of the excitation light and the maximum intensity of the STED light coincide at the focal point of the objective lens. Structures in a sample marked with a fluorescent dye are scanned with the maximum intensity of the excitation light, and an image of the sample is formed by imaging the structures with fluorescence stopping light of different intensities. In this case, the shift between the imaging positions of the structures in the formed image is calculated and adjusted. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2014 / 029978(A1) [Patent Document 2] International Publication No. 2018 / 042056(A1) [Non-patent literature]

[0009] [Non-Patent Document 1] Wei Yan et al.: Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples, Photonics Research, Vol. 5, No. 3, June 2017 [Non-Patent Document 2] Yifan Wang et al.: A 3D Aligning Method for Stimulated Emission Depletion Microscopy Using Fluorescence Lifetime Distribution, Microscopy Research and Technique 77:935-940, August 2014 [Overview of the project] [Problems that the invention aims to solve]

[0010] The fundamental problem of the present invention is to provide a method for correcting aberration-induced imaging errors in an optical system, and a laser scanning microscope having an objective lens and a correction device for correcting aberration-induced imaging errors, which can correct imaging errors for different regions of a sample during the continuous use of an imaging system or laser scanning microscope for measuring or imaging each sample. [Means for solving the problem]

[0011] This problem is solved by a method having the features of claims 1, 3, and 7, and a laser scanning microscope having the features of claim 21, where the method having the features of claim 7 is equivalent in frequency space to the method having the features of claim 1. Dependent claims 2, 4 to 6, and 8 to 20 relate to preferred embodiments of the method of the present invention, and claim 22 is directed to a preferred embodiment of the laser scanning microscope of the present invention.

[0012] This invention utilizes a metric, a measure of the quality of focus when illuminating a sample with light. This metric can be used to correct aberrations that may occur in a microscope. This is achieved by adapting the light distribution for illuminating the sample using an adaptive optical system so that this metric is optimized. To this end, the sample is first illuminated with light according to a given setting of the adaptive optical system, thereby influencing the measurement light from the sample, for example, by guiding the light to emit fluorescence. The measurement signal is then temporally resolved and detected, and divided into at least one preceding and succeeding period for subsequent calculations. From the ratio of measured values ​​of the measurement signal for the two periods, for example, the ratio of preceding and succeeding photons of fluorescence, a degree index (Masszahl), a measure of the quality of focus, is calculated. This degree index is optimized by iterative fitting of the adaptive optical system, such as an adaptive mirror, thereby correcting aberrations. An equivalent degree index can be determined in frequency space.

[0013] The present invention provides a method for correcting aberration-induced imaging errors in an optical system having an objective lens and an adaptive optical system arranged in the optical path through the objective lens, wherein the light and sample are selected such that the light acting on the sample reduces the measurement signal from the sample or approximates the measurement signal to a saturation value from below, where the relative change in the measurement signal depends on the intensity of the light. The measurement signal originating from the focal region of the optical system within the sample is detected over a first period to determine a first measurement and over a second period to determine a second measurement. Over a third period, the selected light is focused by the optical system to the focal region of the sample, where the first period at least partially precedes the second period and / or the second period at least partially follows the first period, and the third period at least partially overlaps the first period and / or the second period and / or an intermediate period between them. From the first and second measurements, a highly monotonically rising or falling function of the relative change in the measurement signal is determined and used as a metric to be optimized by changing the control when controlling the adaptive optical system.

[0014] When carrying out the method of this invention, the aberration-induced imaging error to be corrected affects the focusing of selected light to the focal region of the sample by the optical system. If the imaging error that occurs during this focusing is corrected, this imaging error will also be corrected when the focal region is imaged by the imaging system.

[0015] The aberration-induced imaging error to be corrected is reduced when the selected light is focused to the focal region of the sample by the optical system during the third period. This reduction in the intensity of the selected light from its maximum value occurs without the aberration-induced imaging error to be corrected, i.e., without the so-called Strehr ratio. Correspondingly, the relative change in the measurement signal of the sample caused by the selected light is also reduced.

[0016] From the above relative definitions of the first, second, and third periods, the first measurement will be less affected than the second measurement by the selected light focused in the focal region during the third period.

[0017] Due to the above selection of light and sample, and the above relative definitions of the first, second, and third periods, the first and second measurements similarly depend on the local characteristics of the sample in their respective focal regions. Therefore, for example, if the degree index is determined by calculating the quotient between the second measurement, determined as the integral of the measurement signal over the second period, and the first measurement, determined as the integral of the measurement signal over the first period, it is normalized to the initial value of the measurement signal. That is, it becomes independent of the absolute height, i.e., the level of the measurement signal. However, this degree index still depends on the intensity of the selected light in the focal region.

[0018] Therefore, the degree index is comparable beyond the fluctuating levels of the measurement signal. This means that even if the level of the measurement signal fluctuates across adjacent regions of the sample, the degree index can be optimized across adjacent regions of the sample. This allows for continuous optimization of the degree index, particularly when scanning the sample through the focal region. Optimization of the degree index is not only possible when the position of the focal region within the sample remains the same even when the control of the adaptive optical system is changed. However, this does not mean that the metric to be optimized according to the present invention, i.e., the degree index, has the same value for all positions of the focal region within the sample. Rather, this is certainly not the case, because the sample itself is the source of aberrations that vary at least axially along the optical axis of the objective lens, i.e., in the depth direction of the sample. However, the optimized degree index fluctuates slightly between positions of laterally adjacent focal regions within the sample. Therefore, optimization for directly adjacent focal regions within the sample can be initiated by the control of the adaptive optical system, which has achieved optimization of the metric of the present invention at a preceding position of the focal region.

[0019] The dependence of the intensity of the selected light within the focal region on the determination of the degree exponent is ensured by the fact that, when the selected light reduces the measured signal, and when the selected light approximates the measured signal to a saturation value from below, both the first and second measurements do not directly depend on the intensity of the selected light, either linearly or by another non-zero power of the same power.

[0020] From this, the degree index will increase or decrease together with the intensity of the selected light in the focal region in the method of the present invention. Correspondingly, the degree index is maximized or minimized as a metric optimized by the control of the adaptive optical system in order to maximize the intensity of the light in the focal region. This maximum intensity is exactly reached when the aberration-induced imaging error is removed. Therefore, the degree index is suitable as a metric to be optimized in the method of the present invention in order to achieve the goal of correcting the aberration-induced imaging error by changing the control of the adaptive optical system.

[0021] Calculating the quotient of the second measurement value determined as the integral of the measurement signal over the second period and the first measurement value determined as the integral of the measurement signal over the first period, which has already been described as an example, is to normalize to the initial value of the measurement signal, and the calculation of this quotient is suitable for determining the degree index, for example, when the selected light reduces the measurement signal from the sample. However, the reciprocal of this quotient is also suitable as the degree index when the selected light acts on the measurement signal. However, in this case, this degree index is maximized rather than minimized.

[0022] When the selected light approximates the measurement signal from the sample to the saturation value from below, it is advantageous to normalize the absolute value of the change in the measurement signal to the saturation value in order to determine the degree index. This normalization can be approximately performed such that the determination of the degree index is performed by forming the quotient of the first measurement value and the second measurement value.

[0023] In the method of the present invention, the degree index determined from the first measurement value and the second measurement value, which is used as a metric to be optimized by changing the control of the adaptive optical system, does not necessarily have to be the quotient of these two measurement values, especially a direct quotient. Therefore, there is basically no problem even if the denominator or the numerator of the quotient has another factor multiplied by the measurement value or an offset added to the measurement value. Further, the denominator of the quotient may have a difference of measurement values and / or the numerator may have a sum, or vice versa. Any degree index that is a monotonically increasing or decreasing function of the relative change of the measurement signal is generally suitable. It is obvious that this function only needs to have this characteristic in the relevant region of the generated measurement value.

[0024] Any such degree index reaches an extreme value like the direct quotient of the second measurement value determined as the integral of the measurement signal over the second period and the first measurement value determined as the integral of the measurement signal over the first period in the same control of the adaptive optical system.

[0025] It is basically advantageous if the degree index is an increasing and injective function of the relative change of the measurement signal. However, this is not necessarily essential.

[0026] When modifying the control of the adaptive optical system to optimize the metric of the present invention, the metric can be processed as follows according to the present invention: The old degree index is determined from the first and second measured values. The control of the adaptive optical system is modified in one direction. The measurement signal is newly detected over the same first and second periods, and the light is focused on the focal region in the sample over the same third period, thereby determining a new first and second measured value. A new degree index is determined from the new first and second measured values, and the difference between the new degree index and the old degree index is determined. Depending on the direction of the difference, the control of the adaptive optical system is modified again, either in the same direction as before, or in a different direction, i.e., the opposite direction of modification. Based on the fact that the change in the measurement signal is fundamentally dependent on the intensity of the selected light, if the metric of the present invention should be maximized, the control of the adaptive optical system is modified again in the same direction as before if the new degree index is greater than the old degree index, and the direction of modification is reversed when the adaptive optical system is newly controlled if the new degree index is less than the old degree index. If the metric of the present invention should be minimized, the opposite is done.

[0027] A step of detecting a new measurement signal over the same first and second periods, comprising focusing light onto a focal region in the sample over the same third period to determine a new first measurement and a new second measurement, determining a new degree index from the new first and new second measurement, determining the difference between the new degree index and the old degree index, and changing the control of the adaptive optical system in the previous direction of change or the reverse direction of change depending on the direction of the difference, can be performed at least once more, and each new degree index formed during the execution of the previous steps is used as the old degree index during the current execution of the step. The steps can be repeated until the degree index reaches an extremum, i.e., a minimum or maximum value, by controlling the adaptive optical system in each direction of change. As a criterion for reaching an extremum, for example, the difference of the new degree index to the old degree index may fall below a limit value based on the immediately preceding change in the control of the adaptive optical system, or the difference of the new degree index to the old degree index may repeatedly reverse its direction over the immediately preceding iteration of the step.

[0028] Furthermore, the magnitude of the change in the control of the adaptive optical system may depend on the magnitude of the difference between the new degree index and the old degree index in at least one preceding step during each iteration of the step. This prevents the desired extremum of each metric from overshooting due to excessively large changes in the control of the adaptive optical system.

[0029] The measurement signal, which is affected by selected light and integrated to obtain first and second measurement values ​​in the method of the present invention, can be, in particular, measurement light emitted from a sample. Here, this measurement light can be imaged by an optical system onto a detector that detects the measurement light and, if applicable, directly integrates it. When imaging the measurement light onto the detector, aberration-induced imaging errors that need to be corrected by the optical system have an effect. However, depending on the form of detection of the measurement light, this additional effect may be negligible. This is, for example, true in all cases where the detector records the measurement light from the focal region without spatial resolution.

[0030] The direction in which the control of the adaptive optical system is changed when carrying out the method of the present invention can be selected from a variety of directions. These include, in particular, directions in which spherical aberration, defocus, astigmatism, or coma aberration of the optical system can be compensated. Which change in the control of the adaptive optical system contributes particularly significantly to approximating each extreme value of the metric of the present invention depends on the respective optical system and especially the respective sample. In many cases, it is sufficient to compensate for only one of the aforementioned aberrations in order to keep the imaging error of the optical system very small. This can be spherical aberration in particular.

[0031] The light whose intensity in the focal region affects the temporal change of the measurement signal can be selected from either light that exponentially decreases the measurement signal toward zero over time, or light that exponentially approximates the measurement signal toward the saturation value from below over time. For example, if the measurement signal is fluorescence emitted from a fluorescent dye in the sample, the selected light can be excitation light that excites the fluorescent dye in the focal region to a fluorescent state until the intensity of the resulting fluorescence approximates the saturation value.

[0032] When a fluorescent dye is excited to a fluorescent state within the focal region by additional excitation light in addition to light, the selected light can be a stimulating light that stimulates the fluorescent dye to stimulate emission before it emits fluorescence. In this case, the fluorescence remaining after the action of the stimulating light depends nonlinearly on the intensity of the stimulating light. Specifically, the additional excitation light can be focused together with the stimulating light into the focal region within the sample by the optical system. Here, if the intensity distribution of the selected light has a central minimum intensity that coincides with the central maximum intensity of the excitation light in the focal region, then an intensity distribution of excitation and stimulating light exists, similar to that used in STED microscopes. Therefore, the method according to the present invention corrects aberration-induced imaging errors of the imaging system, which negatively affect the spatially narrowly limited point spread function when exciting each sample for fluorescence emission.

[0033] Specifically, if the third period begins before the first period, and the light intensity at the maximum intensity of the stimulation light intensity distribution adjacent to the central minimum intensity is large enough that more than 50%, preferably more than 66%, and more preferably more than 90% of the measured signal originates from the surrounding region of the minimum intensity, and its dimensions are smaller than the diffraction limit in at least one spatial direction for both light and fluorescence wavelengths, then the method of the present invention corrects for aberration-induced imaging errors with respect to the surrounding region of the minimum intensity. Even if this surrounding region surrounds, but does not include, the minimum intensity corresponding to each measured point of the sample in the STED microscope, this also means correcting for aberration-induced imaging errors with respect to each measured point of the sample. On the contrary, this correction is extremely specific to each measured point of the sample because the dimensions of the surrounding region are already smaller than the diffraction limit in the wavelengths of light and fluorescence.

[0034] Since the requirements for excitation and stimulation light in the last embodiment of the method of the present invention fully correspond to the requirements of a STED microscope, the method of the present invention can be performed during a continuous run of STED microscope measurements or STED microscope image acquisition. Specifically, the fluorescent photons recorded for each measurement or image acquisition can be used to carry out the method of the present invention if they are recorded with temporal resolution and can thereby be allocated to first and second periods. Of course, the method of the present invention can also be carried out before using the optical system to image the original sample.

[0035] In embodiments of the method of the present invention, similar to that of a STED microscope, the metric to be minimized is determined as a quotient in which the denominator is the first measured value and the numerator is the second measured value. This means that if the new degree index determined as the quotient is smaller than the old degree index determined as the quotient, the control of the adaptive optical system should be changed again in the same direction as before, because a smaller metric indicates a smaller aberration-induced imaging error.

[0036] This clearly distinguishes it from methods that use the brightness of the recorded image, i.e., the residual fluorescence after the action of the stimulating light, as a metric to be maximized in order to calibrate a STED microscope. Instead, the method of the present invention aims to prevent as much fluorescence emission as possible from the stimulating light across the entire focal region, in the same embodiments as the STED microscope. This requires a maximum intensity of the stimulating light in the focal region such that it can only be achieved if there are no aberration-induced imaging errors. Here, residual fluorescence emission from the minimum intensity of the stimulating light intensity distribution, which may actually increase as aberration-induced imaging errors decrease, does not impair the function of the method of the present invention.

[0037] In the method of the present invention, each first period, each second period, and each third period can be a closed period independent of each other, or they can be combined from temporally separated subperiods. The combination of the third period from subperiods can mean, for example, that the light is directed to the focal region in multiple pulses that are rapidly consecutive and not resolved during the recording of the measurement light. The combination of the first period and / or the second period from subperiods can mean that the measurement light from the sample is recorded in multiple temporally consecutive subperiods, with dead time in the recording of the measurement signal between those periods.

[0038] In a particular embodiment of the method of the present invention, the integration of the first, second, and third periods from partial periods means that the focal region within the sample is shifted between each partial period of the first, second, and third periods, respectively, before the second period, and optionally before further subsequent periods of the first, second, and third periods. In this way, the measurement signal is averaged over the spatial region of the sample during detection and optionally integration. This has been shown to be particularly advantageous in embodiments of the method of the present invention, particularly those analogous to STED microscopy, for averaging the effect of the concentration of fluorescent dyes in the sample on the degree index used as a metric, which fluctuates with high spatial frequencies.

[0039] In other words, in the method of the present invention, the detection and optionally integration of the measurement signal over the same first and second periods can be performed for multiple pixels to obtain the first and second measurements, respectively, where the light is focused to a focal region within the sample over the same third period.

[0040] A laser scanning microscope of the present invention comprising a first light source for excitation light, a second light source for stimulating light, an objective lens, and a corrector for correcting aberration-induced imaging errors, wherein the corrector includes an adaptive optical system in the optical path passing through the objective lens, the corrector is configured to perform the following steps of the method of the present invention: detecting a measurement signal over a first period and a second period to determine a first measurement and a second measurement, wherein stimulating light is focused over a third period to a focal region in the sample as light that reduces the measurement signal when acting on the sample; determining a degree index from the first measurement and the second measurement; and using the degree index as a metric to be optimized by changing the control of the adaptive optical system when controlling the adaptive optical system.

[0041] The adaptive optical system of the laser scanning microscope of the present invention may include, for example, an adaptive mirror and / or a controllable micromirror array and / or an SLM (spatial light modulator). In particular, in embodiments of the method of the present invention similar to a STED microscope, the SLM can be used to simultaneously shape the wavefront of the stimulated light, such that the stimulated light forms an intensity distribution with a central minimum intensity within the focal region.

[0042] A further method of the present invention for correcting aberration-induced imaging errors in an optical system having an objective lens and an adaptive optical system for the optical path passing through the objective lens, wherein a first light, a second light, and a sample are selected such that when acting on the sample, the first light excites a first measurement signal from a component of the sample with a first transition probability that depends on the intensity of the first light by a first power, and the second light excites a first or second measurement signal from the same component of the sample with a second transition probability that depends on the intensity of the second light by a second power, where the first power and the second power differ by at least 1. The first light is then focused by the optical system into the focal region of the optical system in the sample, and the first measurement signal excited by the first light is detected from the focal region over a first period of time to determine a first measurement value. Furthermore, a second beam of light is focused by the optical system onto a focal region within the sample, and the first or second measurement signal excited by the second beam of light is detected over a second period from the focal region of the sample to determine the second measurement. From the first and second measurement values, a highly monotonically increasing or decreasing function of the relative change in the first measurement signal or the relative difference between the first and second measurement signals is determined and used as a metric to be optimized by changing the control when controlling the adaptive optical system.

[0043] In the method of the present invention, the fact that the first and second transition probabilities depend differently on the intensities of the first and second light when exciting the respective measurement signals is used to determine the degree of exponent used as a metric to be optimized when controlling the adaptive optical system. In its simplest form, even in this method of the present invention, the degree of exponent is determined by calculating the quotient between the first and second measurements, which is a very monotonically increasing or decreasing function of the relative change of the first measurement signal or the relative difference between the first and second measurement signals. The first and second transition probabilities depend on the light intensity with first and second powers differing by at least 1, while the intensities of the first and second light both depend equally on aberration-induced imaging error, so this quotient depends on the imaging error. In contrast, all other influences on both measurements enter into a constant factor in the quotient, but this factor does not change due to aberration-induced imaging error when determining the first and second measurements for the same focal region, respectively. On the contrary, this also applies to cases where the first light excites another component of the sample as a second light to emit the first measurement signal, either in the same focal region or in a different focal region. In this method of the present invention, by exciting each measurement signal with the first or second light, but in the same focal region of the same component of the sample, the degree index becomes substantially independent of the observation location of the sample. The measurement signal excited by the second light can be the same measurement signal as the measurement signal excited by the first light. This simplifies the implementation of the method of the present invention, and this may be a second measurement signal, or for example, a measurement signal of a different wavelength.

[0044] Specifically, the first light excites a first measurement signal from a component of the sample using a single-photon process, while the second light excites either the first or second measurement signal from the same component of the sample using a multiple-photon process.

[0045] Further preferred embodiments of this method of the present invention correspond to the embodiments of the method of the present invention described first earlier.

[0046] A further method of the present invention for correcting aberration-induced imaging errors in an optical system having an objective lens and an adaptive optical system on the light path passing through the objective lens, wherein the light and the sample are selected as in the first method of the present invention described above. That is, the light, when acting on the sample, reduces the measurement signal from the sample or approximates it to a saturation value from below, and the relative change in the measurement signal depends on the intensity of the light. Unlike the two methods of the present invention described earlier, in this method the light is optically modulated in time and focused by the optical system to the focal region of the sample. Here, the measurement signal from the focal region of the optical system on the sample is detected in a time-resolved manner. The phase shift between the optical modulation and the signal modulation of the measurement signal is determined to some extent exponentially as an extremely monotonically rising or falling function of the relative change in the measurement signal and is used as a metric to be optimized, i.e., minimized or maximized, by changing the control when controlling the adaptive optical system.

[0047] This method of the present invention is equivalent to the method of the present invention in frequency space described previously. The phase shift between optical modulation and signal modulation depends on aberration-induced imaging error, similar to the quotient between the second and first measurements in the method of the present invention described earlier.

[0048] Therefore, many of the preferred embodiments described above the method of the present invention, which was initially described, are also preferred embodiments of this method of the present invention.

[0049] Advantageous developments of the present invention are derived from the claims, specification, and drawings. The advantages of the features and combinations of features described in the specification are merely examples and can act either alternatively or cumulatively, and these advantages do not necessarily have to be achieved by embodiments of the present invention. This applies with respect to the original application documents and patent disclosures without altering the subject matter of the attached claims. Further features are understood from the drawings, in particular the illustrated geometry and the relative dimensions of the various components, as well as their relative arrangement and working connections. Combinations of features of various embodiments of the present invention, or combinations of features of various claims, are possible and proposed herein, even if they differ from the selected reference relationships of the claims. This also applies to features shown in separate drawings or described in the descriptions of the drawings. These features can also be combined with features of different claims. Similarly, features listed in the claims also apply to further embodiments of the present invention.

[0050] The features described in the claims and specification should be understood to exist in exactly that number or a number greater than the number stated, without the need to explicitly use the adverb “at least.” Therefore, for example, when describing adaptive optical systems, this should be understood to mean that there is exactly one adaptive optical system, two adaptive optical systems, or more adaptive optical systems. The features listed in the claims may be substituted for other features, or may consist of just one feature having each method or each laser scanning microscope.

[0051] The reference numerals included in the claims do not limit the scope of the subject matter protected by the claims. The reference numerals are used solely for the purpose of facilitating the understanding of the claims.

[0052] The present invention will be further described below based on preferred embodiments shown in the drawings. [Brief explanation of the drawing]

[0053] [Figure 1] This figure shows the laser scanning microscope of the present invention. [Figure 2] Figure 1 shows the intensity distribution of excitation and stimulation light in the focal region of the objective lens of the laser scanning microscope. [Figure 3] This is a time histogram of photons of fluorescence light recorded as a measurement signal in a laser scanning microscope. [Figure 4] This figure plots the metric of the present invention against various intentionally induced spherical aberrations (black squares) and the automatically optimized metric according to the present invention (white circles). [Figure 5] The metric of the present invention is plotted against the spherical aberration of the optical system of the laser scanning microscope shown in Figure 1 when imaging beads filled with dye. [Figure 6] This figure shows the half-width at half maximum of the image formed by dye-filled beads, compared to Figure 5, for the same spherical aberration. [Figure 7] This figure shows the fluorescence intensity of dye-filled beads for the same spherical aberration, in comparison with Figure 5. [Figure 8] Figure 1 shows the metric progression of the defocusing of the optical system of a laser scanning microscope according to the present invention. [Figure 9] This figure shows the progress of the metric of the present invention regarding spherical aberration in the optical system of the laser scanning microscope shown in Figure 1. [Figure 10] Figure 1 shows the progression of the metric of the present invention regarding astigmatism in the optical system of a laser scanning microscope. [Figure 11] This figure shows the progression of the metric of the present invention regarding the coma aberration of the optical system of the laser scanning microscope shown in Figure 1. [Figure 12] This figure shows a bright image of a structure marked with a fluorescent dye, and the progression of the metric of the present invention for spherical aberration. [Figure 13]This figure shows a twilight image with the same structure as Figure 12, and the progression of the metric of the present invention for the same spherical aberration. [Figure 14] Figures 12 and 13 show dark images with the same structure, and the progression of the metric of the present invention for the same spherical aberration. [Figure 15] This figure shows the intensity distribution of excitation and stimulation light in the focal region of the objective lens of the laser scanning microscope shown in Figure 1, in another embodiment of the present invention. [Figure 16] This figure shows various temporal sequences of the first period during which the measurement signal is integrated into the first measurement value, the second period during which the measurement signal is integrated into the second measurement value, and the third period during which the selected light is focused into the focal region of each optical system. [Figure 17] This is a flowchart of the main steps of the method of the present invention. [Figure 18] Further, here is a flowchart of the main steps of the present invention. [Figure 19] Here is a flowchart of the key steps of yet another method of the present invention. [Modes for carrying out the invention]

[0054] The laser scanning microscope 1 of the present invention, shown in Figure 1, is based on a STED microscope and, correspondingly, has a first light source 2 for excitation light 3, as well as a second light source 4 for light 5 in the form of stimulating light 6 to remove the excitation by excitation light 3. Each of the two light sources 2 and 4 includes a laser 7 or 8, polarization-maintaining optical fibers 9 or 10, and sighting optics 11 or 12 for excitation light 3 or stimulating light 6 emitted from optical fibers 9 or 10. A SLM 13 for wavefront shaping is further positioned in the optical path of stimulating light 6, thereby forming an optical intensity distribution with a minimum central intensity in the focal region of the objective lens 14. The stimulating light 6 is guided together with the excitation light 3 by a dichroic beam splitter 16 behind the SLM 13 and after a further optical system 15. The excitation light 3 and stimulation light 6 are input-coupled to the objective lens 14 via a deformable mirror 17 as an adaptive optical system 18 and another optical system 19, which focuses both the excitation light 3 and stimulation light 6 into a focal region within the sample 20. The optical systems 15 and 19 are configured such that the deformable mirror 17 and SLM 13 are in a plane conjugate to the entrance pupil of the objective lens 14. The position of the focal region where the excitation light 3 and stimulation light 6 are focused is movable within the sample 20 by the scanner 21. In Figure 1, the scanner 21 is implied to be a movable sample stage. However, the scanner 21 can be configured in other ways, in particular to move to tilt the center of the entrance pupil of the objective lens 14, so that the excitation light 3 and stimulation light 6 move relative to the objective lens 14. Fluorescent light 22, emitted from the focal region of the sample 20 based on the excitation of a fluorescent dye present therein by excitation light, is output coupled from the optical path of excitation light 3 by a second dichroic beam splitter 23, focused into a multimode optical fiber 25 by an optical system 24, and guided through to a time-resolution detector 26. In the laser scanning microscope 1 of the present invention, a deformable mirror 17 is controlled by a control unit 27 as an adaptive optical system 18 arranged in the optical path passing through the objective lens 14, and a measurement signal 28 representing the fluorescent light is supplied to this control unit from the detector 26. Instead of the deformable mirror 17, the adaptive optical system 18 can be configured by an SLM, a micromirror array, or in other ways known to those skilled in the art.

[0055] Figure 2 shows the intensity distributions 29 and 30 of the excitation light 3 and the stimulating light 6 along a cross-section in the x-direction passing through the center of the focal region. The excitation light intensity distribution 29 is the intensity distribution of the diffraction-limited spot. The spatially donut-shaped intensity distribution 30 of the stimulating light has a minimum intensity in the form of a zero point 32 at the center 31, and this zero point is defined by the maximum intensity 33 in the part of Figure 2. At the maximum intensity 33, the intensity of the stimulating light is the saturation intensity I S This exceeds the limit, and the excitation of the fluorescent dye by the excitation light 3 is completely reversed. Correspondingly, the fluorescent light 22 recorded by the detector 26 in Figure 1 can be assigned to the region 34 surrounding the zero point 32, the size of which is far below the diffraction limit. However, this is only true for the fluorescent light 22 that is recorded only when the excitation by the excitation light 3 is removed by the stimulation light 6 outside the region 34 surrounding the zero point 32.

[0056] Figure 3 shows the timing of individual photons of fluorescence 22 striking the detector 26 in Figure 1 after the excitation pulse 35 and the stimulation pulse 36. First, the number of photons recorded per unit time increases sharply during pulse 35 due to excitation by the excitation light 3. Next, this number decreases rapidly during pulse 36 due to the action of the stimulation light. The photons of fluorescence 22 recorded in period 37 after pulse 36 are photons from the region 34 surrounding the zero point 32. In the method of the present invention, the number of photons of fluorescence 22 recorded in period 37 is related to the number of photons of fluorescence 22 recorded during the preceding period 38. This preceding period 38 is also referred to here as the first period, while period 37 is referred to as the second period. According to Figure 3, pulses 35 and 36 are in the first period 38. According to the present invention, associating photons recorded in the second period 37 with photons of fluorescent light 22 recorded in the first period 38 is done by determining the degree index, in particular by calculating the quotient between the photons recorded in the second period 37 and the photons recorded in the first period 38. Hereinafter, the present invention is based on the knowledge that if the control unit 27 controls the adaptive optical system 18 so that the aberration-induced imaging error of the optical system of the laser scanning microscope 1, including the objective lens 14, is accurately compensated by the adaptive optical system 18, then this degree index will take an extreme value. The aberration-induced imaging error of the optical system of the laser scanning microscope 1, as shown in Figure 1, acts on the intensities of the excitation light 3 and stimulation light 6 in the focal region within the sample 20 so that these intensities decrease as the imaging error increases.Therefore, based on the increase in the imaging error, the excitation of the fluorescent dye in the sample 20 decreases along with the intensity of the excitation light, and consequently the fluorescent light 22 also decreases. However, this attenuation is not distinguishable from other attenuations of fluorescence 22, such as attenuation due to spatial variations in the concentration of the fluorescent dye or attenuation due to bleaching of the fluorescent dye. However, if we consider the relative effect of the stimulating light 6 on the fluorescent dye previously excited by the excitation light 3, which is expressed by a degree index determined from the photons recorded in two periods 37 and 38, this effect depends only on the intensity of the stimulating light 6. The fewer photons recorded in the second period 37 than in the first period 38, the more fluorescent dyes that the stimulating light 6 excites before the second period 37.This means that the greater the intensity of the stimulating light, the smaller the aberration-induced imaging error. Referring to Figure 2, the effect of aberration-induced imaging error can also be explained as follows: In the intensity distribution 30 of the stimulating light 6, which has been reduced overall by aberration-induced imaging error, the saturation intensity I. S This is the first time that the value exceeds zero point 32 at a relatively large interval, and it can be explained that, correspondingly, more fluorescence remains in the region 34 surrounding zero point 32.

[0057] The metric plotted in Figure 4 is the quotient obtained by dividing the number of photons of fluorescence 22 recorded in the first period 38 by the number of photons of fluorescence 22 recorded in the second period 37. In this case, the metric increases if relatively fewer photons are recorded in the second period 37 than in the first period 38. This is because the stimulation light 3 has a relatively high intensity and therefore exerts a stronger reduction effect on the fluorescence 22. Illustrated are the effects of six artificially introduced spherical aberrations (black squares) in the laser scanning microscope 1 of Figure 4, which is equipped with an adaptive optical system 18. As the spherical aberration increases, the decrease in the metric compared to the maximum increases, regardless of its sign. This maximum is an artificially introduced zero aberration, and the metric increases significantly from the initial state by the automatic optimization of the metric of the present invention, which is achieved by changing the control of the adaptive optical system 18 by the control unit 27 in Figure 1 (white circle). In Figure 4, the metric was created when a layer of beads filled with 40 nm-sized dye was imaged in two dimensions using the laser scanning microscope 1 shown in Figure 1, and the photons of the fluorescence 22 were recorded in time-resolved form according to Figure 3.

[0058] Figure 5 plots the same metric of the present invention as in Figure 4, with STED images of individual beads filled with 40 nm size dyes taken for various spherical aberrations as intended by the adaptive optical system 18. Figure 6 shows the corresponding full width at half maximum (FWHM) of the images of individual beads, and Figure 7 shows the fluorescence intensity from individual beads, plotted for the same captured aberrations. Comparing Figures 5 to 7, it can be seen that the maximum of the metric of the present invention in Figure 5 is achieved within a remarkably wide minimum of FWHM in Figure 6 and a similarly wider maximum of fluorescence intensity in Figure 7. This explains why, when optimizing the metric of the optical system of the laser scanning microscope 1 in Figure 1 according to the present invention to eliminate aberration-induced imaging errors, the adaptive optical system 18 can be experimentally modified for the purpose of metric optimization without degrading the imaging quality of the laser scanning microscope 1. Of the criteria plotted for spherical aberrations in Figures 5 to 7, the metric of the present invention responds most quickly to aberration-induced imaging errors. Therefore, this metric's extreme value indicates that other criteria have already passed their extreme value, thereby eliminating any perceived degradation in image quality.

[0059] Figures 8 to 11 show the effects of various aberrations on the metric of the present invention in Figures 4 and 5. Here, the metric is globally maximum when each aberration is zero, whether it is defocus in Figure 8, spherical aberration in Figure 9, astigmatism in Figure 10, or coma aberration in Figure 11. Therefore, optimizing the metric is suitable for eliminating imaging errors based on each of these four different aberrations.

[0060] Figures 12 to 14 show images of the same structure at different brightness levels, with the plot of metric assignments corresponding to Figure 4. Specifically, images with a given aberration (black squares) and those with automatic optimization (white circles) are plotted. The difference in brightness of the structure images is due to the different bleaching processes of the fluorescent dyes marking the structures. Regardless of image brightness, the metric consistently shows a maximum value at the same nearly zero spherical aberration artificially introduced, indicating the need for optimization of the metric according to this invention. Therefore, the removal of aberration-induced imaging errors according to this invention is reliably achieved regardless of image brightness.

[0061] Figure 15 shows the alternative intensity distribution 30 of the stimulating light 6 relative to the intensity distribution 29 of the excitation light 3. Specifically, the stimulating light 6 similarly forms a diffraction-limited spot. In this case as well, if the quotient between the leading and trailing photons of the fluorescence is optimized by changing the control of the adaptive optical system 18 in Figure 1, aberration-induced imaging errors can be eliminated. This is similar to the case of the intensity distribution 30 of the stimulating light 6, where the saturation intensity 1 S This also applies if it does not reach that level.

[0062] Figure 16 shows various sequences of the first period 38 in which the measurement signal 28 from the sample 20 is integrated, the subsequent second period 37 in which the measurement signal 28 is newly integrated, and the third period 39 in which the light 5 acting on the measurement signal 28 is focused to the focal region of the sample 20. The third period 39 corresponds to pulse 36 in Figure 3, where the stimulating light 6 is directed at the sample. According to Figure 3, period 37 follows directly from period 38, and period 39 or pulse 36 overlaps with the latter part of period 38. According to Figure 16a), the order of periods 37 and 38 is the same. The third period 39 completely overlaps with the first period 38. According to Figure 16b), the order of periods 37 and 38 is also the same. However, here the third period 39 overlaps with both a part of the first period 38 and a part of the second period 37. According to Figure 16c), the order of periods 37 and 38 is again the same. However, here the third period 39 overlaps only with the second period 37. According to Figure 16d), the third period 39 is located between periods 37 and 38 without overlap. In either case, the light 5 focused on the sample 20 in the third period 39 acts more strongly on the measurement signal integrated over the subsequent period 37 than on the measurement signal integrated over period 38. This is because the light 5 always further reduces the measurement signal 28, which is in the form of fluorescent light 22, as shown in Figure 1, for example. Therefore, the measurement obtained by integrating the measurement signal 28 in the first period 38 is less affected by the light 5 than the second measurement obtained by integrating in the second period 37, if the third period 39 is completely before the first period 38.

[0063] The flowchart 40 of the method of the present invention shown in Figure 17 begins with the selection 41 of light 5 and sample 20, so that light 5, when acting on sample 20, reduces the measurement signal 28 from sample 20 or approximates it to a saturation value from below, and the change in the measurement signal 28 depends on the intensity of light 5. Then, during detection 42, the measurement signal of sample 20 from the focal region of the optical system, where aberration-induced imaging errors are to be corrected, is detected over a first period 38, for example, integrated to determine a first measurement, and then a second measurement is determined over a second period 37. At this time, light 5 is focused by the optical system to the focal region in sample 20 over a third period 39. From the first and second measurements, during determination 43, a new degree index of a new form of quotient is determined, for example, and during determination 44, the difference of the new degree index to the preceding degree index is determined. Depending on the direction of the difference in degree index, when the control of the adaptive optical system 18 is changed 45, the direction of the previous change in the control of the adaptive optical system 18 is maintained or reversed. Steps 42 to 45 are repeated in loop 46. During this iteration, the focal region of the optical system in the sample 20 can be moved, thereby allowing the adaptive optical system 18 to continuously and optimally compensate for aberration-induced imaging errors of the optical system for various positions of the focal region in the sample 20. During the detection 42 of the measurement signal 28 for determining the first and second measurements, the focal region in the sample 20 can also be moved and spatial averaging can be performed. For this purpose, periods 37 to 39 are divided into corresponding subperiods, one of which is assigned to each position of the focal region in the sample 20.

[0064] Figure 18 shows a flowchart 40 of a modified method from the method described based on Figure 17. During selection 47, the first and second lights and the sample are selected such that the first light, when acting on the sample 20, excites a first measurement signal from a component of the sample with a first transition probability that depends on the intensity of the first light by a first power, and the second light, when acting on the sample, excites a second measurement signal from the same component of the sample, i.e., from the same fluorescent dye, with a second transition probability that depends on the intensity of the second light by a second power, where the first power and the second power differ by at least 1. Next, during focusing 48, the first light is first focused by the optical system into the focal region of the optical system in the sample, and the first measurement signal excited by the first light is detected from the focal region over a first period 38 to determine the first measurement value. Next, during further focusing 49, the second light is focused by the optical system to the same focal region in the sample 20, and the second measurement signal excited by the second light is detected from the focal region of the sample over a second period 37 to determine the second measurement. Subsequently, during determination 43, the first degree index is determined, for example, in the form of a new quotient, in the same manner as the method of the present invention described with reference to Figure 7, then during determination 44, the difference between the new degree index and the previous degree index is determined, and during modification 45, the control of the adaptive optical system 18 is changed in the same direction as before or in a different direction. The steps repeated in loop 46 include, here, steps 48, 49 and 43 to 45.

[0065] In a further method of the present invention shown in Figure 19 based on flowchart 40, selection 41 corresponds to selection 41 in Figure 17. However, the subsequent focusing 50 of the selected light is performed by temporally modulating its intensity with light, and the simultaneous detection of the measurement signal 28 is performed by temporal resolution. Next, in determination 51 as a new degree index, the phase shift between the light modulation and the signal modulation of the measurement signal 28 is determined. Then, in determination 44, the difference of this new degree index to the old degree index is determined, and a change 45 in the control of the adaptive optical system 18 is made based on this difference. Here, loop 46 includes steps 50, 51, 44 and 45. [Explanation of Symbols]

[0066] 1. Laser scanning microscope 2. First light source 3. Excitation light 4. Second light source 5 light 6 Stimulating light 7 Lasers 8 lasers 9 Optical Fiber 10 Optical Fibers 11 Collimation optical system 12 Collimation optical system 13 SLM 14 Objective lens 15 Optical system 16 Dichroic Beam Splitter 17 Transformable Mirror 18 Adaptive Optical Systems 19 Optical system 20 samples 21 Scanners 22 Fluorescent light 23 Dichroic Beam Splitter 24 Optical system 25 Multimode optical fiber 26 Detectors 27 Control Unit 28 Measurement signal 29 Light intensity distribution 30 Light intensity distribution 31 center 32 Zero points 33 Maximum strength 34 Surrounding area 35 pulses 36 pulses 37 Second period 38 The first period 39 Third period 40 Flowcharts 41 Selection 42 detected 43 Decision 44 Decision 45 Changes 46 loops 47 Selection 48 Focus 49 clusters 50 clusters 51 Decision I S Saturation intensity

Claims

1. 1. A method for correcting aberration-induced imaging errors in an optical system having an objective lens (14) and adaptive optics (18) in a light path through the objective lens (14), comprising the steps of: - the stimulation light (6) and the sample (20) containing a fluorescent dye that emits fluorescent light (22) when excited into a fluorescent state are selected so as to reduce a measurement signal (28) representative of the fluorescent light (22) emitted from the sample (20) when the stimulation light (6) acts on the sample (20); The reduction is achieved by the stimulation light (6) exciting the fluorescent dye to a fluorescent state in which it emits fluorescent light (22) by the stimulation light (6) plus the excitation light (3), and stimulating the fluorescent dye to stimulated emission before the fluorescent dye emits the fluorescent light (22) from the fluorescent state; a relative change in the measurement signal (28) representative of the fluorescent light (22) is caused by the selected stimulation light (6) and depends on the intensity of the stimulation light (6); a measurement signal (28) indicative of fluorescent light is detected from a focal region within the sample (20) of the optical system over a first time period (38) to determine a first measurement value by integrating a measurement signal (28) indicative of fluorescent light (22) emitted from the sample (20) during said first time period (38); a measurement signal (28) representative of the fluorescent light is detected from a focal region within the sample (20) of the optical system over a second time period (37) to determine a second measurement value by a new integration of the measurement signal (28) representative of the fluorescent light (22) emitted from the sample (20) during said second time period (37); - the stimulation light (6) is focused by the optical system onto a focal region of the sample (20) for a third period (39); - said first period (38) precedes said second period (37) and / or said second period (37) follows said first period (38); - said third period (39) at least partially overlaps in time with said first period (38) and / or said second period (37) or is an intermediate period between said first and second periods; - from the first and second measurements, an exponent is determined to some extent as a strictly monotonically rising or falling function of the relative change in the measurement signal (28) and is used as a metric to be optimized, i.e. maximized or minimized, when controlling the adaptive optical system (18) by changing the control (45) so as to maximize the intensity of the stimulation light (6) in the focal region.

2. When the stimulation light (6) reduces a measurement signal (28) representative of the fluorescent light (22) emitted from the sample (20), the absolute value of the change in the measurement signal (28) is normalized to an initial value of the measurement signal in order to determine an extent index.

2. The method of claim 1 .

3. 3. The method according to claim 1, wherein the first measurement value is determined such that it indicates a first integral over the first period (38) of the measurement signal (28) and the second measurement value is determined such that it indicates a second integral over the second period (37) of the measurement signal (28).

4. The method according to claim 1 , wherein the degree index is determined as a quotient of the second measurement value and the first measurement value.

5. - determining the staleness index, - changing the control of the adaptive optical system (18) in one direction, - detecting a new measurement signal (28), whereby the stimulation light (6) is focused again onto a focal region within the sample (20), - determining the degree index of novelty; - determining the difference between said new degree index and said old degree index, 5. The method according to claim 1, further comprising: - depending on the direction of the difference, a new modification of the control of the adaptive optical system (18) is performed in the previous direction of modification or in a different direction of modification.

6. - a new detection of a measurement signal (28), the stimulation light (6) being newly focused onto a focal region within said sample (20); - determining a novel degree index; - determining (44) the difference between said new degree index and said old degree index; a step (45) of modifying the control of said adaptive optical system (18) in a new direction of modification, either in the previous direction or in a different direction, depending on the direction of said difference, A control of the adaptive optical system (18) is performed at least once more or in the direction of change several times until the degree index reaches an extreme value in the control, and one of the new degree indexes determined during one of the previous executions of the step is used as the old degree index during the current execution of the step. The method according to claim 5, characterized in that

7. 7. The method according to claim 1, wherein the fluorescent light (22) detected as the measurement signal (28) is measurement light emitted from the sample (20), which is imaged onto a detector (26) by the optical system.

8. The direction in which the control of the adaptive optical system (18) is changed is - spherical aberration, - Defocus, - astigmatism, -Coma aberration 8. The method according to claim 1, wherein the direction of change is selected from the directions of change for which compensation is possible.

9. A method according to any one of claims 1 to 8, characterized in that the excitation light (3) is focused by the optical system to a focal region within the sample (20).

10. The method according to claim 9, characterized in that the intensity distribution of the stimulation light (6) has a central minimum intensity in the focal region that coincides with the central maximum intensity of the excitation light (3).

11. A method according to claim 9 or 10 when directly citing claim 4 and claim 5 or 6, characterized in that if the new degree index determined as a quotient is smaller than the old degree index determined as a quotient, and if the quotient has the first measurement value in the denominator and the second measurement value in the numerator, the control of the adaptive optical system (18) is changed again in the previous direction of change.

12. 2. The method according to claim 1, characterized in that the first period (38), the second period (37) and, if present, the third period (39) are each a closed period independent of one another or are integrated from partial periods separated in time from one another.

13. 13. The method according to claim 1, wherein detection of the measurement signal (28) is performed for a number of pixels and a stimulation light (6) is focused on a focal region within the sample (20) to determine the degree index.

14. A laser scanning microscope (1), comprising: a first light source (2) for excitation light (3), a second light source (4) for the stimulation light (6), an objective lens (14), a correction device including an adaptive optical system (18) in the light path through said objective lens (14) for correcting aberration-induced imaging errors; In a laser scanning microscope having 14. A laser scanning microscope, characterized in that the correction device is configured to carry out the method according to any one of claims 1 to 13.

15. The adaptive optical system (18) comprises: - an adaptive mirror (17), and / or - a controllable micromirror array, and / or - has a spatial light modulator (SLM), 15. The laser scanning microscope (1) according to claim 14, characterized in that it has 16. A method for correcting an aberration-induced imaging error in an optical system having an objective lens (14) and an adaptive optical system (18) in a beam path through the objective lens (14), comprising: the excitation light (3) and the sample (20) containing a fluorescent dye which emits fluorescent light (22) when excited into a fluorescent state are selected in such a way that, when the excitation light (3) acts on the sample (20), it excites the fluorescent dye into a fluorescent state until it approaches a saturation value, and in that fluorescent state the fluorescent dye emits fluorescent light (22), thereby causing a measurement signal (28) representative of the fluorescent light (22) emitted from the sample (20) to approach a saturation value from below, the relative change in the measurement signal (28) representative of the fluorescent light (22) is caused by the selected excitation light (3) and depends on the intensity of the excitation light (3); a measurement signal (28) indicative of fluorescent light is detected from a focal region within the sample (20) of the optical system over a first time period (38) to determine a first measurement value by integrating a measurement signal (28) indicative of fluorescent light (22) emitted from the sample (20) during said first time period (38); a measurement signal (28) representative of the fluorescent light is detected from a focal region within the sample (20) of the optical system over a second time period (37) to determine a second measurement value by a new integration of the measurement signal (28) representative of the fluorescent light (22) emitted from the sample (20) during said second time period (37); - excitation light (3) is focused by said optical system onto a focal region of said sample (20) for a third period (39); - said first period (38) precedes said second period (37) and / or said second period (37) follows said first period (38); said third period (39) overlaps at least in part in time with said first period (38) and with said second period (37); - from the first and second measurements, an exponent is determined to some extent as a strictly monotonically rising or falling function of the relative change in the measurement signal (28) and is used as a metric to be optimized, i.e. maximized or minimized, when controlling the adaptive optical system (18) by modifying the control (45) so as to maximize the intensity of the excitation light (3) in the focal region.

17. The method described in claim 16, characterized in that when the excitation light (3) causes a measurement signal (28) indicating the fluorescent light (22) emitted from the sample (20) to approximate a saturation value from below, the absolute value of the change in the measurement signal (28) is normalized to the saturation value to determine an extent index.

18. A method as described in claim 16 or 17, characterized in that the first measurement value is determined such that it indicates a first integral over the first period (38) of the measurement signal (28), and the second measurement value is determined such that it indicates a second integral over the second period (37) of the measurement signal (28).

19. A method according to any one of claims 16 to 18, wherein the degree index is determined as a quotient of the second measurement value and the first measurement value.

20. - determining an age index, - changing the control of the adaptive optical system (18) in one direction, - detecting a new measurement signal (28), whereby the excitation light (3) is again focused onto a focal region within the sample (20), - determining the degree index of novelty; - determining the difference between said new degree index and said old degree index, 20. The method according to any one of claims 16 to 19, characterized in that depending on the direction of the difference, a new modification of the control of the adaptive optical system (18) is performed in a previous or another direction of modification. a step of newly detecting a measurement signal (28), in which the excitation light (3) is newly focused in a focal region within the sample (20); - determining a novel degree index; - determining (44) the difference between said new degree index and said old degree index; a step (45) of modifying the control of said adaptive optical system (18) in a new direction of modification, either in the previous direction or in a different direction, depending on the direction of said difference, A control of the adaptive optical system (18) is performed at least once more or in the direction of change several times until the degree index reaches an extreme value in the control, and one of the new degree indexes determined during one of the previous executions of the step is used as the old degree index during the current execution of the step.

21. The method of claim 20, wherein:

22. A method according to any one of claims 16 to 21, wherein the fluorescent light (22) detected as the measurement signal (28) is measurement light emitted from the sample (20), and the measurement light is imaged onto a detector (26) by the optical system.

23. The direction in which the control of the adaptive optical system (18) is changed is - spherical aberration, - Defocus, - astigmatism, -Coma aberration 23. The method according to claim 16, wherein the direction of change is selected from the directions of change for which compensation is possible.

24. The method described in claim 16, characterized in that the first period (38), the second period (37), and, if present, the third period (39), are each a closed period independent of each other or are integrated from subperiods separated in time from each other.

25. A method described in any one of claims 16 to 24, wherein detection of the measurement signal (28) is performed for multiple pixels and excitation light (3) is focused to a focal region within the sample (20) to determine the degree index.

26. A laser scanning microscope (1), comprising: a first light source (2) for excitation light (3), a second light source (4) for the stimulation light (6), an objective lens (14), a correction device including an adaptive optical system (18) in the light path through said objective lens (14) for correcting aberration-induced imaging errors; In a laser scanning microscope having 26. A laser scanning microscope, characterized in that the correction device is configured to carry out the method according to any one of claims 16 to 25.

27. ​​The adaptive optical system (18) comprises: - an adaptive mirror (17), and / or - a controllable micromirror array, and / or - has a spatial light modulator (SLM), 27. The laser scanning microscope (1) according to claim 26, characterized in that it has

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