Laser scanning microscope and method for correcting imaging errors particularly in high-resolution scanning microscopy

A laser scanning microscope (SR-LSM) and a method for correcting imaging errors in a laser scanning microscope. The SR-LSM includes an illumination device for providing an illumination spot; a scanner for moving the illumination spot to consecutive scanning positions over a sample to be examined; an adaptive optics unit for controlling a wavefront of the illumination spot with a control device and a detector for determining a spatially resolved imaging spot emitted by the sample. An evaluation unit is provided for determining a point-spread function (PSF) of the imaging spot at each scanning position, whereby a wavefront correction signal determined from the point-spread function (PSF) of a scanning position is supplied to the control device of the adaptive optics unit or is used in digital post-processing of the microscope image (e.g. by means of deconvolution).

FIELD OF THE INVENTION

The invention relates to a laser scanning microscope and to a method for correcting imaging errors, preferably in high-resolution scanning microscopy, particularly in scanning confocal microscopy.

BACKGROUND OF THE INVENTION

Various approaches are known from the prior art for increasing the lateral and axial resolution of such laser scanning microscopes even beyond the diffraction limit of the illumination light, and correcting imaging errors in the process.

Initially, a distinct contrast and resolution improvement was attained with confocal scanning microscopy. In so doing, a laser is used, which illuminates an object in the focal plane and excites fluorescence molecules at all points there. The fluorescence light is imaged on a pinhole in the image plane, only the light directly reaching the focal plane being detected. With confocal imaging, the lateral resolution (which is still diffraction-limited) can be greater by a factor of 1.4 than in conventional microscopy. This depends on the size of the pinhole. For this reason, the size of the pinhole must be optimized here to the size of the spot to be imaged. A pinhole that is too small reduces the amount of usable light, too large a pinhole leaves too much light outside the focal plane and admits too much scattered light.

Methods like InM and 4Pi microscopy improve the axial resolution through the use of two objective lenses with high numerical aperture and superposition of the images in the detection plane either by wide field or confocal laser fluorescence configuration, or by the use of multiple excitation light sources, patterns due to interference being projected onto the sample. The lateral resolution remains unchanged in the process.

Other scanning high-resolution methods are RESOLFT (reversible saturable optical (fluorescence) transitions) microscopy, wherein especially sharp images are obtained. Instead of using conventional objective lenses and diffracted beams, a resolution far beyond the diffraction limit is obtained, down to the molecular scale. RESOLFT microscopy overcomes this diffraction limit by temporarily switching the dyes into a condition wherein they are no longer able to respond with a (fluorescence) signal following illumination.

Special methods of non-scanning optical microscopy, more precisely of fluorescence microscopy, are known as the PALM (photoactivated localization microscopy) method or the STORM (stochastic optical reconstruction microscopy) method. They rely on light-controlled on-and-off switching of fluorescence in individual molecules. In the process, switching on and off is accomplished beyond a certain time interval, during which several individual images can be taken. By means of a subsequent computer calculation, the position of individual molecules can be defined with a resolution beyond the optical resolution limited described by Ernst Abbe.

A microscopy method with increased resolution is known from EP 2 317 362 A1, wherein the illumination or the illumination pattern is shifted, with respect to the detection, with an accuracy exceeding the achievable optical resolution, and several images are taken and evaluated during the shift.

Moreover, it has long been known to use adaptive optics, that is, optically active components, for wavefront modulation. The adaptive optics deliberately alters the phase and/or the amplitude of the light in such a manner that both shifting and forming of the focus in the space, as well as correction of aberrations, if any, can be accomplished. An axial shift of the is achieved by a change of the wavefront. Here, an axial shift of the focus corresponds to a spherical change of the wavefront, a lateral shift to tilting of the wavefront. Aberrations in the beam path are also compensated by changing the wavefront. These manipulations are carried out in an aperture plane of the beam path with the aid of deformable mirrors.

Such adaptive optics systems are described for example in EP 1 253 457 B1, US 7 224 23 B2 or JP 2008 026643 A.

The use of adaptive optics for correcting, for example, sample-induced wavefront errors (including wavefront errors due to the sample carrier and the immersion medium), is known from a multitude of publications.

The problem in implementing adaptive optics is always that a control signal is required for the adaptive optics. It is also necessary to initially specify the wavefront error before it can be removed. Generally, the wavefront error is not known.

Two solution approaches exist in the prior art: in the first solution, an additional measuring system is brought into the microscope so as to directly measure the wavefront error, e.g. by means of an ordinary and known Shack-Hartmann sensor. US 2004/0223214 A1 shows for example a microscope with a Hartmann-Shack wavefront sensor. From the shape of the wavefront, the aberrations which were caused by scattering of the light in the sample can be defined. Depending on performance (correction degrees of freedom of the correction element), various effects can thus be corrected. In elements with very many degrees of freedom, such as an SLM (spatial light modulator) for example, this corresponds to the number of controllable pixels. With such elements, not only aberrations (slowly varying wavefronts) of the system and of the sample, but also high-frequency components (scattered light) can be corrected, to the extent that these can still be measured at the wavefront sensor. In a particular image segment, depending on the scan position, this sometimes very high-frequency wavefront is thus corrected at the element. Consequently, the diffraction-limited performance of the system can also be attained in media or scattering samples, and particularly also for non-vanishing system aberrations of the microscope.

As a consequence, valuable photons only available in a finite number must be for a sensor; these photons are subsequently no longer available for the actual measurement.

In the second solution, the wavefront error is defined iteratively directly from the LSM signal, i.e. the wavefront is optimized until the LSM signal is optimal. For this purpose, a large number of iteration cycles is needed; 10 to 30 cycles are reported in the literature.

Fluorophors fade and can only emit about 50,000 photons. In both prior art solutions, therefore, the disadvantage arises that many of the few photons must be “sacrificed” for determining the control signal for the adaptive mirror.

In conventional scanning microscopes (other than STED), as a matter of principle, diffraction-limited imaging of the (ideally point-like) spot as a so-called Airy disk takes place, which is defined by a point response or point image function, or point-spread function—PSF. The PSF expresses how an idealized, point-like object is imaged by a system. What is problematic is that suitable sensors capable of imaging the Airy disk in the sub-mm range at the desired resolution are not currently available, and other techniques are either very expensive or very slow and are thus not suitable for commercial use.

In conventional scanning microscopy, the imaging spot is evaluated pixel-wise, i.e. for each spot position exactly one pixel is evaluation or one pixel of the total image results from each spot position. Here, only the overall beam intensity (integral) captured by the detector is defined and converted into grey levels. If applicable, suitable pixel or information superposition can take place for small scanning steps. Contrast improvement can be attained here by increasing the grey-scale resolution (color depth). The PMTs (photomultiplier tube) or PMT arrays employed exhibit internal noise at certain amplification ratios, with the result that signal quality declines.

In all these solutions, the problem typically arises that the aberrations vary at different positions on the sample and with different focus positions. These are unknown for the most part, or must be determined by elaborate measurements.

Due to the limited photon yield in fluorescence microscopy, additional measurement of the actual PSF has not been common (or only in wide-field microscopy) up until now.

In a laser scanning microscope, the theoretical PSF can be interfered with by various deviations of the system from the ideal condition or of the sample to be observed. The signal thus becomes interference-prone (noise, signal-to-noise ratio . . . ) and the maximum resolution is reduced.

An object of the invention is to create a scanning microscope and a method for location-dependent aberration correction in the preferably high-resolution scanning microscope.

This is achieved by a laser scanning microscope with the features of claim1and by a method with the features of claim6.

Advantageous modifications and exemplary embodiments are presented in the sub-claims.

The aberrations depend on various influential quantities. The aberrations of the optics of a scanning microscope are known and can be suitably corrected. Sample- and environmentally-dependent refractive index fluctuations, temperature fluctuations and various cover glass thicknesses vary to that effect according to the sample, but are largely constant for each sample. Properties of typical biological samples are often locally only slowly varying. One exception to this is imaging in deep samples, where stronger location-dependence exists due to scattering and a greater correction effort is consequently required.

SUMMARY OF THE INVENTION

The present invention solves the aforementioned problem. The SR-LSM signal (SR-LSM: super resolution laser scanning microscope), unlike an ordinary LSM signal, contains not only the photon count, but also information regarding the lateral intensity distribution of the PSF.

The integral over the intensity measured at the detector gives information regarding the grey value at the location (scanning position x, y), which is accomplished by the setting at the scanning mirror.

The form, or lateral resolution, of the PSF can now be used to define the aberrations in the system, which represent the deviation of the measured values compared to the ideal PSF without aberrations and, in a preferred embodiment, are corrected by means of an element (adaptive optics unit). To this end, the signals of the individual detectors of the detector array are evaluated. In alternative embodiments, the correction can also be implemented in software by means of digital post-processing of the microscope image, e.g. using deconvolution.

The PSF determined in this manner thus also contains the information regarding the wavefront error. Thus, conclusions can ideally also be reached from the PSF regarding the wavefront error. This can be accomplished purely by calculation, i.e. by iterating an assumed wavefront error until the calculated PSF is similar to the measured PSF. Using the correction value thus determined by calculation, the adaptive mirror is controlled in the preferred embodiment. In the ideal case, the wavefront error is already corrected in the second measurement. Adjoining field points always have similar wavefront errors, so that the second measurement can already take place at an adjoining field point (scanning position). The PSF then measured can then deviate from the ideal PSF due to the slight spatial variation of the wavefront error; from this slight deviation, an easily adjusted wavefront correction can again be determined by calculation.

At the next scanning position, the slightly corrected wavefront correction is then used for measuring. In iterative continuation of the method, it consequently follows that no photons need be “sacrificed” for the signal registration for controlling the adaptive optics, and the slowly varying wavefront errors that are present can nevertheless be corrected.

A laser scanning microscope according to the invention initially includes, in known fashion and arrangement, the components of an illumination device; scanner; adaptive optics unit; and detector.

Thus, the laser scanning microscope includes an illumination device for providing an illumination spot, a scanner for moving the illumination spot over a sample to be examined at sequential scanning positions, an adaptive optics unit for controlling a wavefront of the illumination spot with a control device, and a detector for capturing a spatially resolved imaging spot emitted by the sample.

According to the invention, the detector includes an evaluation unit for determining a point-spread function (PSFAbb) of the imaging spot in each scanning position. In the process, the point-spread function of the current scanning position is evaluated starting from the current, one or more prior or adjoining scanning positions, so as to control the adaptive optics unit with a control device.

Now the peculiarity of the detector is that it actually captures an imaging spot with spatial resolution very quickly in each scanning position. This means that the wavefront belonging to this PSF is determined from the two-dimensional image of the Airy disk of the imaging spot by means of a suitable evaluation. As the ideal PSF is known, not only a light yield (as before), but also aberrations such as for example defocus, coma, astigmatism can be extracted from this for each scanning position.

The resolution increase at the LSM with this special detector can be explained the total PSF of the LSM system, which contributes decisively to image formation. This is calculated from the product of the excitation PSF and detection PSF convolved with the transfer function of the pinhole
PSFTotal(x,y)=PSFIllumination(x,y)·[PSFDetection(x,y)*TPinhole(x,y)].
The maximum resolution is achieved if the transfer function of the pinhole corresponds to a delta function. Then the maximum resolution is achieved. With this transfer function, however, hardly any light goes through the detector. Consequently, in practice an expanded pinhole is always so as to obtain sufficient photons for a suitable signal-to-noise ratio. A resolution loss always accompanies this, however. In the SR LSM, the array detector is situated at the location of the confocal pinhole. Each pixel of the detector array corresponds to a very small pinhole. Despite this, altogether, no light is excluded. From the evaluation of the intensities at each pixel, the maximum resolution of the detector can now be improved. This procedure is explained in DE 10 2012 204 128 A1, not pre-published, the disclosure content whereof is included here in its full scope.

Here, what matters for the algorithm for extracting the correction wavefront is only the pixel- or spatially-resolved PSF.

In order to achieve sub-pixel resolution at this scale, the imaging spot is supplied to a detector array (PMT array) by means of an optical deflection element which subdivides the imaging spot into subpixels (fiber optics, micro-mirror array).

Preferably, the detector is an array of individual detectors. PMTs (photomultiplier tubes) or APDs (avalanche photodiodes) are especially suitable. The individual tubes of the PMTs have a diameter of about 0.8 to 2 mm. There are embodiments with8,16,32,64and more channels (tubes). In the arrangement in an array, a separation comparable to the microplates (MCP) is present between the tubes.

The optical deflection element preferably has only small separations between the individual elements (optical fibers, micro-mirrors), making it possible to better resolve the image information of the imaging spot. The light quantities of these individual elements are then directly supplied to the inputs of the individual detectors.

An especially preferred deflection element has at its input a bundle of optical fibers (alternatively, an array of micro-mirrors which can have different inclinations, facetted mirror, DMD or adaptive mirror), which are densely packed into a (nearly) circular cross-section. The cross-section must be matched to the imaging spot. The number of individual elements is to be selected according to the detector array used. Advantageously, a 9×9 PMT array can be used.

An output of the deflection element is configured so as to supply the light quantities of the individual elements to the individual detectors.

With the help of the two-dimensionally resolved Airy disk according to the invention, aberrations (even those of higher order) can be detected and the image representation can be adjusted (corrected) to the ideal PSFideal.

Preferably, the information of the Airy disk can be used, directly or after integration, in a suitable control loop for directing the adaptive optics unit to the next scan position, so as to thereby compensate optical aberrations caused by the sample.

In the process it is crucial that, for each scan position of the imaging spot captured in an individual image, capture occurs at a resolution which is at least twice as high, considering the imaging scale, as a full width at half maximum of the diffraction-limited individual image.

According to the invention, the use of adaptive optics is accomplished in synchronization with the scanning system. Thus, for each scanning position, a local deviation is compensated through the use of a specific correction function. The PSF is thereby improved, which in turn leads to an improved signal-to-noise ratio. Consequently, the option is available, not present in the wide field, of capturing even location-dependent aberrations by correcting the wavefront in every field (scanning) position.

The advantages of the invention are particularly considered to be that a rapid intensity measurement is possible using the novel detector (SR-LSM). This offers the singular possibility of accomplishing a rapid measurement of the PSF for a scan setting. In the process, the correction of the wavefront can be accomplished immediately (“on the fly”), or for example even in post-processing (subsequent deconvolution with the measured PSF). For both methods, it is important to know the PSF.

The signal of the detector is used for defining the deviations of the measured PSFAbbfrom an ideal PSF at each scanning position and making them available for correction. In particular, the wavefront correction can include the wavefront measurement of the previous scanning position, which in slowly varying samples makes possible a quicker convergence of the algorithm and thus adversely affects scanning speed even less. To this end, a control device for the adaptive optics unit is configured in such a way that a correction function is determined by comparison of the PSF of the imaging spot at a scanning position with an ideal PSFideal.

An evaluation unit of the detector array (e.g. 64- or 81-channel PMT array) determines the PSF of the imaging spot with spatial resolution at the resolution of the detector array.

By obtaining this information, not available up until now for an imaging spot, it is now possible in principle to also detect for the first time location-dependent (scanner setting-dependent) aberrations and to correct them by applying fast adaptive optics.

From this information, at least the focus of the PSF is defined.

Based on the location of the focus, a focus deviation in the lateral or axial direction can for example be determined.

In addition, the determination of higher-order imaging errors, such as astigmatism, coma or various distortions or aberration-typical symmetry alterations of the PSF, is optional.

Subsequently, a wavefront correction signal is determined from the PSF and the adaptive optics unit, known in as such and, controlled accordingly, so as to compensate the detected deviations in each scanning position (on the fly).

The wavefront correction signal is transmitted in a forward loop to the adaptive optics unit, so as for example to influence the wavefront signal in an adjoining or in the current scanning position.

While the scan optics unit scans, the adaptive optics unit is initially controlled with the laser scan signal of the previous position. In the process, the system learns and the wavefront correction signals determined are stored in a look-up table, preferably for each scan position. With each scan, the correction data can then be refined and the look-up table updated. The look-up table is also available in particular for possible post-processing (deconvolution).

The wavefront correction signal is triggered in such a way that it is imprinted at respective scan position, either of the illumination (NDD), of the detection PSF (detection only), both (common path), whereby a stepwise approximation of a nearly distortion-free PSF is accomplished.

At a first scanning position (n=1, m=1), a first wavefront error W(1,1)is determined from the spatially resolved PSF. This wavefront error is for example represented as a sum of Zernike wavefront errors.
Wn,m=Σi(Ai;n,m·Zi),
where i=4 . . . 25 for e.g. 25 Zernike polynomials;

and A(i;n,m) as the amplitude of the i-th Zernike wavefront error at the scanning position (n, m). The signal at the first scanning position must not be corrected in most applications; this image information is more or less “dispensed with.”

At the second scanning position, n=2, m=1, correction is performed using the wavefront error W(1,1). The real wavefront error W(2,1)deviates slightly from the correction value entered. The difference of the measured PSF at scanning position (2,1) is used to define a better wavefront correction value. The amplitudes Ai;n,mare slowly-varying functions of the scanning positions (n,m). The field pattern of the amplitudes can usually be represented with low-order polynomials, e.g.
Ai;n,m=ΣkBi,k·Fk
with amplitudes Bi,kand field pattern basis polynomials Fk, such as e.g. Fk=2(x−1) for a linear progression in x and constant in y. Thus the wavefront error is represented in a double series expansion:
Wn,m=ΣiΣk(Bi,k·Fk)*Zi

Thus the probable wavefront error at the next field point (n+1;m) or (n,m+1) can be calculated in advance from measurements known in advance, and the adaptive mirror controlled with this pre-calculated correction value.

In the simplest case, the gradients are defined from wavefront errors calculated at two adjoining measurement points, so as to estimate the wavefront error at the next field point by linear extrapolation.

After several field points, higher field pattern polynomials can also be carried along.

Of course, all known evaluations can be carried out with the detectors.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1shows schematically a super resolution laser scanning microscope1(hereafter abbreviated SR-LSM), which is configured for microscopy of a sample P placed in a sample carrier2. The SR-LSM1is controlled by a control device, not shown,36and includes an illumination beam path B and an imaging beam path D. The illumination beam path B illuminates a spot in the sample P. The imaging beam path D images radiation captured/reflected in the sample in diffraction-limited fashion.

The LSM1is constructed in a known manner. A laser3is coupled into a mirror5through optics4. The laser beam B is deflected by the mirror5through an angle of reflection onto the emission filter6, where it is reflected and deflected onto a scanner7. The scanner7provides for the scanning movement of the laser beam B over the sample P. The scanner7is advantageously equipped with an adaptive optics unit17for wavefront modulation. The adaptive optics unit17can also, however, be integrated at other locations in the illumination beam path B of the LSM1and be constructed in a known fashion.

The laser beam B is focused by means of a scan lens8and a tube lens9through an objective lens10into a spot11in the sample P.

Fluorescence radiation excited in the spot11(imaging beam path D) passes through the objective lens10and optics9,8back to the scanner7. Following the scanner in the imaging direction, a stationary light beam D is present. Emission filters6and12are positioned a known fashion in the imaging beam path D, so as to select the fluorescence radiation from the spot11with respect to its wavelength. An optics unit13provides for imaging the spot11as an imaging spot14(two-dimensional Airy disk) in a detection plane15at a certain size. The detection plane15is a conjugate plane or aperture plane of the plane of the spot11. A detector captures the imaging spot14with spatial and intensity resolution.

A control device36controls all components of the LSM1, particularly the scanner7, detector16and an adaptive optics unit17.

The detector16includes, in the embodiment shown, fiber optics as a non-imaging optical redistribution element, particularly a bundle of optical fibers18. Here, each individual optical fiber18represents a pixel of the imaging spot14. An input19of the bundle is positioned in the detection plane15. Here, the optical fibers18are arranged tightly pressed or packed together so that a circle-like or nearly circular structure is formed which can completely capture the imaging spot14.

The output ends of the optical fibers18are connected as output with the inputs of a detector array20. The connection can be made fixed or releasable, with a plug for example. In this embodiment, the detector array20includes photomultiplier tubes (PMTs) or avalanche photodiodes (APDs) in the same number as require by the pixels in the detection plane15. The PMTs operate sufficiently fast to be able to process the data captured by them in one and the same scan step.

A particular advantage of this arrangement consists in that the detector array20, which is larger in terms of installation volume than the cross-section of the input19of the redistribution element or the fiber optics, can be located outside the detection plane15and can also have an arbitrary shape/arrangement of the individual tubes or elements, depending on the available installation space. If it were desired to use the detector array20directly in the imaging plane, then the imaging spot must be accordingly enlarged to the size of the detector array, which often presents a technical problem.

An evaluation unit21is connected downstream of the detector array20, wherein the spatially- and intensity-resolved data of the detector array20are evaluated and processed. In the process, two-dimensional spatially resolved Airy disks are captured, which represent a PSF a focus position. In the process, the light quantity of a portion of the Airy disk is detected in each pixel of the detector. Thus radiation intensities can be captured with spatial resolution with the detector array20, as indicated in Detail A (source Wikipedia: point spread function).

It is possible, knowing the PSF for various first- and higher-order aberrations, to calculate the type and magnitude of the aberrations present by means of a polynomial definition, and to subtract them out by means of a control loop. One skilled in the art knows the calculations required for this purpose and can implement suitable control routines and evaluation routines. In determining a grey value from the detected total light quantity for the final image pixel of the microscope image, these aberrations can be taken into account. If required, the Airy disks provide additional information in the sub-pixel domain, which can be significant for the adjoining pixels in the microscope image and are accordingly taken into account there.

The evaluation unit21therefore provides pixel data to an image processing unit, not shown. In addition, the data of the evaluation unit21are compared with an ideal PSF (Detail A) and the resulting correction wavefront delivered to the control of the adaptive optics unit17, to be used at the next scan position for on-the-fly image correction.

A preferred embodiment of the SR-LSM is schematically shown inFIG. 2, wherein a separate adaptive optics unit is present. The microscope1serves in the manner described above for imaging the sample P, which is positioned in the sample holder2, on the confocal detector16. The detector16is constructed as described in the preceding figure and equipped with an optical redistribution element, not shown.

Illumination of the sample is also accomplished in known fashion by means of the laser source3which, with an adaptive mirror22, is controllable in focal and lateral position and for correcting aberrations and is movable over the sample by means of the scanner7. Imaging is accomplished in known fashion by means of the objective lens10and the tube lens9into an intermediate image S. From there, the light continues through the scan lens8into a conjugate aperture plane T, in which the scanner7is located. A second aperture plane U is generated by a first relay optics unit23, in which the adaptive mirror22is located.

Another conjugate aperture plane, or an intermediate image V, is formed between the conjugate aperture plane U and a first relay optics unit23. The intermediate image is imaged in the manner described inFIG. 1, by means of second relay optics unites24,25, on the detector16. This delivers a signal to the evaluation unit21. From the spatially resolved PSF determined here, a correction signal is determined which is provided to a control device26for controlling the adaptive mirror22, thereby affecting the wavefront of the illumination beam path.

An optional connectable relay optics unit27with adaptive optics can be provided.

FIG. 3shows another embodiment of an SR-LSM1according to the invention in schematic view with a second (additional) adaptive optics unit. The SR-LSM1has the same basic design as that described earlier. Identical reference symbols refer to identical components.

A repeated description of these parts is therefore dispensed with here. Here, the SR-LSM1includes a second scan mirror28in the aperture plane U, which is (optionally) also configured as an adaptive mirror. The second relay optics unit27is placed as a connectable relay with a second adaptive mirror29in a third conjugate aperture W. This second relay optics unit27includes a common use optics unit30and separate optics units31. One skilled in the art knows the construction of such optics units, so an extensive description is dispensed with here.

The adaptive mirror22,28,29is preferably a deformable mirror, the distortion of which can be controlled in such a way that a wavefront error that is present can be compensated. The system can also be designed in such a way that for example the first adaptive mirror22,28is a rapidly deformable mirror for changing the focus position and spherical aberration, while the second deformable mirror29is for correcting non-axially-symmetric wavefront errors such as coma or astigmatism. Modular system expansion can consequently be accomplished, depending on variants in equipment.

In the evaluation unit21, a detector signal of a first scan position is analyzed and first wavefront deformation determined, by guessing for example. From the knowledge of an wavefront signal, a correction signal can be determined, which is supplied to the control device The adaptive mirror or mirrors22,28,29are controlled using the correction signal for a second scanning position. At the detector16, the signal or the image of the PSF is now captured for the second scanning position and compared to the first detector signal or the ideal PSF. Beginning with the signal of the second scanning position, a new wavefront distortion and the associated correction signal can be determined and the adaptive mirror consequently controlled for the following scanning position. This iterative process usually converges very rapidly. It is to be expected that, after three to eight cycles, the detector signal corresponds closely to the ideal PSF.

The iterative process can occur while the scanner7is being directed to the next scanning position. Adjoining scanning positions in the sample usually have very similar wavefront errors. The control device26for the adaptive mirror(s)22,28,29can be coupled with the transducer for the scan mirror7.

Following definition of the first wavefront correction at a first scanning position, the scan mirror7moves to an adjoining second scanning position. For correcting, the wavefront distortions of the first scanning position are set. Based on the measurement at the current, second scanning position, the transducer determines a new, second wavefront correction, which is applied at an adjoining third scanning position. Consequently, during rapid measurement, the wavefront correction “limps” slightly behind the ideal correction.

The wavefront corrections associated with the scanning positions can be stored in a table as correction coefficients so that, during other measurements on the same or similar samples, the wavefront distortions need not be determined again, and an obvious starting point for optimization is present.

FIG. 4shows two examples for spatially resolved images of a PSF (FIGS. 4(A) and 4(B)) and associated intensity distributions for various aberrations (FIGS. 4(C) and 4(D)). It is obvious from the figures how different aberrations, due to their symmetry, can be recognized from the shape of the envelope of the PSF. A PSF is shown inFIG. 4(A)with the coma imaging error, inFIG. 4(B)a PSF with the defocus imaging error.

FIGS. 4(B) and 4(C)show intensity profiles of the sensor signal for a section in the x-axis as the solid curve40and as a dotted line41for a section through the y-axis.

Asymmetry difference between the axial sections40,41is clearly recognizable inFIG. 4(C). InFIG. 4(D), it can be seen that high intensities are present that are axially symmetric but distant from the center.

For detailed evaluation, all possible (naturally also eccentric) sections through the image of the PSF can be carried out and evaluated, so as to also detect and appropriately compensate higher-order aberrations.

FIG. 5shows by way of example how an imaging spot14is detected as a PSF with spatial resolution with a detector array20consisting of for example 9×9 individual detectors42. Here, different grey values represent different measured intensities of the individual sensors. InFIG. 4(a), a non-axisymmetric aberration is clearly recognizable, whileFIG. 4(b)shows a nearly ideal PSF.