METHOD FOR CALIBRATING A PHASE MASK AND MICROSCOPE

Description

[0001] The invention relates in a first aspect to a method for calibrating a phase mask, in particular an SLM, in a beam path of a Light sheet microscope. In a second aspect, the invention relates to a light sheet microscope, which is particularly suitable for carrying out the method according to the invention.

[0002] A generic microscope for examining a sample has the following components: at least one light source for emitting illuminating light, at least one microscope objective for directing the illuminating light onto the sample and optical means for directing detection light from the sample onto a camera, a phase mask arranged in the beam path, the camera for measuring light in a detection beam path and a control and evaluation unit for controlling the phase mask and the camera and for evaluating light measured by the camera.

[0003] Structured illumination is required in various microscopy applications. For example, light-sheet microscopy requires illumination resembling a thin sheet. This means that a so-called light sheet must be created that is as thin as possible in a first transverse direction, wide in a second transverse direction, and as long as possible in the direction of light propagation. To achieve this, Bessel beams can be coherently superimposed, for example. It is also known to specifically utilize interference effects between individual Bessel beams to create an extended and structured light sheet.

[0004] Also known are so-called Sinc 3< beams, which allow a cuboid-shaped light sheet in the sample with only small secondary maxima.

[0005] Another application for phase masks, particularly spatial light modulators (SLMs), is laser scanning microscopy, where a three-dimensional volume is scanned with an illumination focus. In fluorescence microscopy, certain techniques are used to specifically optically bleach regions of a sample. This also requires appropriate spatial structuring of the light used for bleaching. A prominent application of structured illumination is superresolution microscopy, in which the point spread function is specifically shaped. For example, in the STED method, the point spread function used for illumination has the shape of a doughnut.

[0006] The aforementioned examples of structured illumination can be realized using phase masks, such as spatial light modulators (SLMs). There are essentially two different types of SLMs, which differ in the liquid crystals used. Nematic SLMs allow a maximum continuously adjustable phase shift of 0 to 6π, but are comparatively slow. They typically have refresh rates of around 60 Hz. Special nematic SLMs can achieve repetition rates of up to 500 Hz. High diffraction efficiencies of over 90 percent can be achieved with nematic SLMs.

[0007] Ferroelectric SLMs can only switch between the phase shift state of 0 and the phase shift state of π. The advantage of ferroelectric SLMs is their high speed. Refresh rates of up to 4 kHz can be achieved. However, the diffraction efficiency is comparatively low at around 10 percent.

[0008] What both types of SLMs have in common is that the achievable phase shift depends on the wavelength of the incident laser light.

[0009] A nematic SLM should be calibrated to exhibit a continuous phase shift from 0 to 2π. However, this phase shift can only be adjusted for one design wavelength. As the SLM is irradiated with light of a different wavelength, the phase shift imparted to this light deviates from 2π. At shorter wavelengths, the phase shift increases; at longer wavelengths, the phase shift decreases.

[0010] The situation is similar with a ferroelectric SLM. Here, too, the phase shift of π is only achieved for one design wavelength. At wavelengths other than this, phase shifts deviating from π are generated.

[0011] These properties of the available SLMs directly impact the generation of the structured illumination examples given above. These beams can only be optimally generated if the phase shift is either continuously adjustable from 0 to 2π, for example, for Bessel and Mathieu beams, or is exactly π, for example, for coherently superimposed Bessel or coherently superimposed Sinc 3< beams.

[0012] An SLM is usually controlled via a control and evaluation unit, typically a PC, which provides the SLM with a grayscale image. The grayscale values of the individual pixels are converted into voltages within the SLM and thus correspond to the phase shifts of the individual SLM pixels. For example, with a bit depth of 8 bits, a gray value of 0 in the image can correspond to a phase shift of 0, and a gray value of 255 can correspond to a phase shift of 2π. As described above, the SLM can only be used optimally if the wavelength-dependent phase shift is calibrated accordingly. This means, for example, that a table is provided to the SLM that assigns the respective voltages to the gray values that result in the desired phase shift.

[0013] The company Holoeye has described a measurement setup for calibrating an SLM in which a laser beam is split into two parts, and each of the two partial beams is directed onto a sub-area of the SLM to be calibrated. Both beams are superimposed on a camera, and the resulting striped interference pattern is recorded. Different gray values are applied to the two sub-areas of the SLM to change the relative phase of the two beams and thus shift the interference pattern on the camera. The shift of the interference maxima on the camera is directly proportional to the relative phase shift. With this calibration method, the SLM is calibrated for a desired wavelength before it is permanently installed in an optical device, such as a microscope. Subsequent calibration is not possible with this method without removing the SLM from the respective device.

[0014] In Zheng Zhang et al.: "Simple method for measuring phase modulation in liquid crystal televisions" (OPTICAL ENGINEERING / September 1994 / Vol. 33 No. 9 / p. 3018), a method for measuring phase modulation in liquid crystal displays (LCTVs) is disclosed. In connection with Figure 1 A setup is described there in which a Ronchi grating with variably adjustable gray levels is used.

[0015] US 2015 / 0323787 A1 relates to a microscope with a phase mask. For optimizing the control of the phase mask, reference is made to the aforementioned publication by Zheng Zhang et al.

[0016] One object of the present invention can be considered to provide a method for calibrating a phase mask in an optical device that is particularly inexpensive to implement. Furthermore, a microscope is to be created in which a phase mask present therein can be calibrated without great effort.

[0017] This object is achieved by the method having the features of claim 1 and by the microscope having the features of claim 12.

[0018] Advantageous variants of the method according to the invention and preferred embodiments of the microscope according to the invention are described below, in particular in connection with the dependent claims and the figures.

[0019] In the method according to the invention for calibrating a phase mask in a beam path of an optical device, the following method steps are carried out: The phase mask is successively controlled with different patterns of gray values, wherein a first gray value of a first subset of segments remains constant and wherein a second gray value of a second subset of segments is varied from one pattern to the next pattern, the phase mask is exposed to light from the optical device, at least a portion of the total intensity of the light in the beam path is measured downstream of the phase mask for the different patterns and a characteristic curve of the measured intensity is obtained as a function of the second gray value, from the characteristic curve a relationship is determined between the second gray value and a phase offset imposed by the phase mask,obtained and a control of the phase mask is calibrated based on the obtained relationship between gray value and phase shift.

[0020] The microscope of the type specified above is further developed according to the invention in that the control and evaluation unit is configured to control the phase mask successively with different patterns of gray values, wherein a first gray value of a first subset of segments remains constant and wherein a second gray value of a second subset of segments is varied from one pattern to the next pattern, to control the camera for measuring at least part of the total intensity of the light in the beam path downstream of the phase mask for the different patterns, to form a characteristic curve of the measured intensity as a function of the second gray value, to obtain from the characteristic curve a relationship between the second gray value and a phase shift which is imposed by the phase mask, and to calibrate a control of the phase mask on the basis of the obtained relationship between the gray value and the phase shift.

[0021] For the purposes of this description, a phase mask is understood to be an optical device with a plurality of controllable segments, each of which imposes a defined phase shift on passing or reflected light, which varies depending on the control of the corresponding segment. In principle, phase masks can be used in which the segments are divided only in one spatial direction. An example of this would be a phase mask with strip-shaped segments.

[0022] Phase masks with a two-dimensional structure are particularly preferred. Such phase masks can be referred to as 2D phase masks and can, in particular, have a pixel structure with pixel rows and pixel lines. The individual adjustable or controllable segments of the phase mask are therefore individual pixels. The possibilities for beam manipulation are particularly diverse with such two-dimensional phase masks.

[0023] In principle, ferroelectric SLMs can be used as phase masks. Nematic spatial light modulators (SLMs) are typically used as phase masks due to their variable adjustment options.

[0024] Calibration of a phase mask is the process in which a defined assignment of a control, for example a digital control value, to a desired phase shift of the respectively controlled segment of the phase mask is provided.

[0025] In principle, all devices that use phase masks for the controlled and defined micro-influence of light can be considered as optical devices.

[0026] The main areas of application for the method according to the invention will be in microscopes, where the illumination light, in particular, but also the detection light, is to be structured in a variety of ways.

[0027] For example, the optical device according to the invention can be a light-sheet microscope. Advantageous applications of the method, which are not part of the invention, also exist for photomanipulators for the micromanipulation of dyes as optical devices.

[0028] In the context of this description, the term "gray value" refers to the control value applied to a specific segment of the phase mask. In particular, this can be a digital control value, for example, with a bit width of 8 bits. This value can therefore assume values from 0 to 255. Control means that a specific control value is applied to a specific segment or a specific pixel. This pixel is thereby placed in a state in which it imposes a specific phase shift on passing or reflected light. How large this phase shift is for a specific control value is not initially clear. The aim of the method according to the invention is to determine this.

[0029] A pattern of gray values is understood to be certain combinations of control values for the various segments, in particular pixels, of the phase mask used.

[0030] A subset of segments is a specific subset of segments that can, in principle, be distributed arbitrarily on the phase mask and, in particular, do not have to be adjacent.

[0031] For the purposes of describing the present invention, the term "light from the optical device" refers to light that is to be manipulated by the phase mask during the operation of the optical device. For example, in the case of microscopes, this refers to the illumination light applied to a sample.

[0032] According to the invention, at least a portion of the total intensity of the light in the beam path downstream of the phase mask is to be measured for the different patterns. This specifically means that a portion of the beam cross-section is evaluated in terms of intensity. In particular, the portion of the total intensity can be the light of at least one diffraction order, for example, the zeroth or the first diffraction order.

[0033] The term characteristic curve of the measured intensity as a function of the second gray value refers to the totality of the value pairs (set second gray value, intensity measured for this gray value).

[0034] A key element of calibration is determining the actual relationship between a specific gray value and the phase shift actually caused by that gray value. Knowing this relationship ultimately allows the phase mask control to be calibrated based on this relationship between gray value and phase shift.

[0035] A significant advantage of the present invention is that the phase masks can, in principle, also be calibrated while installed in the respective optical device, in particular in a microscope. Such subsequent calibration may be necessary, for example, if a microscope is operated at different temperatures or if a control unit of the SLM, for example, an FPGA (Field Programmable GateArray) must be replaced due to a defect. Aging of an SLM can also alter the original relationship between gray value and phase shift, which may also necessitate recalibration. These subsequent calibrations can be performed using the method according to the invention and can be performed on the microscope according to the invention without having to remove the respective phase mask. Another significant advantage of the present invention in this context is that complex adjustments in the beam path of the optical device are not necessary because the phase mask to be recalibrated does not have to be removed.

[0036] In principle, there is considerable flexibility in the patterns applied to the phase masks. The key is that the control for a first group or subset of segments remains unchanged, and that the control for a second subset of segments changes from one pattern to the next.

[0037] In principle, the patterns can also be non-periodic. However, the patterns are particularly preferably periodic in at least one spatial direction in the plane of the phase mask.

[0038] For example, the set patterns of gray values can be patterns for creating a Dammann grid.

[0039] In particularly preferred variants of the method according to the invention, the second gray value is varied across the entire dynamic range of the phase mask. Preliminary work leading to the present invention has shown that calibration can then be performed based on comparatively simple mathematical relationships.

[0040] In principle, separate beam deflection devices can be provided for calibration purposes, with which the illumination light used for the calibration measurements is directed to the camera.

[0041] In a variant not forming part of the invention, a special fluorescent sample can be used for calibration measurements, particularly in the case of a laser scanning microscope or a wide-field microscope.

[0042] To implement the method according to the invention, however, it is fundamentally sufficient if light influenced by the phase mask reaches the camera or a detector in some way. According to the invention, light reflected from a slide or coverslip is used for the calibration measurements. The measures required to perform the calibration measurements are then extremely simple.

[0043] According to the invention, any filters that may be present in the detection beam path for the calibration measurements are removed from the beam path. The invention has recognized that the relationship between the gray value and the phase shift can be determined comparatively easily in a particularly preferred variant of the method according to the invention, in which light of the zeroth diffraction order is blocked with a spatial filter downstream of the phase mask and then an integrated intensity of the beam path is measured downstream of the spatial filter. The relationship between the second gray value and the phase shift can then be determined by comparison with I = const * 1 − cos φ be obtained.

[0044] In a corresponding variant of the microscope according to the invention, a diaphragm for blocking light of the zeroth diffraction order is provided downstream of the phase mask.

[0045] A similarly simple relationship exists if only light of the zeroth diffraction order is measured for calibration. The relationship between the second gray value and the phase shift can then be determined by comparing it with I = const * 1 + cos φ be obtained.

[0046] A corresponding embodiment of a microscope according to the invention is characterized in that an aperture is provided for blocking the light from diffraction orders greater than 0.

[0047] In principle, there can be a separate camera that is used only for the measurements to calibrate the phase mask.

[0048] A particular advantage of the invention, however, is that the same camera used for the actual microscopy measurements can be used to measure the intensity. The equipment required to perform the calibration measurements is therefore extremely minimal.

[0049] The microscope according to the invention can be configured in particular to carry out the method according to the invention.

[0050] According to the invention, the phase mask provides structured illumination in the form of, for example, an intensity grating. The phase mask can expediently be arranged in a plane optically conjugate to a sample plane. Such a setup is suitable, for example, for a wide-field microscope. The acquired microscopy data can then be subjected to SIM or apotomic processing. On the SLM, for example, a striped phase pattern can be displayed consisting of lines that alternately have a gray value of 0 and 128. For an ideally calibrated SLM, this corresponds to a phase shift of 0 or π. By removing the laser blocking filters, the back reflection can be measured. The back reflection can come from a separate mirror (not part of the invention) or from a slide or coverslip.

[0051] In other applications, phase masks are used for wavefront manipulation. For example, aberrations can be corrected. For these purposes, the phase mask is preferably arranged in a plane optically conjugate to the rear objective pupil.

[0052] Further advantages and features of the present invention are explained below with reference to the accompanying figures, in which: Figure 1: a schematic representation of a microscope according to the invention; Figure 2: an exemplary representation of a beam path in a beam shaping module of a microscope according to the invention; Figure 3: a first schematic representation of an exemplary phase mask for explaining the method according to the invention; Figure 4: a second schematic representation of the phase mask from Figure 3to explain the method according to the invention; Figure 5: a first diagram to explain the method according to the invention and Figure 6: a second diagram to explain the method according to the invention.

[0053] An embodiment of a microscope 100 according to the invention and variants of the method according to the invention are described with reference to Figures 1 to 6 described. Identical and equivalent components are generally identified by the same reference numerals in the figures.

[0054] Figure 1shows a schematic diagram of an inverted light sheet microscope, which has, as essential components, a light source 10, for example a laser module, for emitting illumination light 11, a microscope objective 30 for directing the illumination light 11 onto a sample 48 to be examined, and further optical means 40, 50, 64 for directing detection light 54 from the sample 48 to a camera 62, 66. In order to generate the beam shape of the illumination light 11 suitable for light sheet microscopy, a beam shaping module 20 is provided, which is arranged immediately downstream of the light source 10.

[0055] The excitation light 11 then passes through a scanner 12 into the microscope objective 30 and from there via a meniscus lens 40 onto the sample 48. The sample 48 is located in the Figure 1In the example shown, the sample slide 44 is arranged in a bowl-shaped slide, which is transparent to both the excitation light 11 and the detection light 54, which is emitted by the sample 48 upon irradiation with excitation light 11, in particular fluorescent light. The sample 48 can be surrounded by water 46. The slide 44 is held by a sample table 42, which can be positioned in a known manner in all three spatial directions x, y, z. In the example shown, the light sheet is irradiated onto the slide 44 at an angle of 45 degrees to the normal direction of the slide.

[0056] A piezomechanism 32 is provided for positioning the microscope objective 30 in a direction along its optical axis.

[0057] The detection optics include the meniscus lens 40 and a detection lens 50 as essential components. A piezoelectric mechanism 52 is provided for positioning the detection lens 50. After passing through the detection lens 50, the detection light 54 strikes a beam splitter 64 and then reaches either a first camera 62 or a second camera 66. The beam splitter 64 can, for example, be a color splitter, so that different dyes can be observed with the first camera 62 and the second camera 66, respectively.

[0058] The structure of the beam forming unit 20 with further details is shown in Figure 2The incoming light 21, which can in particular have a Gaussian beam profile, first reaches a first cylindrical lens 22, which together with a second cylindrical lens 23 forms a first telescope. A phase mask 80, in particular a nematic SLM, is arranged in a collimated part of the beam path. After reflection by the phase mask 80, the light passes via a fourth lens 24 and a fifth lens 26, which form a second telescope, and finally reaches the exit plane 27. Between the third lens 24 and the fourth lens 26 is an aperture 25. The aperture 25 can in particular be a circular disk-shaped aperture, with which only the zeroth diffraction order is blocked out in the central region of the beam profile. In an alternative variant, the aperture is a pinhole aperture, which precisely transmits only the zeroth diffraction order. The phase mask is arranged in a plane optically conjugate to the exit plane 27.For the in . Figure 1 In the illustrated embodiment, this is preferably an intermediate image plane. This means that plane 27 is optically conjugated to the sample plane of the microscope objective 30.

[0059] In principle, however, the beam shaping module 20 can also be positioned in a microscope such that the plane 27 and thus also the plane in which the phase mask 80 is arranged lie in pupil planes, i.e. in planes that are conjugated to a rear objective pupil.

[0060] To control the SLM 80, the microscope 100 includes a control and evaluation unit 70, typically a PC. Using the control and evaluation unit 70, the phase mask 80 can be controlled with various patterns Gi(x,y), Gj(x,y) of gray values G.

[0061] In the method according to the invention for calibrating the phase mask 80, the following steps are now carried out: the phase mask 80 is controlled sequentially with different patterns Gi(x,y) of gray values G. In this case, a first gray value G1 of a first subset 91 of segments remains constant and a second gray value G2 of a second subset 92 of segments is varied from one pattern Gi(x,y) to the next pattern Gj(x,y). With reference to the schematic Figures 3 and 4 This will be explained in more detail. Figures 3 and 4 each show the same phase mask 80, which is controlled with different patterns of gray values. Figure 3 the phase mask 80 is provided with the pattern Gi(x,y) and in Figure 4controlled with the pattern Gj(x,y). The patterns Gi(x,y) and Gj(x,y) each have a regular stripe shape, meaning they are periodic in the x-direction. In principle, such a pattern could be realized with a phase mask that has stripe-shaped segments. However, 2D phase masks with pixels arranged in rows and columns are typically used. For example, SLMs with resolutions of up to 1280 x 1024 are available.

[0062] The patterns Gi(x,y) and Gj(x,y) are characterized by the fact that the gray value of a first subset 91 of segments does not change. This means that the areas 91 in Figure 3 and Figure 4 each have the same gray value G1. The second gray value G2, which is used to control the segments of a second subset 92, differs in the pattern Gj(x,y) from Figure 4 compared to the pattern Gi(x,y) from Figure 3 . This is shown schematically by a different hatching of the areas 92 in the Figures 3 and 4indicated.

[0063] However, it is not necessary to apply a periodic pattern to the phase mask. Any aperiodic pattern can also be used, since such patterns also contain higher spatial frequencies and lead to the desired diffraction effects.

[0064] One possibility that the inventive method can be used with the Figure 1 The aim of the present invention is to carry out the setup shown schematically by coupling the light beam modulated by means of the phase mask 80 (in the example described, this is a light sheet) directly into the detection beam path using a deflection mirror 49, which is arranged at the same location as the sample 48, and thus to image the light sheet directly onto one of the cameras 62, 66.

[0065] Instead of the deflection mirror 49, a special fluorescent sample 48 can also be used.

[0066] An even simpler option, which even requires no additional components in the microscope, is to use a reflection on the slide 44. Due to the geometry described above, the light from the light sheet is reflected from the slide 44 directly into the detection objective 50.

[0067] In principle, it is also possible to decouple the beam from the beam path before entering the sample 48 or before hitting the slide 44 and direct it to a separate camera. Such a separate camera could also be permanently installed in the microscope, with the decoupled beam toward this separate camera being achieved using a beam splitter.

[0068] If, as described above, the zeroth diffraction order is masked out, i.e., if the aperture 55 is a circular disk-shaped aperture, only higher diffraction orders interfere on the camera 62 or 66, and a periodic intensity grating is visible. However, this intensity grating on the camera as such plays no role in the actual calibration. What is essential is that the intensity I of the total light incident on the camera is measured. This is the total intensity of the beam path minus the masked light of the first diffraction order. According to the invention, these intensities are now measured for a plurality of different patterns Gi(x,y), wherein, as described, the first gray value G1 of a first subset 91 of segments remains constant and the second gray value G2 of a second subset 92 of segments is changed.

[0069] Particularly preferably, the second gray value G2 is varied over the entire dynamic range of the phase mask 80.

[0070] From the measurement data, a characteristic curve I(G2) of the measured intensity (I) is obtained as a function of the second gray value (G2). Figure 5 shows an example of such a characteristic curve, in which the intensity normalized to the maximum value Imax is plotted against the gray value G2.

[0071] The total intensity of the light incident on camera 62 or 66 is: I = const 1 − cosφ , where φ is the phase shift actually imposed by the phase mask 80. With this relationship, the phase shift φ as a function of the second gray value G2 can be obtained from the intensity measured as a function of G2. The dependence of the phase shift φ on the set gray value G2 is for the measurement data of the Figure 5 in the diagram in Figure 6 where the phase shift φ is plotted against the gray value G. Figure 6 can be considered as the result of the calibration of the phase mask 80 and in principle provides the desired information on how the phase mask 80 must be controlled in order to achieve a certain desired phase shift φ. For example, the information of the Figure 6 be stored in a table in the control and evaluation unit 70.

[0072] To implement the method according to the invention, it is not necessary to mask out the zeroth diffraction order. If the total intensity of the beam, including the zeroth diffraction order, is measured, the modulation depth of the intensity, i.e., the difference between the maximum and minimum intensity in the camera image, must be measured instead of the total intensity incident on the camera.

[0073] It is also possible to use only the zeroth diffraction order light for the measurement. The measured intensity I is then related to the phase shift φ as follows: I = const 1 + cosφ .

[0074] Finally, the phase mask 80 does not necessarily have to be positioned in an intermediate image plane. In principle, the phase mask 80 can be positioned in any plane. For example, if the phase mask 80 is positioned in a pupil plane, the spatial frequency spectrum of the grayscale pattern can be seen on the camera. In this case, the intensity measurement is performed, for example, precisely in the camera image area corresponding to the first diffraction order or, as described above, the zeroth diffraction order.

[0075] In principle, the measurement data from Figure 6can be further processed, for example, into so-called gamma values, and programmed directly into the SLM control system. However, these additional steps no longer concern the actual calibration procedure, but rather the implementation in a special device.

[0076] In a similar way to the context of Figure 1While described for a light sheet microscope, the method according to the invention can also be applied to a laser scanning microscope in which an SLM is used for beam shaping. Here, too, the SLM can in principle be positioned in an intermediate image plane. However, in a laser scanning microscope the SLM is usually arranged in a pupil plane for wavefront modulation. If the filters in the detection beam path, which ensure that no excitation radiation reaches the camera during actual measurement operation, are removed, the back reflection can be measured. The back reflection can come from a specially installed mirror or, as described above for the light sheet microscope, from the specimen slide. Alternatively, a special fluorescent sample, for example a thin fluorescent layer, can also be used here.

[0077] In a laser scanning microscope, patterns that create Dammann gratings can be used as patterns for the gray values. By modulating the gray value and measuring the intensities of at least one diffraction order as a function of the gray value, the phase shift as a function of the gray value can be determined from the relationship I = const 1 + cosφ . be determined.

[0078] The phase mask, especially the SLM, can also be part of a photomanipulator that is used to selectively switch dyes on and off, for example in a cell nucleus.

[0079] To correct aberrations, the phase mask, in particular the SLM, can also be used in the detection beam path. For example, the SLM can be positioned in a pupil shared by the illumination beam path and the detection beam path. The inventive method for calibrating the SLM can then be used as described in connection with Figure 1described. If the SLM is located in a pupil that is only used by the detection beam path, a back reflection, for example from the specimen slide, can be measured by removing laser blocking filters, as with a laser scanning microscope. Both the illumination and detection sides can be focused on the specimen slide. In this case, the SLM is fully illuminated in the pupil. If the SLM is controlled with a periodic pattern, the spatial frequency spectrum of this pattern can be seen on the camera. As described above, the intensity of the first diffraction order can then be measured and used to calibrate the SLM.

[0080] The present invention provides a novel method for calibrating phase masks, particularly in microscopes. The key advantage of this method is that it can, in principle, be performed in-situ on an operational apparatus without having to dismantle the SLM. Complex adjustment work on the optics is therefore no longer necessary. List of reference symbols

[0081] 10Light source 11Illumination light / excitation light 12Scanner 20Beam shaping unit 21Incoming beam, in particular Gaussian beam 22First lens, for example cylindrical lens 23Second lens, for example cylindrical lens 24Third lens 25Pinhole 26Fourth lens 27Plane optically conjugate to the plane of the phase mask 30Microscope objective 32Piezo mechanics 40Meniscus lens 42Sample stage 44Slide 46Water 48Sample, in particular fluorescent sample 49Separate beam deflection means 50Detection objective 52Piezo mechanics 54Detection light 62Camera 64Beam splitter 66Camera 64Beam splitter 70Control and evaluation unit 80Phase mask 91First subset of segments 92Second subset of segments 100Microscope GGray value G1first gray value G2second gray value Gi(x,y), Gj(x,y) different patterns of gray values G I measured part of the intensity of the light in the beam path I(G2) characteristic curve of the measured intensity as a function of the second gray value G2 LSM laser scanning microscope SLM spatial light modulator φ(G) relationship between the second gray value G2 and a phase shift φ imposed by the phase mask 80,

Claims

1. Method for calibrating a phase mask, in particular a spatial light modulator (SLM), in a beam path of a light-sheet microscope (100) for examining a sample, wherein the light-sheet microscope comprises at least one light source (10) for emitting illumination light (11), at least one microscope objective (30) for guiding the illumination light (11) onto the sample (48), and optical means (40, 50, 64) for guiding detection light (54) from the sample (48) onto a camera (62, 66), wherein the phase mask (80) to be calibrated is arranged in the beam path between the light source (10) and the at least one microscope objective (30) of the light-sheet microscope, wherein in the method the following method steps are performed: the phase mask (80) is actuated successively with different patterns (Gi(x,y)) of grey levels (G), wherein a first grey level (G1) of a first partial quantity (91) of segments remains constant, and wherein a second grey level (G2) of a second partial quantity (92) of segments is varied from one pattern (Gi(x,y)) to the next pattern (Gj (x,y)), at least one part (I) of the total intensity of the light in the beam path is measured downstream of the phase mask (80) for the different patterns (Gi(x,y)) and a characteristic (I(G2)) of the measured intensity (I) is obtained in dependence on the second grey level (G2), a relationship (φ(G)) between the second grey level (G2) and a phase shift (φ), which is imprinted by the phase mask (80), is obtained from the characteristic (I(G2)), and an actuation of the phase mask (80) is calibrated on the basis of the obtained relationship (φ(G)) between grey level (G2) and phase shift (φ), characterized in that the phase mask (80) for the calibration measurements is exposed to light from the light source (10), in that light reflected at an object carrier (44) for holding a sample or at a cover slip is used for the measurements for the calibration, wherein either the detection beam path is free of filters for blocking illumination light or filters for blocking illumination light in the detection beam path are removed from the beam path for the measurements for the calibration, and in that a structured illumination in the form of an intensity grid is provided by the phase mask (80).

2. Method according to claim 1, characterized in that the phase mask (80) is a 2D phase mask with pixel rows and pixel lines.

3. Method according to claim 1 or 2, characterized in that the phase mask (80) is a nematic spatial light modulator (SLM).

4. Method according to any one of claims 1 to 3, characterized in that the patterns (Gi(x,y)) are periodic in at least one spatial direction (x, y).

5. Method according to any one of claims 1 to 4, characterized in that the patterns (Gi(x,y)) are patterns for producing a Dammann grating.

6. Method according to any one of claims 1 to 5, characterized in that the second grey level (G2) is varied over the entire dynamic range of the phase mask (80).

7. Method according to any one of claims 1 to 6, characterized in that the illumination light used for the measurements for the calibration is guided via separate beam deflection means (49) to a camera (62, 66) which is used for the measurements for the calibration.

8. Method according to any one of claims 1 to 7, characterized in that light of the zero order of diffraction is blocked using a spatial filter downstream of the phase mask (80), in that an integrated intensity (I) of the beam path is measured downstream of the spatial filter, and in that the relationship (φ(G)) between the second grey level (G2) and the phase shift (φ) is obtained by a comparison with I = const*(1-cos φ).

9. Method according to any one of claims 1 to 8, characterized in that only light of the zero order of diffraction is measured for the calibration, and in that the relationship (φ(G)) between the second grey level (G2) and the phase shift (φ) is obtained by a comparison with I = const*(1+cos φ).

10. Method according to any one of claims 1 to 9, characterized in that a separate camera is present, which is used only for the measurements for the calibration of the phase mask (80).

11. Method according to any one of claims 1 to 9, characterized in that the camera (62, 66) that is also used for the actual microscopy measurements is used for measuring the intensity (I).

12. Light-sheet microscope for examining a sample and operationally set up for carrying out the method according to any one of claims 1 to 11, comprising: at least the light source (10) for emitting illumination light (11), at least the microscope objective (30) for guiding the illumination light (11) onto the sample (48), and optical means (40, 50, 64) for guiding detection light (54) from the sample (48) onto a camera (62, 66), a phase mask (80) arranged in the beam path, the camera (62, 66) for measuring light in a detection beam path, an object carrier (44) for holding the sample, or a cover glass, and a control and evaluation unit (70) for actuating the phase mask (80) and the camera (62, 66) and for evaluating the light measured by the camera (62, 66), wherein the control and evaluation unit (70) is set up for actuating the phase mask (80) successively with different patterns (Gi(x,y)) of grey levels (G), wherein a first grey level (G1) of a first partial quantity (91) of segments remains constant, and wherein a second grey level (G2) of a second partial quantity (92) of segments is varied from one pattern (Gi(x,y)) to the next pattern (Gj(x,y)), actuating the camera (62, 66) for measuring at least one part (I) of the total intensity of the light in the beam path downstream of the phase mask (80) for the different patterns (Gi(x,y)), forming a characteristic (I(G2)) of the measured intensity (I) in dependence on the second grey level (G2), obtaining from the characteristic (I(G2)) a relationship (φ(G)) between the second grey level (G2) and a phase shift (φ), which is imprinted by the phase mask (80), and calibrating an actuation of the phase mask (80) on the basis of the obtained relationship (φ(G)) between grey level (G2) and phase shift (φ), characterized in that light reflected at the object carrier (44) or at the cover slip is used for the measurements for the calibration, in that the detection beam path is free of filters for blocking illumination light, or in that filters for blocking illumination light are removed from the detection beam path for the measurements for the calibration, and in that the phase mask (80) provides a structured illumination in the form of an intensity grid.

13. Light-sheet microscope according to claim 12, characterized in that the phase mask (80) is arranged in a plane which is optically conjugate to a sample plane.

14. Light-sheet microscope according to claim 12, characterized in that the phase mask (80) is arranged in a plane which is optically conjugate to a rear objective pupil and which in particular is used by an excitation beam path and the detection beam path.

15. Light-sheet microscope according to any one of claims 12 to 14, characterized in that a stop for blocking light of the zero order of diffraction is present downstream of the phase mask (80).

16. Light-sheet microscope according to any one of claims 12 to 14, characterized in that a stop for blocking light of orders of diffraction greater than 0 is present.