Method and microscope with correction device for correcting imaging errors induced by hue differences

Abstract

To correct for chromatic aberration-induced imaging errors in an optical system with a lens (14) and an adaptive optics device (18), light (5) and a sample (20) are selected such that light (5) reduces the measurement signal (28) from the sample (20) when acting on it, wherein the relative change in the measurement signal (28) depends on the intensity of light (5). The measurement signal (28) from the focused region of the optical system in the sample (20) is detected during the first and subsequent second time periods (38, 37) to determine the first and second measurement values. During a third time period (39) overlapping with the first and / or second time periods, light (5) is focused onto the focused region using the optical system. A metric for the relative change in the measurement signal (28) is determined from the first and second measurement values ​​and used as an indicator to be optimized in the manipulation of the adaptive optics device (18).

Description

Technical Field

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

[0002] On the one hand, imaging errors induced by chromatic aberration can be attributed to the optical system itself, for example, if the optical system is not sufficiently achromatic. On the other hand, the sample area located in front of the focusing area of ​​the optical system may also cause potential aberrations.

[0003] The imaging error to be corrected caused by chromatic aberration in the optical system plays a role not only when the light intensity distribution is projected onto the corresponding sample with the aid of a lens, but also when the sample is imaged with the aid of a lens. Background Technology

[0004] Correction for imaging errors induced by chromatic aberration, attributable to the inhomogeneity of optical properties of samples, particularly thicker samples, is known from Wei Yan et al.'s paper, "Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples" (Photonics Research, Vol. 5, No. 3, June 2017). Here, a method called COAT (Coherent Optical Adaptive Technique) is used, in which phase distortion caused by the corresponding sample is measured and compensated. A SLM (Spatial Light Modulator) is used not only to measure but also to compensate for phase distortion. With this SLM, in STED microscopy, the phase wavefront of the stimulus light is also modulated so that the stimulus light focused into the sample has a ring-shaped (Donut-) phase front. Intensity distribution. Phase distortion caused by the sample is measured by applying a phase pattern to the beam of stimulus light using a SLM, where phase shifts in different regions are modulated at different frequencies. The intensity of the spot (to which the beam of stimulus light is subsequently focused) is recorded in a time-resolved manner and analyzed by Fourier transform. Thus, in practice, phase shifts that compensate for chromatic aberration-induced imaging errors are searched in each region of the phase pattern applied using the SLM. Specifically, the intensity of the spot is detected by scanning gold nanoparticles as a spot and recording the stimulus light scattered by them. Accordingly, the corresponding STED microscope must be operated in scattered light mode, and the gold nanoparticles must be positioned on the sample at the location where chromatic aberration-induced imaging errors should be compensated. In practice, the gold nanoparticles are only positioned on the upper and lower sides of the sample to demonstrate the working principle of the method.

[0005] A method for correcting aberrations in STED microscopy using adaptive optics is disclosed in WO 2014 / 029978 A1, which uses an index combining image sharpness and image brightness. This index is maximized or minimized by manipulating an optical modulator, which involves an SLM or deformable mirror. Image sharpness can only be determined after the sample is imaged into the image. Image brightness depends on the concentration and density of the fluorescent label used to label the structures of interest in the sample. Therefore, the corresponding maximum or minimum value of the index combining image brightness and image sharpness has only local validity and cannot be compared with other maximum or minimum values ​​in other regions of the sample.

[0006] A method for aligning the focusing of a ring-shaped stimulus beam and a spot-shaped excitation beam to each other is known from Yifan Wang et al.'s paper, "A 3D Aligning Method for Stimulated Emission Depletion Microscopy Using Fluorescence Lifetime Distribution" (Microscopy Research and Technology 77:935-940, August 2014), in which the offset between the STED image and the lifetime image of the fluorescent nanobeads is determined and eliminated. Specifically, this involves determining and compensating for the offset between the centroid of the normalized spatial intensity distribution of fluorescence from the nanobeads and the normalized spatial lifetime intensity distribution of fluorescence from the nanobeads.

[0007] As known from WO 2018 / 042056 A1, to properly adjust an STED microscope, the maximum intensity of the excitation light and the minimum intensity of the STED light coincide at the focal point of the lens. The sample is scanned at the maximum intensity of the excitation light to obtain sample images of structures labeled with fluorescent dye (Fluoreszenzfarbstoff) to produce images of the structures with varying intensities of fluorescent blocking light. Then, the offset between the positions of the structures in the resulting images is calculated and compensated. Summary of the Invention

[0008] The present invention is based on the following objective: to describe a method for correcting imaging errors induced by chromatic aberration in an optical system and a laser scanning microscope having a lens and a correction device for correcting imaging errors induced by chromatic aberration, which enables the correction of imaging errors for different regions of a sample when the imaging system or laser scanning microscope is used continuously to measure or image a corresponding sample.

[0009] The objective of this invention is achieved by a method having the features of claims 1, 3, and 7, and by a laser scanning microscope having the features of claim 21. Here, 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 according to the invention, and claim 22 relates to a preferred embodiment of the laser scanning microscope according to the invention.

[0010] This invention uses an index that represents a measure of the quality of focusing when a sample is illuminated with light. This index can be used to correct for aberrations, such as those that may occur in a microscope. This can be achieved by using an adaptive optics system to match the distribution of light used to illuminate the sample in such a way that the index is optimized. For this purpose, the sample is first illuminated with light using a given setting of the adaptive optics, thereby affecting the measurement signal from the sample in such a way that, for example, the light causes the emission of fluorescence radiation. The measurement signal is then detected in a time-resolved manner and divided into at least one early time period and a late time period for subsequent calculations. The index representing the quality of focusing is calculated from the ratio of the measured values ​​of the measurement signal in the two time periods, such as the ratio of early and late photons of fluorescence. This index is optimized by repeated matching of the adaptive optics, such as an adaptive mirror (Spiegel), thereby correcting for aberrations. An equivalent index can be determined in the frequency space.

[0011] In the method according to the invention for correcting chromatic aberration-induced imaging errors in an optical system, the optical system has a lens and an adaptive optics device arranged in a beam path passing through the lens, selecting light and a sample in such a way that the light, when acting on the sample, either reduces or approaches a saturation value from the sample, wherein the relative change in the measurement signal depends on the intensity of the light. Measurement signals originating from the focused region of the optical system in the sample are detected during a first time period to determine a first measurement value and during a second time period to determine a second measurement value. During a third time period, the selected light is focused onto the focused region in the sample by means of the optical system. Here, the first time period is at least partially earlier than the second time period and / or the second time period is at least partially later than the first time period, and the third time period overlaps at least partially in time with the first time period and / or the second time period and / or the intermediate time period in between. A metric value is determined from the first and second measurement values, which is a strictly monotonically increasing or decreasing function of the relative change in the measurement signal, and is used as an indicator when manipulating the adaptive optics device, which can be optimized by changing the manipulation.

[0012] When performing the method according to the invention, chromatic aberration-induced imaging errors to be corrected come into play when the selected light is focused onto a focal region in the sample using an optical system. If the imaging error occurring in this focusing is corrected, the imaging error is also corrected when the focal region is imaged using an imaging system.

[0013] Then, when the selected light is focused onto the focal region in the sample using an optical system during the third time period, the chromatic aberration-induced imaging error to be corrected has the following effect: the intensity of the selected light decreases relative to its maximum value without the chromatic aberration-induced imaging error to be corrected, i.e., the so-called Strehl-Zahl number. Accordingly, the relative change in the measurement signal from the sample caused by the selected light also decreases.

[0014] The relative definitions of the first, second, and third time periods mentioned above result in the first measurement being less strongly affected by the selected light focused into the focal region during the third time period compared to the second measurement.

[0015] Due to the aforementioned selection of light and sample, and the relative definitions of the first, second, and third time periods, the first and second measurements depend in the same way on the local characteristics of the sample in the respective focused regions. Therefore, for example, determining the metric by forming a quotient between the second measurement (determined as the integral of the measurement signal during the second time period) and the first measurement (determined as the integral of the measurement signal during the first time period) results in normalization to the initial value of the measurement signal, i.e., resulting in independence from the absolute height, i.e., the level of the measurement signal. However, this metric still depends on the intensity of the light selected in the focused region.

[0016] Therefore, the metric can be compared across fluctuations in the measurement signal level. This means that even when the level of the measurement signal fluctuates in adjacent regions of the sample, the metric can be optimized across these adjacent regions. Thus, the metric can be continuously optimized, especially when scanning the sample with a focused region. Optimization of the metric can be achieved not only when the position of the focused region in the sample remains constant across changes in the control of the adaptive optics. However, this does not mean that the metric to be optimized according to the invention, i.e., the metric, has the same value for all positions of the focused region in the sample. On the contrary, this is certainly not the case, because the sample itself is the cause of aberrations that change at least along the axial direction of the lens optical axis, i.e., in the depth of the sample. However, between laterally adjacent positions of the focused regions in the sample, the optimized metric will only fluctuate slightly, so optimization at directly adjacent positions of the focused regions in the sample can begin with the control of the adaptive optics, which in earlier positions of the focused region has already led to optimization of the metric according to the invention.

[0017] The dependence on the intensity of the selected light in the focused region is not lost due to the determination of the metric values ​​in the following way: not only when the selected light reduces the measurement signal, but also when the selected light causes the measurement signal to approach a saturation value from below, both the first and second measurements are neither linearly nor directly dependent on the intensity of the selected light in any other equal non-zero power (Potenz).

[0018] Therefore, in the method according to the invention, the metric value either increases or decreases with the intensity of the selected light in the focused region. Accordingly, the metric value is maximized or minimized as an indicator to be optimized by manipulating the adaptive optics to maximize the intensity of light in the focused region. This maximum intensity is reached precisely when the imaging error induced by chromatic aberration has been eliminated. Thus, the metric value in the method according to the invention is suitable as an indicator to be optimized by changing the manipulation of the adaptive optics to achieve the goal of correcting the imaging error induced by chromatic aberration.

[0019] When the selected light reduces the measurement signal from the sample, the formation of the quotient of the second measurement value (determined as the integral of the measurement signal during the second time period) and the first measurement value (determined as the integral of the measurement signal during the first time period), as given by example (this formation results in normalization to the initial value of the measurement signal), is, for example, suitable for determining a metric. However, the reciprocal of this quotient is also suitable as a metric for measuring the effect of the selected light on the measurement signal, and in this case, it should not be minimized but maximized.

[0020] If the chosen light causes the measurement signal from the sample to approach its saturation value from below, it is advantageous to normalize the absolute change in the measurement signal to the saturation value in order to determine the metric. This normalization can be approximated by determining the metric by forming a quotient between the first and second measurements.

[0021] However, in the method according to the invention, the metric determined by the first and second measurements (which serves as an indicator for optimization by altering the manipulation of the adaptive optics) need not be a quotient, especially not a direct quotient of the two measurements. Thus, it is essentially harmless if the denominator or numerator of the quotient also has another factor multiplied by the measurement or an offset added to the measurement. Furthermore, the denominator of the quotient can have the difference between the measurements and / or the numerator of the quotient can have the sum of the measurements, and vice versa. Generally, any metric is suitable if it is a strictly monotonically increasing or decreasing function of the relative change in the measured signal. Here, it is understood that the function only needs to possess these properties within the relevant range of the observed measurements.

[0022] Under the same operation of the adaptive optics device, each of these metrics reaches an extreme value, such as the quotient of the second measurement value determined as the integral of the measurement signal during the second time period and the first measurement value determined as the integral of the measurement signal during the first time period.

[0023] It is advantageous in principle if the metric is a continuous and therefore injective function of the relative change of the measured signal. However, this is by no means necessary.

[0024] When modifying the operation of the adaptive optics to optimize the metric according to the invention, the process can be specifically performed as follows: An old metric is determined from a first and a second measurement. The operation of the adaptive optics is changed in a changing direction. The measurement signal is detected again during the same first and second time periods, wherein light is focused into a focal region in the sample during the same third time period to determine new first and new second measurements. A new metric is determined from the new first and new second measurements, and the difference between the new and old metric is determined. Depending on the direction of the difference, the adaptive optics is changed again in the previous changing direction or in a different changing direction, i.e., particularly in a changing direction opposite to the previous changing direction. If the metric according to the invention should be maximized due to the dependence of the change in the measurement signal on the intensity of the selected light, then when the new metric is greater than the old metric, the operation of the adaptive optics should be changed again in the previous changing direction, and thus when the new metric is less than the old metric, the changing direction should be switched when the operation of the adaptive optics is changed again. The opposite is true when the metric according to the invention should be minimized.

[0025] The steps of detecting the measurement signal again during the same first time period and the same second time period (wherein, during the same third time period, light is focused into a focal region in the sample to determine new first and new second measurements), determining new metric values ​​from the new first and new second measurements, determining the difference between the new and old metric values, and changing the control of the adaptive optics again in the previous or different change direction according to the direction of the difference can be performed at least once more, wherein the new metric values ​​formed in the previous execution of the steps are used as the old metric values ​​in the current execution of the steps respectively. The mentioned steps can be repeated so frequently that the following control of the adaptive optics is achieved in the corresponding change direction, under which the metric value reaches an extreme value, i.e., either a minimum or a maximum value. As a criterion for reaching an extreme value, for example, the following can be considered: the difference between the new and old metric values ​​is lower than the limit value associated with the last change of control of the adaptive optics, or the difference between the new and old metric values ​​repeatedly switches its direction on the last repetition of the steps.

[0026] Furthermore, in each repetition of the step, the magnitude of the change in the control of the adaptive optics can depend on the magnitude of the difference between the new and old metric values ​​in at least one previous step. This avoids overshooting of the sought extrema of the corresponding metric due to excessively drastic changes in the control of the adaptive optics.

[0027] In the method according to the invention, the measurement signal can in particular be measurement light emitted from a sample, which is affected by selected light and integrated to obtain first and second measurement values. Here, the measurement light can be imaged onto a detector by means of an optical system, which detects the measurement light and integrates it directly if necessary. When the measurement light is imaged onto the detector, the chromatic aberration of the optical system induces an imaging error to be corrected. However, depending on how the measurement light is detected, this additional effect may be negligible. For example, this applies to all cases where the detector records measurement light from the focal region without spatial resolution.

[0028] When performing the method according to the invention, the direction of change in the manipulation of the adaptive optics can be selected from a variety of directions. This includes, in particular, directions capable of compensating for spherical aberrations, defocus, astigmatism, or coma of the optical system. Which change in the manipulation of the adaptive optics provides a particularly large contribution to approaching the corresponding extreme value of the indices according to the invention depends on the specific optical system and, in particular, the specific sample. Generally, compensating for only one of these aberrations is sufficient to keep the imaging error of the optical system very small. This is especially likely to be spherical aberration.

[0029] The light whose intensity in the focused region affects the measurement signal over time can be selected from light that causes the measurement signal to decrease exponentially to zero over time, or light that causes the measurement signal to approach a saturation value exponentially from below over time. For example, if the measurement signal is fluorescence emitted by a fluorescent dye in the sample, the selected light could be an excitation light that excites the fluorescent dye in the focused region into a fluorescent state until the intensity of the fluorescence produced by the fluorescent dye approaches a saturation value.

[0030] If a fluorescent dye is excited into a fluorescent state by an excitation light attached to the light in the focal region, and the fluorescent dye emits fluorescence from this fluorescent state as measurement light, the selected light can be a stimulus light, which stimulates the fluorescent dye to be excited and emitted before the fluorescent dye emits fluorescence. Then, the remaining fluorescence after the stimulus light has taken effect depends non-linearly on the intensity of the stimulus light. Specifically, the additional excitation light and the stimulus light can be focused together in the focal region of the sample by the optical system. Here, if the intensity distribution of the selected light has a minimum intensity at the center that coincides with the maximum intensity at the center of the excitation light in the focal region, then there exists an intensity distribution of excitation and stimulus light as it is also used in STED microscopy. Thus, the method according to the invention achieves correction for imaging errors induced by chromatic aberration in the imaging system, which negatively affect the effective point tailing function when the corresponding sample is excited to emit fluorescence.

[0031] Then, specifically, if, during the execution of the method according to the invention, the third time period begins before the first time period and the intensity of the light in the intensity maximum adjacent to the central intensity minimum in the intensity distribution of the stimulus light is so high that more than 50%, preferably more than 66%, or even more preferably more than 90%, of the measured signal originates from the surrounding environment of the intensity minimum, then the method according to the invention precisely corrects for chromatic aberration-induced imaging errors in this surrounding environment of the intensity minimum, the size of which in at least one spatial direction is smaller than the diffraction limit in both the light and fluorescence wavelengths. Although this surrounding environment surrounds the intensity minimum (which corresponds to the sample points measured separately in the STED microscope) but does not itself include it, this also means correcting for chromatic aberration-induced imaging errors for the separately measured sample points. This correction is even highly specific for the separately measured sample points because the size of the surrounding environment is already smaller than the diffraction limit in the wavelengths of light and fluorescence.

[0032] Since the requirements for excitation and stimulation light when performing the embodiments of the method described last here according to the invention can completely correspond to the requirements in an STED microscope, the method according to the invention can be performed during continuous STED microscopic measurements or during STED microscopic image recording. Specifically, if photons are recorded in a time-resolved manner, so that photons can be allocated to first and second time periods, then the fluorescence photons recorded for the corresponding measurement or image recording can be used to perform the method according to the invention. Obviously, the method according to the invention can also be performed before the optical system is used for actual imaging of the sample.

[0033] In an embodiment of the method according to the invention, similar to that of the STED microscope, the metric determined as the quotient (the first measurement is in the denominator of the quotient and the second measurement is in the numerator of the quotient) should be used as the index to be minimized. This means that when the corresponding new metric determined as the quotient is less than the corresponding old metric determined as the quotient, the control of the adaptive optics should be changed again in the previous direction of change, because the smaller index indicates a smaller imaging error induced by chromatic aberration.

[0034] Therefore, the difference becomes apparent from methods that, in order to adjust the STED microscope, take the brightness of the recorded image, i.e., the intensity of the fluorescence remaining after the stimulus light has taken effect, as the metric to be maximized. In contrast, the method according to the invention, in its embodiment similar to the STED microscope, aims to prevent fluorescence emission as much as possible throughout the focused area. This requires the maximum intensity of the stimulus light in the focused area, as it is achieved only when there are no chromatic aberration-induced imaging errors. Here, the emission of residual fluorescence from the minimum intensity distribution of the stimulus light (which may even increase as chromatic aberration-induced imaging errors decrease) does not interfere with the function of the method according to the invention.

[0035] In the method according to the invention, the corresponding first time period, the corresponding second time period, and the corresponding third time period can be independently closed time periods or composed of temporally spaced partial time periods. The third time period being composed of partial time periods can, for example, mean that light is directed into the focused region in the form of multiple very rapid, sequential pulses, which are indistinguishable when recording the measurement signal. The first time period and / or the second time period being composed of partial time periods can mean that the measurement signal from the sample is recorded in multiple temporally sequential partial time periods, with dead time intervals for recording the measurement signal between these partial time periods.

[0036] In a particular embodiment of the method according to the invention, the first, second, and third time periods being composed of partial time periods means that, before the second partial time periods of the first, second, and third time periods, and before any subsequent partial time periods, the focused area shifts slightly within the sample between each partial time period of the first, second, and third time periods. In this way, the measurement signal is averaged over the spatial region of the sample during detection and, if necessary, integration. This has proven particularly advantageous in embodiments of the method according to the invention, particularly in near-STED microscopy, to average the effect of fluorescent dye concentrations fluctuating at high spatial frequencies within the sample on the metric used as an indicator.

[0037] In other words, in the method according to the invention, detecting and integrating the measurement signal during the same first and second time periods (wherein, focusing the light onto the focal region in the sample during the same third time period) can be performed for multiple image points to obtain corresponding first and second measurement values.

[0038] In a laser scanning microscope according to the invention, having a first light source for excitation light, a second light source for stimulation light, a lens, and a correction device for correcting imaging errors induced by chromatic aberration (the correction device including an adaptive optics element in the beam path passing through the lens), the correction device is configured to perform the following steps of the method according to the invention: detecting a measurement signal during a first time period and a second time period, wherein, during a third time period, the stimulation light is focused as light that reduces the measurement signal when acting on the sample into a focal region in the sample to determine a first measurement value and a second measurement value; determining a metric value from the first measurement value and the second measurement value; and using the metric value as an indicator to be optimized by changing the manipulation of the adaptive optics element.

[0039] The adaptive optics of the laser scanning microscope according to the invention may, for example, have adaptive mirrors and / or tunable micromirror arrays and / or SLMs (Spatial-Light-Modulators). In particular, the SLM can also be used in a method according to the invention, in an embodiment similar to an STED microscope, in such a way that the stimulus light is configured with an intensity distribution having a central minimum intensity value in the focused region.

[0040] In another method according to the invention for correcting chromatic aberration-induced imaging errors in an optical system, the optical system has a lens and adaptive optics in a beam path passing through the lens, wherein a first light, a second light, and a sample are selected such that the first light, when acting on the sample, has a first conversion probability. A first measurement signal is excited from a component of the sample, the first conversion probability depending on the intensity of the first light with a first power of 1, and a second light, when acting on the sample, excites a first or second measurement signal from the same component of the sample with a second conversion probability depending on the intensity of the second light with a second power of 2, wherein the first and second powers differ by at least one. The first light is then focused onto a focal region of the optical system within the sample using an optical system, wherein the first measurement signal excited by the first light from the focal region is detected during a first time period to determine a first measurement value. Furthermore, a second light is focused onto a focal region of the sample using an optical system, wherein the first or second measurement signal excited by the second light from the focal region of the sample is detected during a second time period to determine a second measurement value. A metric is then determined from the first and second measurement values, the metric being a strictly 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, and serves as an indicator that can be optimized by changing the control when manipulating the adaptive optics.

[0041] In the method according to the invention, the different dependencies of the first and second conversion probabilities on the intensities of the first and second light when the corresponding measurement signals are excited are fully utilized to determine a metric to be used as an optimization index when manipulating the adaptive optics. In its simplest form, the metric is also determined in the method according to the invention by forming a quotient between the first and second measurement values, which is a strictly 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. Since the first and second conversion probabilities depend on the light intensity with a difference of at least one first and second power, and the intensities of the first and second light depend in the same way on the imaging error induced by chromatic aberration, this quotient depends on the imaging error. Conversely, all other effects on the two measurement values ​​are incorporated into a constant factor of the quotient, which does not change with the imaging error induced by chromatic aberration when determining the first and second measurement values ​​for the same focal regions respectively. This principle also applies even when the first light excites other components of a sample in the same or even different focal regions to emit the first measurement signal compared to the second light. Since, in the method according to the invention, the corresponding measurement signal is excited by a first light or a second light, and is excited by the same components of the sample in the same focal region, the measurement value becomes substantially independent of the observation position of the sample. The measurement signal excited by the second light can be the same measurement signal excited by the first light, which simplifies the execution of the method according to the invention, but it can also involve a second measurement signal, for example, involving measurement light of a different wavelength.

[0042] Specifically, the first light can excite a first measurement signal of a component of the sample through a single-photon process, while the second light can excite a first or second measurement signal of the same component of the sample through multi-photon excitation.

[0043] Other preferred embodiments of the method according to the present invention correspond to the embodiments of the method according to the present invention first described above.

[0044] In another method according to the invention for correcting chromatic aberration-induced imaging errors in an optical system, the optical system has a lens and an adaptive optics device in the beam path passing through the lens, selecting light and a sample in the same manner as in the method according to the invention first described above, i.e., selecting such that the light, when acting on the sample, causes the measurement signal from the sample to either decrease or approach a saturation value from below, wherein the relative change in the measurement signal depends on the intensity of the light. Unlike the two methods according to the invention previously described, in this method, the light is provided with temporal optical modulation of its intensity and focused by means of the optical system onto a focal region in the sample. Here, the measurement signal from the focal region of the optical system in 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 then determined as a metric, which is a strictly monotonically increasing or decreasing function of the relative change in the measurement signal, and serves as an indicator when manipulating the adaptive optics device, which can be optimized by changing the manipulation, i.e., minimized or maximized.

[0045] The method according to the invention is equivalent in frequency space to the method according to the invention first described. The phase shift between optical modulation and signal modulation depends on the imaging error induced by chromatic aberration in a manner similar to the quotient derived from the second and first measurements in the method according to the invention first described above.

[0046] Therefore, most of the preferred embodiments of the method according to the invention described above are also preferred embodiments of the method according to the invention.

[0047] Advantageous extensions of the invention are derived from the patent claims, the specification, and the drawings. The advantages of features and combinations of features mentioned in the specification are merely exemplary and may occur alternatively or cumulatively, without necessarily requiring the acquisition of these advantages by embodiments according to the invention. Without altering the subject matter of the appended patent claims, the following applies to the disclosure of the original application and the patent: additional features can be extracted from the drawings, particularly the illustrated geometry and the relative dimensional relationships between the components, their relative arrangement, and effective connections. Combinations of features from different embodiments of the invention or from different patent claims may also differ from and be inspired by the selected referencing relationships in the patent claims. This also applies to features shown in separate drawings or mentioned in their description. These features may also be combined with features from different patent claims. Similarly, for further embodiments of the invention, features listed in the patent claims may be omitted.

[0048] The quantity of features mentioned in the patent claims and specification should be understood as exactly that quantity or a greater quantity than mentioned exists, without explicitly using the adverb "at least". Thus, for example, if referring to adaptive optics, this should be understood as exactly one, two, or more adaptive optics devices being present. Features listed in the patent claims may be supplemented by other features, or may be unique features possessed by the corresponding method or the corresponding laser scanning microscope.

[0049] The reference numerals included in the patent claims do not indicate any limitation on the scope of the subject matter protected by the patent claims. These reference numerals are used solely for the purpose of making the patent claims easier to understand. Attached Figure Description

[0050] The invention is further described and illustrated below based on the preferred embodiments shown in the accompanying drawings.

[0051] Figure 1 A laser scanning microscope according to the present invention is shown.

[0052] Figure 2 Showing according to Figure 1 The intensity distribution of excitation and stimulation light in the focal region of the lens of a laser scanning microscope.

[0053] Figure 3 The histogram of fluorescence photons recorded as a measurement signal in a laser scanning microscope is shown over time.

[0054] Figure 4 The graphs show the index according to the invention on different spherical aberrations (black squares) induced in a targeted manner and on the optimization according to the invention (white circles) of the index in free run.

[0055] Figure 5 The index according to the present invention is used when imaging dye-filled spheres. Figure 1 The pattern on the spherical aberration of the optical system of a laser scanning microscope.

[0056] Figure 6 Showing with Figure 5 Compared to the half-value width of an image of a sphere filled with dye on the same spherical aberration.

[0057] Figure 7 Showing with Figure 5 Compared to the fluorescence intensity of a small sphere filled with dye on the same spherical aberration.

[0058] Figure 8 The indicators according to the present invention are shown in accordance with Figure 1The process of defocusing changes in the optical system of a laser scanning microscope.

[0059] Figure 9 The indicators according to the present invention are shown in accordance with Figure 1 The process of spherical aberration changes in the optical system of a laser scanning microscope.

[0060] Figure 10 The indicators according to the present invention are shown in accordance with Figure 1 The process of astigmatism change in the optical system of a laser scanning microscope.

[0061] Figure 11 The indicators according to the present invention are shown in accordance with Figure 1 The process of change in coma of the optical system of a laser scanning microscope.

[0062] Figure 12 A bright image of a structure labeled with a fluorescent dye is shown, along with the corresponding changes in spherical aberration according to the indices of the invention.

[0063] Figure 13 Shown with Figure 12 The same less bright image of the same structure and the related changes in the index according to the invention on the same spherical aberration.

[0064] Figure 14 Shown with Figure 12 and 13 Dark images of the same structure and the related changes in the indices according to the invention on the same spherical aberration.

[0065] Figure 15 In another embodiment of the invention, according to Figure 1 The intensity distribution of excitation and stimulation light in the focal region of the lens of a laser scanning microscope.

[0066] Figure 16 The different time sequences are shown: a first time period for integrating the measurement signal to form a first measurement value, a second time period for integrating the measurement signal to form a second measurement value, and a third time period for focusing the selected light onto the focal region of the corresponding optical system.

[0067] Figure 17 This is a flowchart of the basic steps of the method according to the present invention.

[0068] Figure 18 This is a flowchart of the basic steps of another method according to the present invention.

[0069] Figure 19 This is a flowchart of the basic steps of another method according to the present invention. Detailed Implementation

[0070] exist Figure 1 The laser scanning microscope 1 according to the invention shown is based on a STED microscope and accordingly has a second light source 4 in addition to the first light source 2 for excitation light 3. The second light source 4 has light 5 in the form of stimulation light 6, which is used to eliminate the excitation caused by the excitation light 3. Each of the two light sources 2 and 4 includes a laser 7 or 8, a polarization-maintaining fiber 9 or 10, and a collimating optics 11 or 12 for the excitation light 3 or stimulation light 6 emitted from the fiber 9 or 10. A wavefront shaping SLM 13 is also arranged in the beam path of the stimulation light 6 so that the stimulation light 6 forms an intensity distribution with a central minimum intensity in the focusing region of the lens 14. After the SLM 13 and another optics 15, the stimulation light 6 is combined with the excitation light 3 by means of a dichroic beam splitter 16. Excitation light 3 and stimulation light 6 are coupled into lens 14 via deformable mirror 17 (acting as adaptive optics 18) and another optics 19. This lens focuses the excitation light 3 and stimulation light 6 together into a focal region within sample 20. Optics 15 and 19 are configured such that deformable mirror 17 and SLM 13 lie in a plane conjugate to the entrance pupil of lens 14. The position of the focal region focused by excitation light 3 and stimulation light 6 can be shifted within sample 20 using scanner 21. Figure 1 In the diagram, scanner 21 is shown as a movable sample stage. However, scanner 21 can also be constructed differently, and in particular, it can be constructed to displace excitation light 3 and stimulation light 6 relative to lens 14, especially to tilt the center of the entrance pupil of lens 14. Fluorescence 22 emitted from the focal region in sample 20 due to the excitation of a fluorescent dye located there by means of excitation light 3 is coupled out from the beam path of excitation light 3 by means of a second dichroic beam splitter 23, focused into multimode fiber 25 by means of optics 24, and guided through the multimode fiber to time-resolved detector 26. In the laser scanning microscope 1 according to the invention, deformable mirror 17, as an adaptive optics device 18 arranged in the beam path passing through lens 14, is controlled by controller 27, to which the detector 26 transmits a measurement signal 28 displaying fluorescence. Instead of deformable mirror 17, adaptive optics device 18 can also be constructed by SLM, micromirror array, or other methods known to those skilled in the art.

[0071] Figure 2 The intensity distributions 29 and 30 of the excitation light 3 and stimulation light 6 along the x-direction through the center of the focusing region are shown. The intensity distribution 29 of the excitation light is the intensity distribution of the diffraction-limited spot. The spatially observed annular intensity distribution 30 of the stimulation light has a minimum intensity value at center 31 in the form of a zero point 32, which is determined according to... Figure 2The cross-section is bounded by the maximum intensity of 33. Within the maximum intensity of 33, the intensity of the stimulus light is higher than the saturation intensity I. S This saturation intensity leads to complete reduction via excitation light 3 caused by the fluorescent dye. Accordingly, based on... Figure 1 The fluorescence 22 recorded by detector 26 can be assigned to the surrounding environment 34 of zero point 32, the size of which is much smaller than the diffraction limit. However, this applies only to fluorescence 22 that is recorded only if the stimulation light 6 has eliminated the excitation caused by the excitation light 3 outside the surrounding environment 34 of zero point 32.

[0072] Figure 3 This shows that after the excitation pulse 35 and the subsequent stimulation pulse 36, individual photons of fluorescence 22 are injected into the circuit according to... Figure 1 The time at detector 26. First, during pulse 35, the number of photons recorded per unit time increases sharply due to excitation caused by excitation light 3. Then, during pulse 36, the number of photons recorded per unit time decreases sharply due to the effect of stimulation light. Then, the photons recorded by fluorescence 22 in time period 37 after pulse 36 are photons from the surrounding environment 34 at zero point 32. In the method according to the invention, the number of photons recorded by fluorescence 22 in time period 37 is correlated with the number of photons recorded by fluorescence 22 in an earlier time period 38. This earlier time period 38 is also referred to herein as the first time period, and time period 37 as the second time period. According to Figure 3 Pulses 35 and 36 fall within the first time period 38. This correlation between the photons recorded by fluorescence 22 in the second time period 37 and the photons recorded in the first time period 38 is, according to the invention, achieved by determining a metric (Maβzahl), particularly by forming a quotient between the photons recorded in the second time period 37 and the photons recorded in the first time period 38. Here, the invention is based on the knowledge that the metric has an extreme value when the controller 27 manipulates the adaptive optics 18 such that the chromatic aberration-induced imaging error of the optical system of the laser scanning microscope 1, including the lens 14, is exactly compensated by the adaptive optics 18. According to... Figure 1The chromatic aberration-induced imaging error of the optical system of the laser scanning microscope 1 affects the intensity of the excitation light 3 and the stimulation light 6 in the focused region of the sample 20 in such a way that the intensity decreases with increasing imaging error. Therefore, due to the increased imaging error, the excitation of the fluorescent dye in the sample 20, and thus fluorescence 22, also decreases with increasing excitation light intensity. However, this regression is not different from other regressions in the intensity of fluorescence 22, such as those caused by spatial changes in the concentration of the fluorescent dye or by bleaching of the fluorescent dye. However, if we consider the relative effect of stimulation light 6 on the fluorescent dye previously excited by excitation light 3, illustrated by the measure determined by the photons recorded in the two time periods 37 and 38, this relative effect depends only on the intensity of stimulation light 6. Compared to the photons in the first time period 38, fewer photons are recorded in the second time period 37. The stimulation light 6 excites more fluorescent dyes before the second time period 37, which is equivalent to the following: higher stimulation light intensity and smaller chromatic aberration-induced imaging errors. (Reference) Figure 2 The effect of imaging error induced by hue difference can also be explained in this way: when the intensity distribution 30 of the stimulus light 6 is reduced overall due to imaging error induced by hue difference, it only exceeds the saturation intensity I at a greater distance from zero point 32. S And correspondingly, more fluorescence from the surrounding environment 34 at 0.32 remains.

[0073] exist Figure 4 The index plotted is the quotient of the number of photons of fluorescence 22 recorded in the first time period 38 divided by the number of photons of fluorescence 22 recorded in the second time period 37. Therefore, when relatively fewer photons are recorded in the second time period 37 compared to the first time period 38, this index increases because the stimulation light 3 produces a stronger reduction effect on fluorescence 22 due to increased intensity. This is illustrated in a device with adaptive optics 18... Figure 4 The effect of six artificially introduced spherical aberrations (black squares) in the laser scanning microscope 1. Regardless of their sign, increased spherical aberration leads to an increase in the decrease of this index relative to its maximum value. Furthermore, by means of... Figure 1 The controller 27 manipulates a variant of the adaptive optics 18, and through the free-running optimization of this index according to the invention, the maximum value can be significantly increased relative to the initial case (white circle) when the artificially introduced aberrations are zero. For Figure 4 With the help of Figure 1 The index was created using a laser scanning microscope 1, in the case of 2D-STED recording of a layer of dye-filled microspheres of 40 nm in size, wherein time-resolved recordings were performed according to... Figure 3 The photons of fluorescence 22.

[0074] Figure 5 Is and in Figure 4 The same graph according to the present invention is used, wherein STED images of individual 40 nm dye-filled spheres are recorded for different spherical aberrations specifically introduced by means of adaptive optics 18. Figure 6 The corresponding half-width of the image for each ball is shown, and Figure 7 The fluorescence intensity of each sphere is shown, plotted on the same introduced aberration. Figures 5 to 7 The comparison shows that Figure 5 The maximum value of the index according to the present invention is in Figure 6 The minimum value of the half-value width is significantly wider and within the range of... Figure 7 The fluorescence intensity is reached within a wider range of maximum values. This is why the indicators are optimized according to the present invention for the elimination of [the fluorescence intensity]. Figure 1 When the optical system of the laser scanning microscope 1 experiences imaging errors induced by chromatic aberration, the control of the adaptive optics device 18 can be experimentally modified with the goal of optimizing performance without degrading the imaging quality of the laser scanning microscope 1. Figures 5 to 7 Among the standards plotted on spherical aberration, the index according to the invention reacts most quickly to imaging errors induced by chromatic aberration, thus indicating that the index has deviated from its extreme value in a identifiable manner before deviations from the extreme value can be seen in other standards and thus before the image quality deteriorates.

[0075] Figures 8 to 11 Showing according to Figure 4 and Figure 5 The influence of different aberrations on the indicators according to the present invention. Here, not only according to Figure 8 In the defocus, and according to Figure 9 In spherical aberration, and according to Figure 10 As astigmatism, and according to Figure 11 In the coma aberration, the global maximum value of the index is shown when the corresponding aberration is zero. Therefore, index optimization is applicable to eliminating imaging errors based on each of these four different aberrations.

[0076] exist Figures 12 to 14 The images shown below have different brightness levels and correspond to the same structure. Figure 4The relevant graphs of the index are plotted, that is, once with pre-given aberrations (black squares) and once under free-run optimization (white circles). The different brightness levels of the structure's image are based on different degrees of bleaching with a fluorescent dye used to mark the structure. Regardless of the image brightness, the index always shows its maximum value at the same, artificially introduced near-zero spherical aberration, and optimization of the index according to the invention is successful. Therefore, the elimination of chromatic aberration-induced imaging errors according to the invention is reliably performed independent of image brightness.

[0077] Figure 15 The diagram shows an alternative intensity distribution 30 for the stimulus light 6, relative to the intensity distribution 29 of the excitation light 3. Specifically, the stimulus light 6 also constructs a diffraction-limited spot. Even so, by changing according to Figure 1 The manipulation of the adaptive optics device 18 to optimize the quotient of early and late photons of fluorescence leads to the elimination of chromatic aberration-induced imaging errors. Furthermore, as in the intensity distribution 30 of the stimulus light 6, even when the stimulus light 6 does not reach the saturation intensity I... S This also applies.

[0078] Figure 16 The diagram illustrates the different sequences of a first time period 38 for integrating the measurement signal 28 of sample 20, a second time period 37 after further integrating the measurement signal 28, and a third time period 39 for focusing the light 5 acting on the measurement signal 28 onto the focal region of sample 20. The third time period 39 corresponds to... Figure 3 Pulse 36, in which the stimulation light 6 is directed onto the sample. According to Figure 3 Time period 37 directly follows time period 38, and time period 39 or pulse 36 overlaps with the portion following time period 38. According to... Figure 16 a) The order of time periods 37 and 38 is the same. However, the third time period 39 completely overlaps with the first time period 38. According to... Figure 16 (b) The order of time periods 37 and 38 is again the same. However, here, the third time period 39 overlaps not only with a portion of the first time period 38 but also with a portion of the second time period 37. According to Figure 16 c) The order of time periods 37 and 38 is again the same. However, here, the third time period 39 only overlaps with the second time period 37. According to Figure 16 d) The third time period 39 is arranged between time periods 37 and 38 without overlap. In any case, the light 5 focused on sample 20 in the third time period 39 has a stronger effect on the measurement signal integrated over the subsequent time period 37 than on the measurement signal integrated over time period 38, because light 5 is always further reduced, for example, according to Figure 1The measurement signal 28 is in the form of fluorescence 22. Therefore, only when the third time period 39 is completely before the first time period 38, is the measurement value obtained by integrating the measurement signal 28 in the first time period 38 not less affected by light 5 than the second measurement value obtained by integrating the measurement signal 28 in the second time period 37.

[0079] exist Figure 17 The flowchart 40 of the method according to the invention shown in the figure begins with the selection 41 of light 5 and sample 20, such that when light 5 acts on the sample, it either reduces the measurement signal 28 from sample 20 or approaches a saturation value from below, wherein the change in measurement signal 28 depends on the intensity of light 5. Then, at detection 42, the measurement signal from the focused region in sample 20 of the optical system (which will correct for imaging errors induced by chromatic aberration of the optical system) is detected and integrated, for example, in a first time period 38 to determine a first measurement value, and then detected and integrated, for example, in a subsequent second time period 37 to determine a second measurement value, wherein, in a third time period 39, light 5 is focused in the focused region in sample 20 by means of the optical system. In determination 43, a new metric value is determined from the first and second measurement values, for example, in the form of a new quotient, and then, in determination 44, the difference between the new metric value and the earlier metric value is determined. Depending on the direction of the difference in the metric value, when changing 45 the operation of the adaptive optics 18, the direction of the earlier change in the operation of the adaptive optics 18 is retained or switched. Steps 42 to 45 are repeated in loop 46. During these repetitions, the focused area of ​​the optical system in sample 20 can be shifted to optimally compensate for chromatic aberration-induced imaging errors of the optical system with adaptive optics system 18 for different positions of the focused area in sample 20 consecutively. Even during the detection of measurement signal 28 at 42 to determine the first and second measurements, the focused area in sample 20 can be shifted to perform spatial averaging. For this purpose, time periods 37 to 39 are divided into corresponding sub-time periods, one of which is assigned to a corresponding position of the focused area in sample 20.

[0080] Figure 18 Showing relative to based Figure 17The method described is a modified flowchart 40. In selection 47, the first light, the second light, and the sample are selected such that the first light, when acting on the sample 20, excites a first measurement signal of a component of the sample with a first conversion probability, and the second light, when acting on the sample, excites a second measurement signal of the same component, i.e., in particular the same fluorescent dye, with a second conversion probability. The first conversion probability depends on the intensity of the first light by a first power, and the second conversion probability depends on the intensity of the second light by a second power, wherein the first and second powers differ by at least one. Then, in focusing 48, the first light is first focused into the focused region of the optical system in the sample using an optical system, wherein the first measurement signal 38 excited by the first light from the focused region is detected during a first time period 38 to determine a first measurement value. Then, in another focusing 49, the second light is focused into the same focused region in the sample 20 using an optical system, wherein the second measurement signal excited by the second light from the focused region in the sample is detected during a second time period 37 to determine a second measurement value. Then, in determination 43, the second measurement signal is determined in conjunction with the measurement signal based on the first light. Figure 7 The method according to the invention, as described herein, determines, for example, a new metric value in the form of a new quotient, and then, in determination 44, determines the difference between the new metric value and the earlier metric value, and in change 45, changes the operation of the adaptive optics device 18 in the same or different direction as before. Here, the steps repeated in loop 46 include steps 48, 49, and 43 to 45.

[0081] exist Figure 19 In another method according to the invention shown in flowchart 40, selection 41 corresponds to... Figure 17 Selection 41. However, the subsequent focusing 50 of the selected light is performed with temporal light modulation of its intensity, and the simultaneous detection of the measurement signal 28 is performed in a time-resolved manner. Then, in determination 51, the phase shift between the light modulation and the signal modulation of the measurement signal 28 is determined as a new metric. In determination 44, the difference between this new metric and the old metric is then determined, and the operation of the adaptive optics device 18 is changed 45 according to this difference. Here, loop 46 includes steps 50, 51, 44, and 45.

[0082] List of reference numerals

[0083] 1. Laser scanning microscope

[0084] 2 First Light Source

[0085] 3. Excitation light

[0086] 4 Second Light Source

[0087] 5 Light

[0088] 6. Stimulating light

[0089] 7. Laser

[0090] 8. Laser

[0091] 9 optical fibers

[0092] 10 optical fibers

[0093] 11 Collimating Optical Devices

[0094] 12 Collimating Optical Devices

[0095] 13 SLM

[0096] 14 lenses

[0097] 15 Optical Components

[0098] 16 Dihedral Beam Splitter

[0099] 17 Deformable Reflector

[0100] 18 Adaptive Optics

[0101] 19 Optical Devices

[0102] 20 samples

[0103] 21 Scanners

[0104] 22 Fluorescence

[0105] 23 Dichroic Beam Splitter

[0106] 24 Optical Devices

[0107] 25 multimode fiber

[0108] 26 detectors

[0109] 27 Controller

[0110] 28 Measurement Signal

[0111] 29. Light Intensity Distribution

[0112] 30 Light Intensity Distribution

[0113] 31 Center

[0114] 32 midnight

[0115] 33 Maximum strength value

[0116] 34 Surrounding Environment

[0117] 35 pulses

[0118] 36 pulses

[0119] 37 Second time period

[0120] 38 First Time Period

[0121] 39 Third time period

[0122] 40 Flowchart

[0123] 41 choices

[0124] 42 Detection

[0125] 43 Confirmed

[0126] 44 Confirmed

[0127] 45 Changes

[0128] 46 cycles

[0129] 47 choices

[0130] 48 Focus

[0131] 49 Focus

[0132] 50 Focus

[0133] 51 Confirmed

[0134] I S Saturation intensity

Claims

1. A method for correcting chromatic aberration-induced imaging errors in an optical system having a lens (14) and an adaptive optics (18) in a beam path passing through the lens (14). - in, The light (5) and the sample (20) are selected in such a way that when the light (5) acts on the sample (20), it reduces the measurement signal (28) from the sample (20). The relative change of the measurement signal (28) depends on the intensity of the light (5). The measurement signal (28) is fluorescence (22) emitted by the fluorescent dye in the sample (20). Wherein, the light (5) is a stimulating light (6), which stimulates the fluorescent dye to emit fluorescence (22) before the fluorescent dye emits fluorescence. The fluorescent dye is excited into a fluorescent state in the focal region of the optical system in the sample (20) by the excitation light (3) attached to the light (5), and the fluorescent dye emits fluorescence (22) from the fluorescent state. - Wherein, during a first time period (38), a measurement signal (28) from the focused region of the optical system in the sample (20) is detected in order to determine a first measurement value in such a way that the first measurement value represents the first integral of the measurement signal (28) over the first time period (38). - During the second time period (37), a measurement signal (28) from the focused area of ​​the optical system in the sample (20) is detected to determine a second measurement value such that the second measurement value represents the second integral of the measurement signal (28) over the second time period (37). - During the third time period (39), the light (5) is focused into the focal region of the sample (20) using the optical system. - wherein the first time period (38) is at least partially earlier than the second time period (37) and / or the second time period (37) is at least partially later than the first time period (38), and the third time period (39) overlaps at least partially in time with the first time period (38) and / or with the second time period (37) and / or with an intermediate time period located between the first time period and the second time period, such that the first measurement is less strongly affected by the light (5) focused into the focal region in the third time period (39) compared to the second measurement, and, - Wherein, a metric value is determined by the first measurement value and the second measurement value, wherein the metric value is the quotient of the second measurement value and the first measurement value, the metric value is a strictly monotonically increasing or decreasing function of the relative change of the measurement signal (28), the metric value is used as an indicator when manipulating the adaptive optics device (18), the indicator can be maximized or minimized by changing (45) the manipulation in order to maximize the intensity of the light (5) in the focused region, wherein the direction of the change in the manipulation of the adaptive optics device (18) is selected from those that can compensate for: - Spherical aberration, - Defocus, - Astigmatism, - coma The change of direction.

2. The method according to claim 2, characterized in that, The fluorescence (22) is imaged onto the detector (26) using the optical system.

3. The method according to claim 1 or 2, characterized in that, - Determine the old metric. - Change the manipulation of the adaptive optics device (18) in a change of direction. - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine the difference between the new and old metrics, and, - Depending on the direction of the difference, the manipulation of the adaptive optics device (18) is changed again in either the previous change direction or another change direction.

4. The method according to claim 3, characterized in that, The following steps: - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine (44) the difference between the new and old measures, and, - Depending on the direction of the difference, the manipulation of the adaptive optics (18) is changed again (45) in either the previous change direction or another change direction. The operation is performed at least once more or so many times until the following manipulation of the adaptive optics (18) is achieved in the direction of change: the metric value reaches an extreme value in the manipulation, wherein one of the new metric values ​​determined in one of the previous executions of the step is used as the old metric value when the step is currently performed.

5. The method according to claim 1, characterized in that, The additional excitation light (3) is focused into the focal region in the sample (20) by means of the optical system.

6. The method according to claim 5, characterized in that, The intensity distribution of the light (5) has a minimum intensity value at the center, which coincides with the maximum intensity value at the center of the excitation light (3) in the focusing region.

7. The method according to claim 5 or 6, characterized in that, If the new metric value determined by the quotient is less than the corresponding old metric value determined by the quotient, wherein these quotients have a corresponding first metric value in the denominator and a corresponding second metric value in the numerator, then the manipulation of the adaptive optics device (18) is changed again in the direction of change up to the present.

8. The method according to claim 1 or 2, characterized in that, The first time period (38), the second time period (37) and the third time period (39) are each a closed time period independently of each other, or are composed of partial time periods that are separated from each other in time.

9. The method according to claim 1 or 2, characterized in that, The measurement signal (28) is detected for multiple image points in order to determine the corresponding metric value, wherein the light (5) is focused into the focal region in the sample (20).

10. A method for correcting chromatic aberration-induced imaging errors in an optical system having a lens (14) and an adaptive optics (18) in a beam path passing through the lens (14). - in, The light (5) and the sample (20) are selected such that when the light (5) acts on the sample (20), the measurement signal (28) from the sample (20) approaches a saturation value from below. The relative change of the measurement signal (28) depends on the intensity of the light (5). The measurement signal (28) is fluorescence (22) emitted by the fluorescent dye in the sample (20). In this process, the fluorescent dye is excited into a fluorescent state in the focal region of the optical system in the sample (20) by the light (5) until it approaches the saturation value, at which point the fluorescent dye emits fluorescence (22) from the fluorescent state. - Wherein, during a first time period (38), a measurement signal (28) from the focused region of the optical system in the sample (20) is detected in order to determine a first measurement value in such a way that the first measurement value represents the first integral of the measurement signal (28) over the first time period (38). - During the second time period (37), a measurement signal (28) from the focused area of ​​the optical system in the sample (20) is detected to determine a second measurement value such that the second measurement value represents the second integral of the measurement signal (28) over the second time period (37). - During the third time period (39), the light (5) is focused into the focal region of the sample (20) using the optical system. - wherein the first time period (38) is at least partially earlier than the second time period (37) and / or the second time period (37) is at least partially later than the first time period (38), and the third time period (39) overlaps at least partially in time with the first time period (38) and / or with the second time period (37) and / or with an intermediate time period located between the first time period and the second time period, such that the first measurement is less strongly affected by the light (5) focused into the focal region in the third time period (39) compared to the second measurement, and, - Wherein, a metric value is determined by the first measurement value and the second measurement value, wherein the metric value is the quotient of the second measurement value and the first measurement value, the metric value is a strictly monotonically increasing or decreasing function of the relative change of the measurement signal (28), the metric value is used as an indicator when manipulating the adaptive optics device (18), the indicator can be maximized or minimized by changing (45) the manipulation, so as to maximize the intensity of the light (5) in the focused region, wherein the direction of the change in the manipulation of the adaptive optics device (18) is selected from those that can compensate for: - Spherical aberration, - Defocus, - Astigmatism, - coma The change of direction.

11. The method according to claim 10, characterized in that, The fluorescence (22) is imaged onto the detector (26) using the optical system.

12. The method according to claim 10 or 11, characterized in that, - Determine the old metric. - Change the manipulation of the adaptive optics device (18) in a change of direction. - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine the difference between the new and old metrics, and, - Depending on the direction of the difference, the manipulation of the adaptive optics device (18) is changed again in either the previous change direction or another change direction.

13. The method according to claim 12, characterized in that, The following steps: - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine (44) the difference between the new and old measures, and, - Depending on the direction of the difference, the operation of the adaptive optics device (18) is changed again (45) in either the previous change direction or another change direction. The operation is performed at least once more or so many times until the following manipulation of the adaptive optics (18) is achieved in the direction of change: the metric value reaches an extreme value in the manipulation, wherein one of the new metric values ​​determined in one of the previous executions of the step is used as the old metric value when the step is currently performed.

14. The method according to claim 10 or 11, characterized in that, The first time period (38), the second time period (37) and the third time period (39) are each a closed time period independently of each other, or are composed of partial time periods that are separated from each other in time.

15. The method according to claim 10 or 11, characterized in that, The measurement signal (28) is detected for multiple image points in order to determine the corresponding metric value, wherein the light (5) is focused into the focal region in the sample (20).

16. A method for correcting chromatic aberration-induced imaging errors in an optical system having a lens (14) and an adaptive optics (18) in a beam path passing through the lens (14). - in, The first light (5), the second light, and the sample (20) are selected such that the first light (5), when acting on the sample (20), excites a first measurement signal (28) from a component of the sample (20) with a first conversion probability, the first measurement signal (28) being a measurement light emitted from the sample (20), the first conversion probability depending on the intensity of the first light (5) by a first power, and the second light, when acting on the sample (20), excites either the first measurement signal or the second measurement signal (28) from the same component of the sample (20) with a second conversion probability, the second measurement signal being a measurement light of another wavelength, the second conversion probability depending on the intensity of the second light by a second power, wherein the first power and the second power differ by at least 1, wherein the first light (5) excites the first measurement signal (28) from a component of the sample (20) through a single-photon process, while the second light excites either the first measurement signal or the second measurement signal (28) from the same component of the sample (20) through a multi-photon process. - Wherein, the first light (5) is focused into the focal region of the optical system in the sample (20) by means of the optical system, wherein a first measurement signal (28) excited by the first light (5) from the focal region is detected during a first time period (38) in order to determine a first measurement value in such a way that the first measurement value shows the first integral of the measurement signal (28) over the first time period (38). - Wherein, the second light is focused into the focused region in the sample (20) by means of the optical system, wherein, during the second time period (37), a first or second measurement signal (28) excited by the second light from the focused region in the sample (20) is detected in order to determine a second measurement value in such a way that the second measurement value shows a second integral of the measurement signal (28) over the second time period (37). - Wherein, a metric is determined by the first measurement and the second measurement, wherein the metric is the quotient of the second measurement and the first measurement, the metric is a strictly monotonically increasing or decreasing function of the relative change of the first measurement signal (28), or the metric is a strictly monotonically increasing or decreasing function of the relative difference between the first measurement signal and the second measurement signal (28), the metric is used as an indicator when manipulating the adaptive optics (18), the indicator can be maximized or minimized by changing (45) the manipulation in order to maximize the intensity of the light (5) in the focused region, wherein the direction of the change in the manipulation of the adaptive optics (18) is selected from those that can compensate for: - Spherical aberration, - Defocus, - Astigmatism, - coma The change of direction.

17. The method according to claim 16, characterized in that, The measurement light is imaged onto the detector (26) using the optical system.

18. The method according to claim 16 or 17, characterized in that, The first time period (38) and the second time period (37) are each a closed time period, or are composed of time periods that are separated from each other in time.

19. A method for correcting chromatic aberration-induced imaging errors in an optical system having a lens (14) and an adaptive optics (18) in a beam path passing through the lens (14). - in, The light (5) and the sample are selected in such a way that when the light (5) acts on the sample (20), it reduces the measurement signal (28) from the sample (20). The relative change of the measurement signal (28) depends on the intensity of the light (5), wherein the measurement signal (28) is fluorescence (22) emitted by the fluorescent dye in the sample (20). Wherein, the light (5) is a stimulating light (6), which stimulates the fluorescent dye to emit fluorescence (22) before the fluorescent dye emits fluorescence. The fluorescent dye is excited into a fluorescent state in the focal region of the optical system in the sample (20) by the excitation light (3) attached to the light (5), and the fluorescent dye emits fluorescence (22) from the fluorescent state. - Wherein, the light (5) is provided with temporal light modulation of the intensity of the light, and the light is focused into a focal region in the sample (20) by means of the optical system. - Detect the measurement signal (28) from the focused area of ​​the optical system in the sample (20) in a time-resolved manner. - Wherein, the phase shift between the light modulation and the signal modulation of the measurement signal is determined as a metric, which is a strictly monotonically increasing or decreasing function of the relative change of the measurement signal (28), and the metric is used as an indicator when manipulating the adaptive optics (18), which can be maximized or minimized by changing (45) the manipulation in order to maximize the intensity of the light (5) in the focused region, wherein the direction of the change in the manipulation of the adaptive optics (18) is selected from those that can compensate for: - Spherical aberration, - Defocus, - Astigmatism, - coma The change of direction.

20. The method according to claim 19, characterized in that, The fluorescence (22) is imaged onto the detector (26) using the optical system.

21. The method according to claim 19 or 20, characterized in that, - Determine the old metric. - Change the manipulation of the adaptive optics device (18) in a change of direction. - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine the difference between the new and old metrics, and, - Depending on the direction of the difference, the manipulation of the adaptive optics device (18) is changed again in either the previous change direction or another change direction.

22. The method according to claim 21, characterized in that, The following steps: - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine (44) the difference between the new and old measures, and, - Depending on the direction of the difference, the manipulation of the adaptive optics (18) is changed again (45) in either the previous change direction or another change direction. The operation is performed at least once more or so many times until the following manipulation of the adaptive optics (18) is achieved in the direction of change: the metric value reaches an extreme value in the manipulation, wherein one of the new metric values ​​determined in one of the previous executions of the step is used as the old metric value when the step is currently performed.

23. The method according to claim 19, characterized in that, The additional excitation light (3) is focused into the focal region in the sample (20) by means of the optical system.

24. The method according to claim 23, characterized in that, The intensity distribution of the light (5) has a minimum intensity value at the center, which coincides with the maximum intensity value at the center of the excitation light (3) in the focusing region.

25. The method according to claim 23 or 24, characterized in that, If the new metric value determined by the quotient is less than the corresponding old metric value determined by the quotient, wherein these quotients have a corresponding first metric value in the denominator and a corresponding second metric value in the numerator, then the manipulation of the adaptive optics device (18) is changed again in the direction of change up to the present.

26. The method according to claim 19 or 20, characterized in that, The measurement signal (28) is detected for multiple image points in order to determine the corresponding metric value, wherein the light (5) is focused into the focal region in the sample (20).

27. A method for correcting chromatic aberration-induced imaging errors in an optical system having a lens (14) and an adaptive optics (18) in a beam path passing through the lens (14). - in, The light (5) and the sample are selected such that when the light (5) acts on the sample (20), the measurement signal (28) from the sample (20) approaches a saturation value from below. The relative change of the measurement signal (28) depends on the intensity of the light (5). The measurement signal (28) is fluorescence (22) emitted by the fluorescent dye in the sample (20). In this process, the fluorescent dye is excited into a fluorescent state in the focal region of the optical system in the sample (20) by the light (5) until it approaches the saturation value, at which point the fluorescent dye emits fluorescence (22) from the fluorescent state. - Wherein, the light (5) is provided with temporal light modulation of the intensity of the light, and the light is focused into the focused region in the sample (20) by means of the optical system. - Detect the measurement signal (28) from the focused area of ​​the optical system in the sample (20) in a time-resolved manner. - Wherein, the phase shift between the light modulation and the signal modulation of the measurement signal is determined as a metric, which is a strictly monotonically increasing or decreasing function of the relative change of the measurement signal (28), and the metric is used as an indicator when manipulating the adaptive optics (18), which can be maximized or minimized by changing (45) the manipulation in order to maximize the intensity of the light (5) in the focused region, wherein the direction of the change in the manipulation of the adaptive optics (18) is selected from those that can compensate for: - Spherical aberration, - Defocus, - Astigmatism, - coma The change of direction.

28. The method according to claim 27, characterized in that, The fluorescence (22) is imaged onto the detector (26) using the optical system.

29. The method according to claim 27 or 28, characterized in that, - Determine the old metric. - Change the manipulation of the adaptive optics device (18) in a change of direction. - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine the difference between the new and old metrics, and, - Depending on the direction of the difference, the manipulation of the adaptive optics device (18) is changed again in either the previous change direction or another change direction.

30. The method according to claim 29, characterized in that, The following steps: - The measurement signal (28) is detected again, wherein the light (5) is refocused onto the focal region in the sample (20). - Determine the new metric. - Determine (44) the difference between the new and old measures, and, - Depending on the direction of the difference, the manipulation of the adaptive optics (18) is changed again (45) in either the previous change direction or another change direction. The operation is performed at least once more or so many times until the following manipulation of the adaptive optics (18) is achieved in the direction of change: the metric value reaches an extreme value in the manipulation, wherein one of the new metric values ​​determined in one of the previous executions of the step is used as the old metric value when the step is currently performed.

31. The method according to claim 27 or 28, characterized in that, The measurement signal (28) is detected for multiple image points in order to determine the corresponding metric value, wherein the light (5) is focused into the focal region in the sample (20).

32. A laser scanning microscope (1), said laser scanning microscope having: - The first light source (2) used to excite light (3). - A second light source (4) for stimulating light (6). - Shot (14) and - A correction device for correcting imaging errors induced by chromatic aberration, the correction device comprising an adaptive optics (18) in the beam path passing through the lens (14). Its features are, The calibration device is configured to perform the method according to any one of claims 1 to 31.

33. The laser scanning microscope (1) according to claim 32, characterized in that, The adaptive optics device (18) has - Adaptive reflector (17) and / or - Manipulable micromirror arrays and / or - Spatial light modulator (SLM).

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