DE102024104898A1 - Microscope and microscopy techniques with improved image quality - Google Patents

Abstract

The invention relates to a microscope with an imaging beam path comprising an objective (4) and imaging a sample (2) from an image field onto a two-dimensional detector (8), and an illumination beam path (B) illuminating the sample (2) through the objective (4), which illumination beam path comprises a light source and is reflected into the imaging beam path (M) via a beam splitter (6) in front of the objective (4), and a measuring device (10) followed by a light modulator (16) comprising at least one intensity-modulating, first line grating structure (26-29), wherein the light modulator (16) is arranged obliquely to a plane conjugated to the focal plane of the objective (4) and is projected onto the sample (2) through the illumination beam path (B) and the objective (4), wherein the at least one intensity-modulating, first line grating structure (26-29) is projected into a measuring area of the sample (2) that only partially fills the image field, and the measuring device (10) has a processor (32) which reads out image data supplied by the detector (8) and divides said image data into measurement data and display data for displaying a sample image based on a position in the image field, wherein the processor (32) selects as measurement data those image data which correspond to the measurement area of the sample (2) and evaluates the measurement data with regard to reflection images of the at least one line grating structure (26-29) projected onto the sample (2).

Description

The invention relates to a microscope with an imaging beam path which images a sample in an image field onto a detector and which comprises an objective, and an illumination beam path which illuminates the sample through the objective and is reflected into the imaging beam path in front of the objective via a main beam splitter, and a measuring device which has a measuring illumination source which is followed by a light modulator which comprises at least one intensity-modulating first line grating structure, wherein the light modulator is arranged obliquely to a plane conjugate to the focal plane of the objective and is projected onto the sample through the illumination beam path and the objective.

To obtain precise images of a sample or sample section using imaging optics, the sample must be positioned precisely in the focal plane of the objective. Defocusing reduces image quality. Aberrations also play a role in image quality, the extent of which depends, for example, on the thickness of a coverslip or components of the sample. It is common practice to place aberration compensation optics, such as a correction ring on the objective, in the beam path and adjust them to suit the current measurement task so that aberrations are minimized as much as possible. Tilting the sample relative to the focal plane of the objective also affects image quality.

1. 1. Bei der sogenannten konfokalen Pinhole-Methode wird Strahlung durch das Objektiv in die Probenebene fokussiert. Aufgrund von Brechzahlunterschieden zwischen Luft/Immersionsflüssigkeit und Deckglas sowie zwischen Deckglas und Probe wird dort ein Teil der Strahlung reflektiert und dann durch ein Pinhole, welches in einer zur Fokusebene des Objektivs konjungierten Ebene platziert ist, mit einem Fotodetektor detektiert. Dessen Signalstärke hängt vom Abstand des Objektivs zu den reflektierenden Grenzschichten ab. Wird ein axialer Scan durchgeführt, lassen sich die beiden Grenzschichten als Signalmaxima identifizieren. Das System bietet den Vorteil, dass es einen absoluten Wert für die Fokuslage der Grenzflächen liefert. Jedoch muss dafür ein axialer Scan durchgeführt werden. Ein weiterer Nachteil liegt darin, dass der Abbildungs- und Autofokusstrahlengang sich relativ zueinander verschieben können, was eine Drift des Ergebnisses der Fokusebenenermittlung zur Folge hat.

1. 2. Bei Triangulationsverfahren wird ein kollimierter Laserstrahl in die Pupillenebene eines Objektivs eingespiegelt und aus dem Verlauf dieses Laserstrahls relativ zum Abbildungsstrahlengang auf die z-Position des von der Probe reflektierten Laserlichts geschlossen. Eine Offset-Linse kann benutzt werden, so dass bei der gewünschten Fokusposition die Bildebene des AF-Systems in der Proben-Deckglas-Grenzfläche liegt und somit das reflektierte Licht scharf in der Mitte eines Liniendetektors abgebildet wird. Eine Fokusdrift führt zu einer Verschiebung des Signals auf dem Liniendetektor, weg von der mit der Offset-Linse eingestellten Position. Im Gegensatz zur konfokalen Pinhole-Detektion hat diese Technik den Vorteil, dass keine z-Stapel-Aufnahme zur Fokuslagenbestimmung nötig ist. Somit kann mit dieser Technik die Fokuslage während eines Messvorganges überwacht und gegebenenfalls korrigiert werden. Da der Mikroskop- und Autofokus-Abbildungsstrahlengang getrennt aufgebaut sind, führen Drifts jedoch zu einer Verschiebung der bestimmten Fokuslage. Bei der Abbildung des Laserlichts in unterschiedlich tief gelegene Ebenen der Probe treten zudem Bildfehler auf, so dass die Fokusmessgüte über einen gegebenen Tiefenschärfebereich stark variiert. Auch treten Schwankungen auf, wenn das Messergebnis am Zentrum oder am Rand der Probe ermittelt wird. Üblicherweise wird ein Triangulationsverfahren deshalb iterativ ausgeführt, was verhältnismäßig zeitraubend ist.
2. 3. Bei abbildenden Verfahren mit Kontrastauswertung wird die Probe mit einer bestimmten Intensitätsverteilung beleuchtet, z.B. indem in eine Feldblendenebene eines Beleuchtungsstrahlengangs ein Gitter gestellt wird. Man nimmt eine Serie von Bildern mit unterschiedlichen Abständen zwischen Abbildungsoptik und Probe auf und ermittelt in dieser Serie das Bild mit dem höchsten Kontrast, dem dann der optimale Fokusabstand zugeordnet ist. Beispiele für eine solche Autofokuseinrichtung mittels Kontrastanalyse eines auf eine Probe projizierten Musters finden sich in der US 5 604 344 A oder der US 6 545 756 B1 . Nachteilig hieran ist es, dass zur Aufnahme der Bildserie verschiedene z-Positionen mit hoher Genauigkeit angefahren werden müssen, was wiederum zeitraubend ist.

With regard to the focus position, in the case of a blurred image, it is important to know by what amount and in which direction the position of the sample relative to the objective needs to be changed, and if necessary, to derive corresponding adjustment commands that can be used for refocusing. The main applications of such focus control using an explicit autofocus system are in monitoring the distance between the sample and the objective. On the sample, this is usually referenced to an immersion coverslip or coverslip-sample interface. The feedback from the focus control is evaluated, and the result, if desired, can be used to readjust the position of the microscope's focal plane. Various options are known for such an autofocus function:

1. 1. In the so-called confocal pinhole method, radiation is focused through the objective lens into the sample plane. Due to differences in refractive index between the air/immersion liquid and the coverslip, as well as between the coverslip and the sample, part of the radiation is reflected and then detected by a photodetector through a pinhole positioned in a plane conjugate to the focal plane of the objective lens. The photodetector's signal strength depends on the distance of the objective lens from the reflective interfaces. If an axial scan is performed, the two interfaces can be identified as signal maxima. The system has the advantage of providing an absolute value for the focal position of the interfaces. However, this requires an axial scan. A further disadvantage is that the imaging and autofocus beam paths can shift relative to each other, resulting in a drift in the result of the focal plane determination.

1. 2. In triangulation methods, a collimated laser beam is reflected into the pupil plane of an objective lens, and the z-position of the laser light reflected from the sample is determined from the path of this laser beam relative to the imaging beam path. An offset lens can be used so that, at the desired focus position, the image plane of the AF system lies at the sample-coverslip interface, and the reflected light is thus sharply imaged in the center of a line detector. Focus drift leads to a shift of the signal on the line detector away from the position set with the offset lens. In contrast to confocal pinhole detection, this technique has the advantage that no z-stack image is required to determine the focus position. Thus, the focus position can be monitored and corrected if necessary during a measurement process. However, since the microscope and autofocus imaging beam paths are constructed separately, drifts lead to a shift in the determined focus position. When the laser light is projected into different depths of the sample, image errors occur, causing the focus measurement quality to vary significantly across a given depth of field. Fluctuations also occur when the measurement result is determined at the center or edge of the sample. Therefore, a triangulation process is usually performed iteratively, which is relatively time-consuming.
2. 3. In imaging techniques with contrast evaluation, the sample is illuminated with a specific intensity distribution, e.g., by placing a grating in the field diaphragm plane of an illumination beam path. A series of images is taken with different distances between the imaging optics and the sample, and the image with the highest contrast is determined from this series, to which the optimal focus distance is then assigned. Examples of such an autofocus device using Kon Contrast analysis of a pattern projected onto a sample can be found in the US 5 604 344 A or the US 6 545 756 B1 The disadvantage of this is that in order to capture the image series, different z-positions must be approached with high precision, which in turn is time-consuming.

A variant of the contrast-evaluating method is the position determination using a confocal slit diaphragm, which is placed in a field stop plane of the illumination beam path and imaged onto the sample. The light reflected from the sample is directed onto a CCD array arranged at an angle relative to the slit diaphragm, and the position on the CCD array at which the reflected light reaches a maximum is determined. This method is very fast, but has problems with contamination on the sample or sample surface, which can lead to intensity fluctuations. Furthermore, a very large adjustment effort is required when imaging the slit onto the CCD array, because the slit must be very narrow to achieve sufficient accuracy. DE 103 19 182 A1 This autofocus system is further developed in that, instead of a slit diaphragm, a line grating structure arranged at the edge of a field diaphragm is projected into the sample plane. The reflections from this grating structure are then detected by an inclined autofocus detector. Only a portion of the grating is sharply imaged on the inclined autofocus detector. A change in the focus position leads to a shift in the sharp image on the detector.

In a further development of the concept of the DE 103 19 182 A1 The line grating structure and the autofocus detector are both reflected into the illumination beam path and at least one of them is inclined. This is DE 10 2006 027 836 A1 The advantages and disadvantages of these methods are similar to triangulation using oblique illumination. Drifts in the beam paths lead to a shift in the determined focus position.

1. 1. Aberrationen eines Abbildungssystems sind Wellenfrontstörungen. Diese lassen sich über interferometrische Messungen mittels eines bekannten Referenzstrahls direkt bestimmen. Die Phaseninformation lässt sich somit in eine einfach zu messende Intensitätsinformation übersetzen (siehe I. Papadopoulos et al., „Scattering compensation by focus scanning holographic aberration probing (F-SHARP)“, Nature Photonics 11 (2), 116-123 (2016 )). Mittels Pupillensegmentation arbeitet der Shack-Hartmann Wellenfrontsensor, welcher jedoch auf räumlich kohärente Abbildungssysteme limitiert ist. Das Konzept lässt sich jedoch auf inkohärente Abbildungssysteme (z.B. ein Fluoreszenz-Mikroskop) übertragen, indem eine Bildverschiebung zwischen einem Referenzbild, aufgenommen mit der maximal möglichen Pupillenfunktion, und einem Bild, welches lediglich mit einem Pupillensegment aufgenommen wurde, vergleichen wird. Dies ergibt die Tip/Tilt-Aberration für jedes Pupillensegment.

There are also various options for determining aberrations and adjusting the aberration compensation optics:

1. 1. Aberrations of an imaging system are wavefront disturbances. These can be directly determined via interferometric measurements using a known reference beam. The phase information can thus be translated into easily measured intensity information (see I. Papadopoulos et al., “Scattering compensation by focus scanning holographic aberration probing (F-SHARP),” Nature Photonics 11 (2), 116-123 (2016 The Shack-Hartmann wavefront sensor uses pupil segmentation, but is limited to spatially coherent imaging systems. However, the concept can be transferred to incoherent imaging systems (e.g., a fluorescence microscope) by comparing the image shift between a reference image acquired with the maximum possible pupil function and an image acquired with only one pupil segment. This yields the tip/tilt aberration for each pupil segment.

1. 2. In bildbasierten Methoden werden mit dem Mikroskop mehrere Bilder mit jeweils unterschiedlichen Aberrationen aufgenommen. Eine Bildmetrik wird benutzt, um die Bildqualität zu bewerten. Da verschiedenen Zernike-Moden unabhängig voneinander eingestellt und gemessen werden, lassen sich die Gesamtaberrationen des Systems (z.B. inverse Wellenfront, die zur Korrektur benötigt wurde) rekonstruieren (siehe D. Débarre et al., „Image based adaptive optics through optimisation of low spatial frequencies“, Optics Express Vol. 15, Nr. 13 (25.06.2007 )).
2. 3. Wie in WO 2022 / 150 396 A1 beschrieben, kann ein mit einem Mikroskop aufgenommenes Bild mittels einer Punktbildverwaschungsfunktion (PSF) von einer Referenzprobe entfaltet werden. Dafür wird der Reflex an der Deckglas-Probe-Grenzschicht einer Referenzprobe benutzt, um die PSF zu berechnen. Diese PSF spiegelt alle im System enthaltenen Aberrationen wider und wird anschließend benutzt, um eine Entfaltung des aufgenommenen Bildes vorzunehmen und so den Einfluss variierender Deckgläser auszugleichen. Da die Aberration auch durch Elemente der Probe, z.B. ein Deckglas oder oberhalb der Fokusebene liegende Probenbestandteile wie Nährlösung etc., beeinflusst werden, ist nachfolgend unter dem Begriff „Aberration“ nicht alleine der Einfluss des Abbildungsstrahlengangs zu verstehen.
3. 4. Die Phaseninformation (d.h. Wellenfront) des Lichts und damit ein direkter Nachweis der eingeführten Aberrationen geht bei der Detektion der Lichtintensität verloren. Phase-Retrieval-Algorithmen rekonstruieren aus Intensitätsmessungen die Phaseninformationen. Dies ist eindeutig möglich, wenn ein überbestimmtes Phasenproblem (phase diversity) erzeugt wird. Dies wird erreicht, indem dieselbe Intensitätsaufnahmen mit unterschiedlichen Phasen (im einfachsten Fall unterschiedlicher Defokussierung, siehe G. Zheng et al., „Characterization of spatially varying aberrations for wide field-of-view microscopy, Optics Express Vol. 21, Nr. 13 (01.07.2013 )) aufgenommen werden. Die rekonstruierten Phaseninformationen lassen sich direkt in Aberrationen (und damit die Ansteuerung von Ausgleichsoptiken) umsetzen. Dabei kann als Basis für den Algorithmus das gesamte Bild oder andere Informationen (z.B. integrierte Bildintensität, siehe Q. Gong et al., „Aberration measurement using axial intensity“, Optical Engineering 33, 4 (1994 )) benutzt werden.

The system aberrations or the wavefront of the entire pupil function result from the parts (see Na Ji et al., “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues,” Nature Method 7, 2 (2010 ) and D. Ancora et al., “Spinning pupil aberration measurement for anisoplanatic deconvolution,” Optics Letters 46, 12 (June 15, 2021 )).

1. 2. In image-based methods, several images are acquired with the microscope, each with different aberrations. An image metric is used to evaluate the image quality. Since different Zernike modes are adjusted and measured independently, the overall aberrations of the system (e.g., the inverse wavefront required for correction) can be reconstructed (see D. Débarre et al., “Image based adaptive optics through optimization of low spatial frequencies,” Optics Express Vol. 15, No. 13 (June 25, 2007 )).
2. 3. As in WO 2022 / 150 396 A1 As described, an image taken with a microscope can be deconvoluted from a reference sample using a point spread function (PSF). For this purpose, the reflection at the coverslip-sample interface of a reference sample is used to calculate the PSF. This PSF reflects all aberrations contained in the system and is then used to deconvolute the recorded image and thus compensate for the influence of varying coverslips. Since aberrations can also be influenced by elements of the sample, e.g. a coverslip or sample components lying above the focal plane such as nutrient solution, etc., the term "aberration" in the following does not only refer to the influence of the imaging beam path.
3. 4. The phase information (ie wavefront) of the light and thus a direct detection of the introduced aberrations is lost during the detection of the light intensity. Phase retrieval algorithms reconstruct the phase information from intensity measurements. This is a clearly possible if an overdetermined phase problem (phase diversity) is created. This is achieved by taking the same intensity images with different phases (in the simplest case, different defocus, see G. Zheng et al., “Characterization of spatially varying aberrations for wide field-of-view microscopy, Optics Express Vol. 21, No. 13 (July 1, 2013 )). The reconstructed phase information can be directly converted into aberrations (and thus the control of compensating optics). The entire image or other information (e.g. integrated image intensity, see Q. Gong et al., “Aberration measurement using axial intensity,” Optical Engineering 33, 4 (1994 )) can be used.

• Nachteil 1: Für Autofokus und Aberrationskorrektur sind i.d.R. eigenständige optische Aufbauten nötig.
• Nachteil 2: Die Drift von Bauteilen in den Abbildungsstrahlengängen von Mikroskop und von Autofokus führen zu einem Wandern des Referenzfokus-Punktes. Folglich kann die Fokusposition nicht auf in einer gewünschten Position gehalten werden.
• Nachteil 3: Im Bereich „Automation“ besteht die Aufgabe meist im Messen von relativ großflächigen Proben, denn die Probenfläche ist um ein Vielfaches größer als die Bildfeldgröße (sog. whole slide imaging), wobei die Probe meist robotergestützt in das Mikroskop eingelegt wird. Im Betrieb ist ein Tool nötig, das es den Nutzern erlaubt, schnell und automatisiert die korrekte Probenlage (Verkippung) über das Experiment hinweg zu überwachen.
• Nachteil 4: Bei der Vermessung von Proben mit hoch-NA-Objektiven durch ein (z.B. 170 µm dickes) Deckglas muss aufgrund der Dickenvarianz der Deckgläser die sphärische Aberration mithilfe einer Aberrationskorrekturoptik (z.B. Korrekturring am Objektiv) ausgeglichen werden. Der Ausgleich der sphärischen Aberration verschiebt jedoch die Lage der Fokusebene. Der Nutzer muss sich also um die sich gegenseitig beeinflussende Einstellung von Aberrationskorrektur und Fokuslage kümmern bzw. diese überwachen oder schnell und automatisiert beurteilen können.
• Nachteil 5: Viele Maßnahmen zur Bestimmung von Aberrationen und/oder Verkippung der Probe machen es nötig, dass eine Vielzahl von einzelnen Aufnahmen gemacht werden müssen, was zeitraubend ist.

The known solutions regarding autofocus and aberration correction have the following disadvantages:

• Disadvantage 1: Autofocus and aberration correction usually require separate optical setups.
• Disadvantage 2: Drift of components in the imaging beam paths of the microscope and the autofocus system causes the reference focus point to drift. Consequently, the focus position cannot be maintained at a desired position.
• Disadvantage 3: In the area of "automation," the task usually involves measuring relatively large samples, as the sample area is many times larger than the image field size (so-called whole-slide imaging). The sample is usually inserted into the microscope using a robot. During operation, a tool is required that allows users to quickly and automatically monitor the correct sample position (tilt) throughout the experiment.
• Disadvantage 4: When measuring samples with high-NA objectives through a coverslip (e.g., 170 µm thick), the spherical aberration must be compensated for using aberration correction optics (e.g., a correction ring on the objective) due to the thickness variance of the coverslips. However, compensating for the spherical aberration shifts the position of the focal plane. Therefore, the user must manage the mutually influencing adjustment of aberration correction and focus position, or monitor them, or be able to quickly and automatically assess them.
• Disadvantage 5: Many measures to determine aberrations and/or tilt of the sample require that a large number of individual images be taken, which is time-consuming.

In the field of automated microscopy, these disadvantages usually do not occur individually, but in combination.

The invention is therefore based on the object of providing an improved measuring function that eliminates the disadvantages of the prior art with regard to image quality, in particular with regard to automated microscopy.

The invention is defined in the independent claims. The subclaims relate to preferred developments.

The microscope has an imaging beam path that images a sample in an image field through an objective lens onto a detector. An illumination beam path illuminates the sample through the objective lens and is reflected into the imaging beam path via a main beam splitter. A measuring device arranged in the microscope has a measurement illumination source, downstream of which is a light modulator comprising at least one intensity-modulating, first line grating structure. It is arranged obliquely to a plane conjugate to a focal plane of the objective lens and is projected via a beam combiner arranged in the illumination beam path into the illumination beam path and from there through the objective lens onto the sample, wherein the at least one intensity-modulating, first line grating structure is projected into a measurement region of the sample that only partially fills the image field.

The measuring system includes a processor that reads image data supplied by the detector and divides it into measurement data and display data based on its position in the image field. The processor selects the image data that corresponds to the sample's measurement range as the measurement data. The processor evaluates this measurement data for back reflections from the grating structures projected onto the sample.

Thus, an image of the sample is displayed simultaneously for evaluating the back reflections based on the display data. This means that the detector on which the sample is imaged is also used as a measurement detector. It is crucial for this that the detector is read out simultaneously for evaluating the light modulator, which is preferably projected at the edge of the image field, and for image display.

The light modulator preferably comprises a central, rectangular field, at at least one edge of which an intensity-modulating, first line grating structure extends. Each of the thus up to The four first line grating structures preferably comprise a sequence of first line elements extending parallel to the respective edge. The up to four first line grating structures are projected onto the sample at the edges of the image field, preferably one at each edge. The light modulator is inclined relative to the conjugate plane such that the first line grating structure(s) are inclined to the conjugate plane.

The measuring device achieves an improvement in image quality in several respects, namely as a focus measurement in the sense of an autofocus function and also through aberration determination and tilt measurement during image display. The measuring device makes it possible, with a single measurement and with one and the same optical elements, to detect a tilt of the sample, to determine aberrations that have not yet been compensated for and, at the same time, to provide an autofocus control signal. The invention thus achieves an improvement in image quality through rapid and simultaneous determination of sample position, focus position as well as aberration and image display. Each of these measures alone already improves image quality. While individual measures are described in isolation below, they can nevertheless be combined to great advantage. However, they always take place during image display.

In one embodiment, the measurement serves the autofocus function, i.e., for monitoring the distance between the sample and the objective lens. A line grating structure is sufficient for this purpose. The measuring device uses the microscope imaging beam path and the microscope detector for detection. Since sample imaging and measurement occur through the same beam path, a focus position that must be maintained cannot be lost due to component drift (e.g., due to temperature changes). In particular, the reference point no longer moves.

Since the microscope detector cannot be tilted, the line grating structure(s) are tilted and projected into the sample plane instead. Preferably, the first line grating structure(s) (all at the same angle) are inclined to the conjugate plane.

The central field is preferably unstructured or, in the case of option 3 below, contains at most only one edge-giving structure.

The central field corresponds to at least 50% or 60% or 70% or 80% or 90% of the area of the image field when imaged in the focal plane.

If four line grating structures are present, the processor can, in one embodiment, determine the tilt of the sample in the microscope. Typically, all areas of the sample that reflect projection images of the line grating structures as reflected images are located in the same plane—even if this plane is tilted relative to the optical axis of the microscope. Tilting shifts the reflected grating signals of the opposing line grating structures differently. From this, the processor determines a tilt angle of the sample. Preferably, the processor determines a relative displacement between reflected images of opposing line grating structures and uses this to determine an indication of a tilt angle of the sample.

To determine aberrations, the following four options, among others, can be used individually or in any combination on the basis of the measuring device in order to adjust an aberration compensation optics arranged in the imaging beam path, adjustable and controlled by the processor, in particular in the form of a correction ring integrated into the lens.

As option 1, in addition to the inclined (first) line grating structures, at least one non-tilted, so-called aberration grating is projected into the focal plane as a second line grating structure. The spacing of the grating lines of this grating is selected so that they are closer together than in the first line grating structures, preferably as close as possible to the resolution limit of the imaging system. There are a variety of ways to implement the aberration grating. In one embodiment, a finer substructure is added to each line of the first line grating structure. In another embodiment, which can be combined, lines of the aberration grating run in addition to at least one of the four first line grating structures and perpendicular to their grating lines. For this purpose, it is therefore preferred that the light modulator has an intensity-modulating, second line grating structure parallel to or overlapping at least one, preferably each first line grating structure, which is formed from second line elements, wherein adjacent second line elements are spaced apart from one another by a distance that is smaller than a distance between adjacent first line elements, and the processor adjusts the aberration compensation optics such that a contrast of the second line grating structure is maximized in an image recorded by the detector. It should be noted that the reflected images originating from the cover glass-sample interface are only imaged aberration-free, i.e., without defocus, if the focal plane of the microscope and the light modulator projection coincide. With this technique, the aberration correction can therefore only be carried out at this one focal plane position in the sample. This may need to be adjusted after each change in aberration correction; consequently, the processor performs an iterative process.

In Option 2, edges are recorded across different defocus positions, and the modulation transfer function (MTF) is determined for each z-position. System aberrations can be determined from the dependence of the MTF value on the z-position for an (arbitrary) spatial frequency. Multiple edges oriented in different directions allow the determination of non-radially symmetric aberrations. The imaged edges could, for example, be realized by a structure in the field of view (square, circle, etc.) or a frame between the gratings and the central field. It is preferred that the lens is controlled by the processor to adjust the focal plane, the light modulator has an edge structure parallel to at least one of the first line grating structures, which runs to the respective edge along which the first line grating structure extends, and the processor sets several different positions of the focal plane on the lens, determines a value of the modulation transfer function for each position from an image contrast of the at least one edge structure, and uses this to determine image aberrations, in particular a spherical aberration.

If one restricts oneself to correcting spherical aberrations, e.g., using a correction ring, the optimal setting for compensating for spherical aberration can be determined from a z-stack image of the edges. When the setting is changed, the resulting known focus shift is compensated for by changing the working distance.

Option 3 uses one of the phase retrieval algorithms mentioned above. If one of the tilted line grating structures is imaged onto the upright detector, the lateral locations of the grating lines represent images from different axial z-planes. A similar result can be generated by tilting the line grating structure around an axis that runs along the line elements. The out-of-focus and in-focus regions now run along the line elements. Consequently, a single image can be captured from different focal planes (corresponding to different defocus positions). In both cases, it is possible to acquire the known line grating structure across different defocus positions using a single image. From this, the phase retrieval algorithm determines the spherical aberrations without having to acquire a z-stack. This allows the aberrations to be corrected in a non-iterative workflow. However, the coupling of the spherical aberrations and the defocus may need to be considered.

Using option 4, spherical aberration can be determined by exploiting the property of spherical aberration that near-axis and far-axis rays are imaged at different focal points. In the pupil of an imaging system, the near-axis and far-axis rays correspond to low and high spatial frequencies, respectively. Two line grating structures with different grating periods are then imaged: the first and second line grating structures. The tilted first and second line grating structures convert the aberration-induced axial offset of the focal position for different spatial frequencies into a lateral displacement of the sharply imaged sections. The spherical aberration compensation optics are now adjusted so that the sharply imaged sections of the first and second line grating structures lie adjacent to one another with respect to the line element extension.

Preferably, the measurement illumination source is designed as an IR radiation source, the first - and if present also the second - line grating structures are projected onto the sample by IR radiation and the main beam splitter transmits a portion of the IR radiation coming from the sample to the detector, as a 50/50 splitter.

The light modulator can be composed of separate first line grating structures. However, the four first line grating structures are preferably applied as absorbing line elements on a planar surface of a transparent substrate (in transmission mode) or as non-reflective line elements on a mirrored substrate (in reflection mode), and the surface is inclined.

The adjustable aberration compensation optics for compensating for defocus and/or aberrations may comprise at least one of the following elements: an Alvarez manipulator, an objective correction ring, an adjustable lens, a deformable mirror, a spatial light modulator (SLM), a micromirror element (MEMS).

• ◯ Kontrast, Signal, Schärfe, SNR sowie Kombinationen davon von Gitterlinien (Aberrationsgitter)
• ◯ Z-abhängige MTFs mehrerer Kanten (falls sphärische Aberrationen ausreichend, reicht eine Kante)
• ◯ Z-abhängige Aufnahme von Linien

The measurement signal evaluated by the processor, which depends on the strength of the aberrations, can be:

• ◯ Contrast, signal, sharpness, SNR and combinations thereof of grid lines (aberration grid)
• ◯ Z-dependent MTFs of multiple edges (if spherical aberrations are sufficient, one edge is sufficient)
• ◯ Z-dependent recording of lines

To the extent that method features are described here, the processor is configured in embodiments to implement these features. To the extent that the operation of the processor is described, analogous method features are also disclosed.

It is understood that the features mentioned above and those to be explained below can be used not only in the combinations indicated, but also in other combinations or in isolation, without departing from the scope of the present invention.

• 1 eine schematische Darstellung eines Strahlengangs eines Mikroskops,
• 2 eine schematische Darstellung der Entstehung eines Reflexbildes im Mikroskop der 1,
• 3 einen Lichtmodulator des Mikroskops der 1 umfassend mehrere erste Liniengitterstrukturen,
• 4 im Mikroskop der 1 detektierte Reflexbilder im Fall eines Defokus,
• 5 im Mikroskop der 1 detektierte Reflexbilder im Fall einer Verkippung der Probe,
• 6 zwei Möglichkeiten zur Realisierung von als Aberrationsgitter dienenden zweiten Liniengitterstrukturen,
• 7 ein erstes Blockschaltbild zum Aberrationsausgleich im Mikroskop der 1,
• 8 Möglichkeiten zur Realisierung von Kantenstrukturen, die zur Aberrationsreduktion eingesetzt werden können,
• 9 ein zweites Blockschaltbild zum Aberrationsausgleich im Mikroskop der 1,
• 10 eine schematische Darstellung der Wirkung einer Verkippung der Liniengitterstrukturen,
• 11 ein drittes Blockschaltbild zum Aberrationsausgleich im Mikroskop der 1 und
• 12 die Auswertung eines Aberrationsgitters gemäß der rechten Variante der 6.

The invention will be explained in more detail below using exemplary embodiments with reference to the accompanying drawings, which also disclose features essential to the invention. These exemplary embodiments are for illustrative purposes only and are not to be interpreted as limiting. For example, a description of an exemplary embodiment with a plurality of elements or components should not be interpreted as meaning that all of these elements or components are necessary for implementation. Rather, other exemplary embodiments may also contain alternative elements and components, fewer elements or components, or additional elements or components. Elements or components of different exemplary embodiments may be combined with one another unless otherwise stated. Modifications and variations described for one of the exemplary embodiments may also be applicable to other exemplary embodiments. To avoid repetition, identical or corresponding elements in different figures are designated by the same reference numerals and are not explained more than once. The figures show:

• 1 a schematic representation of a microscope beam path,
• 2 a schematic representation of the formation of a reflex image in the microscope of the 1 ,
• 3 a light modulator of the microscope 1 comprising several first line grating structures,
• 4 in the microscope of 1 detected reflex images in case of defocus,
• 5 in the microscope of 1 detected reflection images in case of tilting of the sample,
• 6 two possibilities for the realization of second line grating structures serving as aberration gratings,
• 7 a first block diagram for aberration compensation in the microscope of the 1 ,
• 8 Possibilities for realizing edge structures that can be used to reduce aberrations,
• 9 a second block diagram for aberration compensation in the microscope of the 1 ,
• 10 a schematic representation of the effect of tilting the line grid structures,
• 11 a third block diagram for aberration compensation in the microscope of the 1 and
• 12 the evaluation of an aberration grid according to the right variant of the 6 .

1 shows a schematic diagram of a microscope 1 with which a sample 2 is examined. The sample is imaged through an imaging beam path M, which has an objective 4, and via a beam splitter 6 and a tube lens 7 onto a detector 8, preferably designed as a camera. The sample 2 is illuminated in reflected light, i.e. through the objective 4, by means of an illumination beam path B. Furthermore, the microscope 1 comprises a measuring device 10, which has a measurement illumination source 12, which radiates, for example, in the infrared radiation range and differs in this respect from the reflected light source 14 of the illumination beam path B. In 1 IR radiation is shown in dashed lines. The light from the measurement illumination source 12 is reflected into the illumination beam path B by a light modulator 16, which here operates in transmissive mode as an example, but can equally well be designed reflectively, and an optics 17 via a coupling beam splitter 18, so that the light modulator 16 is projected onto the sample 2 by means of an illumination tube lens 19 and the objective 4. The optics 17 can be axially displaceable in order to refocus the light modulator 16 to different settings of the objective 4. For this purpose, the light modulator 16 is arranged in a plane 11 which is conjugate to the focal plane of the objective 4.

The projection images of the light modulator 16 on the sample are, as the 2 shows, are reflected from the sample and reach the detector 8 in the form of reflected images from the light modulator 16. 2 shows the conditions in sample 2 (not shown) with the focal plane 20 and a reflection plane 22 of the measuring system 10. The reflection images are created at the interface and therefore at the reflection plane 22. This forms the reference plane for the measurement. The interface can be: Immersion medium coverslip, coverslip sample, immersion medium sample (if no coverslip is present). Since the light modulator 16, as will be explained, is positioned at an angle to the conjugate plane 11, which is conjugate to the focal plane of the objective 4, the difference between the focal plane 20 and the reflection plane 22, at which the reflection images are formed, causes the reflection images to be indistinctly imaged onto the detector 8, unless the inclination of the light modulator 10 compensates for the difference between the focal plane 20 and the reflection plane 22.

As in 1 As illustrated, the oblique light modulator 16 is illuminated by an IR light source 12 and reflected into the illumination beam path B of the microscope 1 via the preferably dichroic mirror 18. It should be noted here that the beam splitter 6 in the imaging beam path M reflects not only the light from the microscope illumination, but also the AF radiation, e.g., IR, into the focal plane. If the reflected images generated by the sample 2 are to be detected in the IR, which is preferred, the beam splitter 6 must allow IR to pass through to the detector 8.

The design of the light source 12 as an infrared light source is, however, optional, as will be explained below with reference to the 3 becomes clear. 3 shows the light modulator 16 in plan view. The detector 8 is also shown. The light modulator 16 has a square field 24, which is unstructured in this embodiment, along whose edges four line grating structures 26 to 29 extend, each of which is formed as a sequence of line elements aligned transversely to the extension of the respective line grating structure 26 to 29. The line grating structures 26 to 29 are located at the edge of the square field 24 and thus in a region in which optical imaging onto the sensor is usually unnecessary for imaging the sample 2. Therefore, a spectral range that is not in the IR can certainly be used for the measuring device.

3 For clarity, the image field 30 in the sample is shown in a circular form, which, as usual, is a circle inscribed in the square of the detector 8. Only the area within the circle 30 is available for imaging the sample 2, which makes it clear that the line grating structures 26 to 29 are located at the edge of this image field if one imagines the line grating structure projected into the sample 2.

To perform the measurement, the microscope 1 preferably separates the image information from field 24, which represents the image of the sample 2, from the reflected images of the line grating structures 26 to 29 by appropriately processing the image data of the detector 8. No spectral separation takes place. This separation is performed by a processor 32, which controls the operation of the microscope and provides the measuring function and is connected to the detector 8 and the objective 4 for this purpose. Only the image information from the area corresponding to field 24 within the line grating structures 26 to 29 is provided for displaying the image of the sample 2. The edge regions of the image, in which the reflected images of the line grating structures 26 to 29 are located, are, however, separated from the image information to be displayed. This area thus displayed is somewhat smaller than the image field 30 would actually allow. Since a rectangular or square image is ultimately desired, the size difference is actually not significant, because the square of field 24 inscribed in the circular image field 30 is located with its corners not far from circle 30, i.e., from the maximum image field extent. Since image quality regularly decreases toward the edge of image field 30, sacrificing image size by using the slightly smaller field 24 is actually not disadvantageous.

In order to detect the position of the focal plane in microscope 1 with detector 8, which is also responsible for imaging sample 2, the light modulator 16 with its four line grating structures 26 to 29 is tilted. It is generally tilted around an axis corresponding to one of the diagonals drawn through field 24. The tilt is not shown in the top views of light modulator 16 in these figures to keep the illustrations clear. The tilt is necessary because the detector used for the measuring function is detector 8, which is also used for imaging sample 2. Obviously, this detector cannot be tilted.

4 shows on the left a top view of the light modulator 16 and on the right two images of the reflection images 33 to 36 of the light modulator 16 projected into the sample 2. It can be seen that an axial displacement of the sample, namely a defocusing, leads to a lateral displacement of the center of gravity of the sharply reproduced reflection images 33 to 36 of the four line grating structures 26 to 29. In the upper part of the right representation of the 4 The reflected images 33 to 36 are centered in relation to the coordinate system of the detector 8 with respect to their sharply imaged sections (the middle three line elements). If the sample 2 is defocused, the sharp sections in the coordinate system 38 shift from the center of the detector 8. The reflected images 33, 35 and 34, 36 of opposite line grating structures 26, 28 and 27, 29 shift in the same direction. The lateral shift indicates the defocusing. This is due to the tilting of the light modulator 16 and the four Line grating structures 26 to 29 are implemented. The tilt direction of the opposing gratings 26 and 28, or 27 and 29, is irrelevant. They can be tilted differently or identically, as long as they have the same tilt angle to the conjugate plane (also measurable as the tilt angle relative to the optical axis). The co-directional displacement of the sharply imaged sections of the reflex images 33 to 36 of the line grating structures 26 to 29 allows the processor 32 to easily determine the defocus.

5 shows that with the same setup, a tilt of sample 2 can also be determined. In contrast to defocusing, the opposing line gratings then shift in opposite directions. The right-hand representation of the 5 shows this case in the upper illustration for a tilt in the yz-plane and in the lower illustration for a tilt in the xz-plane. In the case of tilt in the yz-plane, the opposing reflected images 33 and 35 shift in opposite directions, while reflected images 34 and 36 do not. In the case of a shift in the yz-plane, reflected images 33 and 35 do not shift, but reflected images 34 and 36 shift in opposite directions. The tilt angle of sample 2 can thus be easily determined quantitatively from the shift of the reflected images on detector 8.

Since the defocus shifts occur relative to the center of the detector coordinate system 38, while the tilt shifts occur symmetrically to the center, defocus and tilt can be determined simultaneously from one image.

The measuring device 10 not only enables the simultaneous detection of defocus and tilt of the sample 2, but also allows aberration determination with a simple modification of the light modulator 16. For this purpose, tilted or non-tilted so-called aberration gratings are projected into the sample plane.

To distinguish them from the previously described line grating structures 26 to 29, if confusion with the aberration gratings is to be feared, these are referred to here as the first line grating structures 26 to 29 and the aberration gratings as the second line grating structures.

The spacing of the line elements of the second line grating structures, ie the aberration gratings, is narrower than in the first line grating structures 26 to 29. In this way, other spatial frequencies are transmitted. Preferably, the spacing of the line grating elements of the aberration gratings is such that it is close to the resolution limit of the microscope 1. There are several possibilities for implementing the aberration gratings. 6 The example shown explains that each line element of the first line grid structure is added a kind of fine structure made up of further line grid elements. This shows 6 purely by way of example using two line elements 40, 42 of the first line grating 27 by further line grating elements 44, 46. Another possibility is to realize the aberration gratings by line gratings 48 to 51, which are arranged, for example, on the outside of the first line grating structures 26 to 29 with respect to the field 24, but run perpendicular to their line elements as far as the line elements of the aberration gratings are concerned.

Using the aberration gratings, for example according to 6 , the aberration can be corrected, for example by adjusting a correction ring, according to the 7 shown block diagram. In a step S1, the sample 2 is inserted. Subsequently, the interface between cover glass 34 and sample 2 is determined in the z-direction. For this purpose, the first line grating structures 26 to 29 are evaluated, as is the case, for example, with 4 explained. Sample 2 is then placed in the desired focal plane position on the determined interface. Next, the current contrast at the aberration gratings is determined. Step S3 checks a termination criterion as to whether the aberration has been corrected (contrast at aberration gratings is above a certain threshold) and the focus position is correct. If this criterion is not met, an aberration compensation optic, for example a correction ring of the objective, is adjusted in step S4 to improve the contrast of the aberration gratings. If the aberration has been reduced in this way in step S4, this inevitably leads to a change in the focal plane, since the aberration-correcting optical elements, for example a correction ring, fundamentally also affect the position of the focal plane. Step S2 is therefore repeated. Steps S2 and S4 are repeated with the intermediate termination criterion of step S3 until its termination criterion is met, ie, both the aberration is minimized, which can be seen from the fact that the aberration gratings are imaged with optimal contrast, and sample 2 is appropriately focused on the reference plane, ie, the reflected images 33 to 36 are symmetrical to the center of the detector 8. In step S5, sample 2 is then examined as desired.

8 shows another option for determining spherical aberration. For this, an intensity edge located at the edge outside the field used for image display is recorded at various defocus positions, and the modulation transfer function (MTF) is determined for each z-position, i.e., defocus position. In principle, one edge illuminated by the measuring radiation source 12 is sufficient for this purpose. Multiple edges oriented in different directions allow the determination of non-radially symmetric aberrations.

The procedure for determining the aberration is described in 9 shown as a block diagram. Again, in step S1, sample 2 is placed into the microscope. Subsequently, in step S6, the MTF is determined as a function of defocusing, ie, a z-stack of images is acquired. From this, the spherical aberration and the target setting of the aberration compensation optics, e.g. a correction ring, are determined. This target setting is set in step S7, and subsequently, the shift of the focal plane caused by the adjustment of the aberration compensation optics is compensated in step S8 by repositioning sample 2 in the z-direction. Then, as shown in the block diagram of the 7 also, the experiment is carried out in step S5. Another option for determining the aberration is to exploit the fact that the lateral locations of the tilted grating lines represent images from different axial z-planes. This results in an image with different defocusings in a single image. In the case of a tilt of the line grating structures transverse to the longitudinal extension of the line grating structure (i.e., perpendicular to the longitudinal extension of the line grating elements), this is evident. But even with a tilt of the line grating structures along the individual line elements, an image of the lines from different focal planes is obtained from a single image, such as 10 based on the first line grid structure 27. If this is tilted in the direction of the tilt direction 56, the individual line elements 40, 42 are defocused at their upper edges 58, for example to negative z-values, focused in the center 60 and at their lower edges 62 (each related to 10 ) defocused again, for example to positive z-values.

Thus, a single image of the tilted line grating structures provides all the prerequisites for the application of a phase retrieval algorithm, as already explained in the general part of the description. The block diagram of the 11 In step S1, sample 2 is placed in the microscope. Subsequently, in step S9, a single image of the tilted line grating structure is taken, with the tilt being able to be parallel to the individual line elements or perpendicular to them (see above). In step S10, the phase retrieval algorithm is executed, and the aberration to be corrected is determined. Subsequently, in step S7, the aberration compensation optics are adjusted. Not shown is a corresponding adjustment of the focal plane in step S8, before the examination of sample 2 can begin in step S5.

A fourth possibility for determining the aberration, here the spherical aberration, shows 12 Here, the inclined line grid structures 26 to 29 are provided with a fine structure, so that the individual line grid elements 40, 42 each have a plurality of significantly more closely spaced line elements 64, 66. In the right part of the 12 The position of the reflected images can be seen. On the left, the condition with insufficiently corrected spherical aberration is shown. The reflected images of the line grating elements 40, 42 are offset from the reflected images of the finer line grating elements 64, 66. This is caused by two gratings with different grating periods, since low and high spatial frequencies are imaged into different focus positions due to the spherical aberration. If, however, the spherical aberration is compensated, as 12 As shown in the right-hand illustration, the sharply focused sections for the coarser line grating structure 27 with its line grating elements 40, 42 in the detector are located on the same y-coordinates as the sharply focused sections for the finer subdivision according to the line elements 64, 66. In this way, the spherical aberration can be compensated for very easily, because the aberration compensation element, for example a correction ring, only has to be adjusted in such a way that the line gratings with the different grating constants are next to each other, i.e. on the same coordinate of the sensor, which designates the longitudinal extent of the line grating structure.

QUOTES CONTAINED IN THE DESCRIPTION

This list of documents submitted by the applicant was generated automatically and is included solely for the convenience of the reader. This list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

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Cited non-patent literature

• I. Papadopoulos et al., “Scattering compensation by focus scanning holographic aberration probing (F-SHARP),” Nature Photonics 11 (2), 116-123 (2016 [0006]
• Na Ji et al., “Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues”, Nature Method 7, 2 (2010 [0007]
• D. Ancora et al., “Spinning pupil aberration measurement for anisoplanatic deconvolution”, Optics Letters 46, 12 (June 15, 2021 [0007]
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• G. Zheng et al., “Characterization of spatially varying aberrations for wide field-of-view microscopy, Optics Express Vol. 21, No. 13 (July 1, 2013 [0007]
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Claims (14)

Microscope with an imaging beam path comprising an objective (4) and imaging a sample (2) from an image field onto a two-dimensional detector (8), and an illumination beam path (B) illuminating the sample (2) through the objective (4), which illumination beam path comprises a light source and is reflected into the imaging beam path (M) via a beam splitter (6) in front of the objective (4), and a measuring device (10) followed by a light modulator (16) comprising at least one intensity-modulating, first line grating structure (26-29), wherein the light modulator (16) is arranged obliquely to a plane conjugate to the focal plane of the objective (4) and is projected onto the sample (2) through the illumination beam path (B) and the objective (4), characterized in that - the at least one intensity-modulating, first line grating structure (26-29) is projected into a measuring region of the sample (2) that only partially fills the image field, and - the Measuring device (10) has a processor (32) which reads out image data supplied by the detector (8) and divides these into measurement data and display data for displaying a sample image based on a position in the image field, wherein the processor (32) selects as measurement data those image data which correspond to the measurement area of the sample (2) and evaluates the measurement data with regard to reflection images of the at least one line grating structure (26-29) projected onto the sample (2). Microscope after Claim 1 , characterized in that the processor (32) selects as display data those image data which correspond to a part of the image field not assigned to the measuring area, and provides the display data for displaying a sample image. Microscope according to one of the above claims, characterized in that the light modulator (16) has a central rectangular field (20), to the edges (22-25) of which an edge region is respectively connected, of which at least one has the intensity-modulating, first line grating structure (26-29), wherein the edge regions and thus the up to four first line grating structures (26-29) are projected onto edges of the image field (18) onto the sample (2). Microscope according to one of the above claims, characterized in that the measuring area extends along at least one edge of a central, preferably rectangular, field of the image field, wherein the display data originate from the field. Microscope according to one of the above claims, characterized in that the measuring range - imaged in the focal plane - corresponds to a maximum of 50% of the area of the image field, preferably a maximum of 40% or a maximum of 30% or a maximum of 20%, particularly preferably a maximum of 10%. Microscope according to one of the above claims, characterized in that - the imaging beam path has an adjustable aberration correction optics controlled by the processor, in particular in the form of a correction ring integrated into the objective, - the light modulator (12) has at least one intensity-modulating, second line grating structure (48-51) which is formed from second line elements (44, 46), wherein adjacent second line elements are at a distance from one another which is smaller than a distance between adjacent first line elements of the first line grating structure (26-29), and - the processor adjusts the aberration correction optics such that a contrast of the second line grating structure (48-51) is maximized in an image recorded by the detector (8). Microscope after Claim 6 , characterized in that the second line grid structure (48-51) is formed parallel to or overlapping the first line grid structure (26-29). Microscope according to one of the above claims, characterized in that - the objective is controlled by the processor for setting the focal plane, - the light modulator (12) has an edge structure (26-29) parallel to at least one of the first line grating structures (26-29) and - the processor sets several different positions of the focal plane on the objective, determines a value of the modulation transfer function for each position from an image contrast of the at least one edge structure and determines image aberrations, in particular a spherical aberration, of the image beam path therefrom. Microscope according to one of the above claims, characterized in that the light modulator (12) has at least one pair of first line grating structures (26-29), both of which are inclined to the conjugate plane and, when projected onto the sample (2), are opposite one another with respect to a center of the image field. Microscope after Claims 9 , characterized in that the light modulator (12) has two pairs of first line grating structures (26-29) lying opposite one another in the pair, so that four first line grating structures (26-29) are projected onto the sample (2) at edges of the image field (18). Microscope after Claims 10 , characterized in that the processor determines a displacement of the reflection images of opposite line grating structures and determines therefrom an indication of a tilt angle of the sample (2). Microscope according to one of the above claims, characterized in that the processor determines spherical aberrations from a single image recording of the reflex image of the at least one line grating structure by means of a phase retrieval algorithm. Microscopy method in which a sample (2) is imaged onto a two-dimensional detector (8) by an imaging beam path (M) comprising an objective (4) and is illuminated by an illumination beam path (B) comprising a light source and reflected into the imaging beam path (M) via a beam splitter (6) in front of the objective (4), wherein a light modulator (16) comprising at least one intensity-modulating, first line grating structure (26-29) is illuminated from a measurement illumination source (12) and projected through the objective (4) onto the sample (2), wherein the light modulator (16) lies obliquely to a plane conjugate to the focal plane of the objective (4) and is projected through the illumination beam path (B) and the objective (4) onto the sample (2), characterized in that - the at least one intensity-modulating, first line grating structure (26-29) is projected into a measurement region of the sample (2) which covers the image field only partially fills, and - during operation of the measuring illumination source (12) image data of the detector (8) are separated into measurement data and display data, wherein the measurement data correspond to the measurement area of the sample (2), but not to image areas which correspond to the edge areas, and the measurement data are evaluated with regard to reflection images of the at least one line grating structure (26-29) projected onto the sample (2), and thus the detector (8) on which the sample (2) is imaged is also used as a measurement detector. Microscopy method according to Claim 13 , characterized in that a microscope according to one of the Claims 1 until 12 is used.