Patent Application
20240385421
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The invention relates to a method and to an apparatus for high resolution microscopy, in particular 3D microscopy (i.e. microscopy for obtaining three-dimensional information from a sample).
In structured illumination microscopy (abbreviated to SIM), which is already generally known, a sample is illuminated in a structured manner and the fluorescent light is detected using a camera. Several images of the sample are acquired, wherein the structured illumination is varied between these images. In this regard, periodic structures with many illumination lines or illumination points are used for the illumination and are slightly displaced laterally on the sample from acquisition to acquisition. Next, a homogeneously illuminated image of the sample can be reconstructed from several of these acquisitions (what are known as raw images), to some extent already with a resolution below the diffraction limit.
For SIM microscopy, until now, 1D line lattices have mostly been used. In order to increase the resolution or in order to obtain good confocality (in the context of the invention, defined as the suppression of fluorescent light which is generated in the sample outside the focal plane), the sample had to be acquired with different orientations of that 1D lattice illumination. Thus, the illumination not only had to be displaced laterally, but it also had to be rotated. This can be technically complicated, and meant that very many raw images had to be acquired in order to reconstruct an image of the sample.
However, with illumination patterns which are modulated in two dimensions, it is possible to obtain images with a higher resolution and/or good confocality with only lateral displacements; see M. Schropp and R. Uhl, “Two-dimensional structured illumination microscopy” in Journal of Microscopy, 2014, 256 23-36.
In almost all SIM systems, a field of view which is the size of the camera sensor (full surface area) is illuminated. In contrast, US 2014/313576 proposes a SIM method in which the sample is illuminated with only a narrow light strip which is modulated with the lattice. The detection of the fluorescent light is carried out here in only a restricted region of the camera (what is known as the “region of interest”, abbreviated to ROI), the position of which is selected to match that of the narrow light strip. Using what are known as rolling shutter (RS) cameras, inter alia, successive acquisitions can be made continuously. In this regard, the modulated illuminating strip is scanned over the sample synchronously with the moving ROI of the rolling shutter camera. Light from planes outside the focus are hereby largely not detected by the camera (in contrast to sheet illumination), because it impinges on the camera outside the active ROI. Because the out-of-focus light is only suppressed in the regions outside the line of illumination, this method is also spoken of as line-confocal detection. With the aid of suitable algorithms, an image can then be computed from several such confocally acquired structured illuminated raw images, with even better suppression of the out-of-focus light.
An often interesting factor in SIM microscopy is the fraction of the sample surface which is illuminated in a raw image, or in other words the density of the illumination points. If the sample is illuminated by the image of a transmitting amplitude lattice, this corresponds to the fraction of the transmitting area of the lattice, what is known as the fill factor. In order to obtain a high out-of-focus light suppression (confocality), then for each raw image, only a relatively small portion of the sample surface, concentrated as far as possible but also distributed, must be illuminated (point pattern). This means that what is known as “crosstalk” of the fluorescence response from different illumination points is minimised. “Crosstalk” in this case means that fluorescent light from one illumination point arrives at a position on the camera sensor which belongs to another illumination point. This crosstalk deteriorates the confocality and the signal-to-noise ratio (abbreviated to SNR).
Various methods can be used to remove the remaining out-of-focus signal from the acquired raw images and the confocal image of the samples can be computed. The simplest method here is certainly the “2D pinholing” method, in which the out-of-focus light is suppressed on the raw images by setting the pixels outside the regions associated with the illumination points to zero. This “2D pinholing” is mentioned, for example, in US 2003/0021016 A1. US 2003/0021016 A1 describes an arrangement in which a diffractive beam splitter generates a plurality of illumination points, so that in order to increase the acquisition rate over the conventional single point confocal microscopy, at each point in time, a plurality of regions of the sample are simultaneously illuminated in parallel. US 2006/0017001 A1 describes a microscopy arrangement in which, instead of a single-point or multiple-point illumination, a line in the sample is illuminated and reproduced on a detector. DE 10 2007 009 550 A describes a SIM microscopy apparatus in which a pattern of illumination points is focussed into a sample.
For both confocality and also to improve the resolution, patterns with primitive vectors “a” and “b” with the same length, i.e. symmetrical 2D patterns, are used here.
The objective of the present invention is to provide a method and an apparatus for high resolution microscopy which is more rapid than conventional methods and which improves the signal-to-noise ratio in the compiled image of the sample. In particular, the method and the apparatus should be suitable for 3D microscopy.
The objective is achieved by a microscopy method in accordance with the objective described above or by a microscope apparatus that addresses these short comings. Advantageous further embodiments form the subject matter of the respective dependent claims.
In a first aspect, the invention concerns a microscopy method, wherein
Accordingly, described here is a microscopy method, wherein an illuminating light (27a) produced by a light source (27) illuminates a sample (32) over an illuminating beam path (24), an objective (20) is disposed in the illuminating beam path (24) in order to focus the illuminating light (27a) into the sample (32), a detector (31) with an adjustable region of interest (12) detects radiation (25a) from the sample (32) over a detecting beam path (25) containing the objective (20), an illumination modulator (17) is disposed in the illuminating beam path (24), optics (28) disposed in the illuminating beam path (24) together with the illumination modulator (17) produce a light strip (3) of the illuminating light (27a) in the sample (32) at the focus of the objective (20), wherein the light strip (3) comprises a plurality of points of illumination (8, 11) and has an axial direction (3a) which is transverse to the illuminating beam path (24) at the location of the sample (32), a scanning unit (29) disposed in the illuminating beam path (24) is controlled in order to displace the light strip (3) of points of illumination (8, 11) in the sample (32) in a scanning direction (4) which is transverse to the direction of propagation of the illuminating beam path (24), characterized in that during a scan of the light strip (3) of the illuminating light (27a) in the sample (32), the illumination modulator (17) produces an illumination pattern (17a) which has a plurality of points of illumination (8, 11) which are disposed in the form of an asymmetrical 2D Bravais lattice (G4, G5, G6) with a first, longer primitive vector (a) and a second, shorter primitive vector (b) and in that the projection of the first primitive vector
Various exemplary details are described with reference to the following figures, wherein:
In a first aspect, the invention concerns a microscopy method, wherein an illuminating light (27a) produced by a light source (27) illuminates a sample (32) over an illuminating beam path (24), an objective (20) is disposed in the illuminating beam path (24) in order to focus the illuminating light (27a) into the sample (32), a detector (31) with an adjustable region of interest (12) detects radiation (25a) from the sample (32) over a detecting beam path (25) containing the objective (20), an illumination modulator (17) is disposed in the illuminating beam path (24), optics (28) disposed in the illuminating beam path (24) together with the illumination modulator (17) produce a light strip (3) of the illuminating light (27a) in the sample (32) at the focus of the objective (20), wherein the light strip (3) comprises a plurality of points of illumination (8, 11) and has an axial direction (3a) which is transverse to the illuminating beam path (24) at the location of the sample (32), a scanning unit (29) disposed in the illuminating beam path (24) is controlled in order to displace the light strip (3) of points of illumination (8, 11) in the sample (32) in a scanning direction (4) which is transverse to the direction of propagation of the illuminating beam path (24),
The axial direction of the light strip is defined here as the axis of the strip perpendicular to the beam path. In a variation of the invention, the scanning unit (29) disposed in the illuminating beam path (24) may also be identical to the mask/illumination modulator.
The term “asymmetrical 2D Bravais lattice” as used in the context of the invention means the following. In general, lattices/structures exist which are only modulated in a single dimension (what are known as line lattices) which will be referred to below as one-dimensional or “ID” lattices—see
The invention is particularly suitable for three-dimensional fluorescence microscopy. It has surprisingly been shown that the use of asymmetrical 2D illumination patterns in combination with a scanned line illumination of the pattern offers considerable advantages. Preferably, the illumination may be selected in a manner such that the axial direction of the light strip has an angle of 0-60° to the longer primitive vector of the Bravais lattice.
The method is particularly advantageous when the light strip is so narrow that its maximum width is that of the shorter primitive vector (b) of the Bravais lattice, or even preferably a maximum of 0.8 times or even a maximum of 0.6 times that of the shorter primitive vector (b) of the Bravais lattice. These measures can ensure that in the direction of the longer primitive vector (a), the light strip in fact illuminates or activates several points of illumination of the Bravais lattice parallel to the shorter primitive vector (b) of the Bravais lattice, but no more than one lattice point is illuminated parallel to the shorter primitive vector (b) of the Bravais lattice at each point in time. This has the advantage that in every direction in space, a contrast-reducing “crosstalk” of the image information (or fluorescent light) contained in neighbouring lattice points is prevented: parallel to the axial direction of the light strip, because of the longer primitive vector, the lattice points are sufficiently widely spaced apart to completely or largely suppress any crosstalk. Transverse to the light strip, i.e. parallel to the shorter primitive vector of the Bravais lattice, the width of the light strip which has been reduced to one row of the lattice points ensures that no more than one lattice point is illuminated at each point in time. By means of these measures, every piece of optical information from the sample can be optimally associated with a specific point of illumination and therefore the contrast can be significantly improved.
At the same time, the invention offers further advantages: it enables the points of illumination transverse to the axial direction of the light strip to be brought significantly closer together than would be conceivable with a symmetrical 2D Bravais lattice. This in turn means that what is known as the fill factor for the illumination modulator becomes significantly larger than with a comparable symmetrical 2D lattice. Because of the higher fill factor, light sources can be used without having to increase their light output to extremes. Thus, the microscopy method in accordance with the invention can be carried out in a cost-effective manner.
A further, decisive advantage is that the microscopy method in accordance with the invention permits significantly shorter acquisition times to be used than in conventional microscopy methods, because fewer acquisitions or raw images of a sample area or sample volume are sufficient. The larger is the 3D volume of the sample to be examined, the more important is this advantage.
In the simplest case of the invention, the projections may lead to a variation in which a first primitive vector (a) of the Bravais lattice (G4, G5, G6) parallel to the axial direction (3a) of the light strip (3) is longer than a primitive vector (b) of the Bravais lattice (G4, G5, G6) transverse to the axial direction (3a) of the light strip (3). The variations in the projections are shown in
The scanning direction (4) may additionally be transverse to the axial direction (3a) of the light strip (3).
Preferably, the first, longer primitive vector of the Bravais lattice is longer than the second, shorter primitive vector of the Bravais lattice by a factor of at least 1.5, more preferably by a factor of 2 to 15. The larger this factor is, then the stronger is the suppression of the crosstalk of information from neighbouring image points, albeit at the expense of a reducing fill factor.
Preferably, the region of interest of the surface detector can be limited to a specific “active region” (also “ROI”). All other regions of the surface detector are ignored or discarded. The region of interest of the surface detector may be synchronised with a displacement of the light strip by means of the scanning unit. This has the advantage of providing what is known as “line-confocal detection”, in which light from outside the region illuminated by the light strip is suppressed by the selective reading of the surface detector. In a variation of the invention, the height of the region of interest is approximately the same (i.e. ±10%) as the laser line/point line.
In a further variation, the region of interest (12) of the surface detector (31) is rectangular with a longer and a shorter side, wherein the longer side is parallel to the axial direction (3a) of the light strip (3) reproduced on the surface detector (31).
In a further variation, the illumination modulator (17) is planar in configuration.
In a further variation, optics (28) are disposed between the light source (27) and the illumination modulator (17) in the illuminating beam path (24), the optics producing a light strip (3) of the illuminating light (27a) on the planar illumination modulator (17). The light strip (3) has an axial direction (3a) transverse to the illuminating beam path (24) at the location of the illumination modulator (17), and the scanning unit (29) is controlled in order to displace the light strip (3) of the illuminating light (27a) on the illumination modulator (17) in a scanning direction (4) which is transverse to the axial direction (3a) of the light strip (3) and transverse to the direction of propagation of the illuminating beam path (24).
In a further variation, the light strip (3) has a width which is a maximum of 50% (for example 10-50%) larger than the FWHM diameter of a point of illumination (8, 11) in the sample (32), and in addition is smaller than the separation of the points in the direction of the shorter primitive vector.
In order to compile an image of a plane or of a 3D volume of the sample, preferably, a plurality of acquisitions are produced, of which each acquisition comprises a scan of the light strip over the illumination modulator. A relative movement between the sample and illumination occurs between two acquisitions. In this regard, these relative movements may either be always in the same direction in space or, however, in alternation or in a specific sequence in a plurality of mutually transverse directions in space. In order to record the image of a 3D volume of the sample, preferably, a plurality of images of a plurality of mutually parallel planes of the sample are acquired. The compilation of the image may be carried out in an analytical unit, for example in an appropriately programmed microprocessor or computer.
The light source may be coherent (i.e. producing coherent light) or incoherent (i.e. producing incoherent light).
Various technical solutions may be considered as the illumination modulator. As an example, a reflective element may be used as the illumination modulator, for example what is known as a Digital Micro Mirror Device (DMD). As an alternative, the illumination modulator may also comprise a transmissive element, for example a spatial light modulator (SLM), diffractive optical elements (DOE), amplitude masks or phase gratings. All of the aforementioned types of illumination modulators may be used independently of one another, whether or not the light source is a coherent or an incoherent light source. In the case of an incoherent light source, the illumination modulator may be configured such that illumination patterns are produced in the form of the asymmetrical 2D Bravais lattice interferometrically, i.e. by interference between the illuminating light of the light source transmitted or reflected by the illumination modulator.
There are a number of possibilities for producing a relative movement between the sample and illumination between two acquisitions: the displacement of the pattern in the sample may, for example, be carried out in the case of DMDs or SLMs by electronic switching of the micro-mirror (DMD) or of the display (SLM). In the case of amplitude masks or phase gratings, a mechanical displacement in one or two directions of the mask/grating is employed in the intermediate image, for example. In addition or as an alternative, a displacement of the illumination pattern in the sample may be obtained with the aid of a (motorized) tilted glass plate in a portion of the beam path.
In an advantageous variation, the illumination modulator has a plurality of mutually parallel cylindrical lenses, wherein a row of apertures in an (amplitude) mask is associated with each cylindrical lens. In this regard, the cylindrical lenses are preferably configured such that illuminating light is concentrated onto the apertures in the mask. This improves the light output of the illuminating light which, put another way, means that less expensive light sources can be used.
In another variation, the illumination modulator has a plurality of mutually parallel cylindrical lenses and is pulse illuminated by the illuminating light. In this regard, the light source itself may be pulsed, for example as a stroboscopic light. In this case, each cylindrical lens is preferably parallel to the shorter primitive vector (b) of the Bravais lattice, i.e. transverse to the light strip illuminating the illumination modulator. In this way, the cylindrical lenses ensure the concentration of the illuminating light along the light strip onto individual, discrete points, their separation from each other defining the longer primitive vector (a) of the Bravais lattice. The combination of the frequency of the pulsed illumination and the scan rate transverse to the light strip (in other words: along the cylindrical lenses) produces the shorter primitive vector (b) of the Bravais lattice. An advantage of this arrangement is that the shorter primitive vector (b) of the Bravais lattice can be adjusted by selecting the scan rate for the light strip and the pulse frequency of the illuminating light.
In a second aspect, the invention concerns a microscopy apparatus (100), comprising
In general, each secondary aspect discussed in connection with the microscopy method in accordance with the invention may also be applied to the microscopy apparatus in accordance with the invention, and vice-versa.
The variations discussed in the first aspect of the invention (i.e. the microscopy method) are equally applicable to the second aspect (i.e. the microscopy apparatus).
The first primitive vector of the Bravais lattice is preferably longer than the second (shorter) primitive vector of the Bravais lattice by a factor of at least 1.5, more preferably in fact longer than the shorter primitive vector by a factor of 2 to 15. The larger this factor is, the stronger will be the suppression of the “crosstalk” between the information from neighbouring points of illumination, albeit at the cost of a reduced fill factor.
Preferably, the surface detector is configured in a manner such that a region of interest of the detector (i.e. an active region, also known as the ROI), can be selected. This means that the surface detector can be operated in a manner such that only the region of interest is acquired and all other regions of the detector are discarded or ignored. This improves the contrast that can be obtained, i.e. the resolution, because scattered light from other regions can be effectively suppressed. The surface detector may, for example, be a (CCD) camera. The surface detector may be suitable for or configured for a rolling shutter operation.
Preferably, the microscopy apparatus has a controller which is configured to synchronise the region of interest of the surface detector with a displacement of the light strip by means of the scanning unit. In this manner, what is known as line-confocal detection is made possible, in which scattered light from regions outside the region of the sample illuminated by the light strip are particularly effectively suppressed. Preferably, the ROI running synchronously with the line scan has a minimum width of one point of illumination and a maximum size which is the spacing of the points in the direction of the shorter primitive vector.
The illumination modulator may be produced in a variety of manners. As an example, the illumination modulator may be an amplitude mask with a structure in the form of the asymmetrical 2D Bravais lattice. As already discussed above, the illumination modulator may be configured reflectively, transmissively or interferometrically. Preferably, in the case of an illumination modulator with a planar phase mask or amplitude mask, this mask is positioned in the intermediate image. The mask can then be illuminated with a line (initially not modulated). The line is modulated into a line of points by means of the mask. The line may, for example, be produced by a Powell lens. The illumination of the sample is then carried out via a scan of the light line (for example with a Galvo scanner) via the mask and focussing into the sample.
In a variation of the invention, the illumination modulator has a plurality of mutually parallel cylindrical lenses. The cylindrical lenses concentrate the illuminating light along the light strip and therefore lead to a particularly high light output. In the case in which the orientation of the cylindrical lenses is parallel to the scanning direction of the light strip over the illumination modulator, the cylindrical lenses have the additional advantage that the focussing properties do not vary during the scan.
In a further variation of the invention, each cylindrical lens is associated with a row of apertures in an (amplitude) mask. The cylindrical lenses then have the advantage that illuminating light is concentrated onto the apertures in the mask and therefore, a particularly high light output is obtained.
In an alternative variation, the light source may be pulsed in order to produce individual points of light during the scan of the light strip parallel to the cylindrical lenses.
The microscopy apparatus may have an evaluation unit which is configured to compile an image of a plane or of a volume of the sample from a plurality of acquisitions, of which each acquisition comprises a scan of the light strips over the illumination modulator.
The microscopy device may have a drive or a displacement unit in order to obtain a relative movement between sample and illumination pattern between two acquisitions. This ensures that after several displacements, acquisitions of each region of the desired volume of the sample are obtained and in total, a complete image can be compiled.
The microscopy apparatus is preferably configured in a manner such that the light strip is narrow enough to produce a maximum of one row of points of illumination of the Bravais lattice at each point in time in a direction transverse to its axial direction.
Preferably, the first, longer primitive vector (a) of the Bravais lattice (G4, G5, G6) is at an angle to the axial direction (3a) of the light strip (3) of 0-60°, particularly preferably of 0-20°.
Advantageous embodiments of the invention will now be discussed in more detail with the aid of the drawings, in which:
Identical components in the figures are consistently provided with identical reference symbols.
As already discussed above,
As will be discussed below, the illumination modulator 17 lies across or even perpendicular to an illuminating beam path. It is illuminated by a comparatively narrow light strip 3 the axial direction 3a of which is parallel to the first, longer primitive vector a of the Bravais lattice G4 of the illumination modulator 17. It can be seen that a width of the light strip 3 perpendicular to its axial direction 3a is approximately as wide as one diameter of the holes 2 or only slightly larger, but in any case smaller than the length of the second primitive vector “b”. The width of the line of light 3 perpendicular to its axial direction 3a is indicated by h.
During the course of the microscopy method, the light strip 3 is scanned in a scanning direction 4, i.e. here, parallel to the shorter primitive vector “b” of the Bravais lattice G4. At point in time t1, starting at the upper edge of the field of illumination 1, during one complete scan, the light strip 3 passes over the entire field of illumination 1 once, i.e. once over the region of the illumination modulator 17 to be illuminated. As a result, a single acquisition of a layer is acquired from the sample 32. Following the acquisition, a relative movement between the illumination modulator 17 and the sample 32 is carried out in a direction of movement 5. A plurality of scans are carried out in stepwise succession and a plurality of acquisitions are made until finally, a surface of the sample 32 has been completely imaged and illuminated.
Part B) of
Part C) of
Telecentric optics with an excitation tubular lens 23 and a scanner ocular lens 26 are located in the illuminating beam path 24. These optical elements 23, 26 produce an intermediate image in an intermediate image plane 30 which is focussed via the objective 20 into the sample 32. An illumination modulator 17 is at the site of the intermediate image plane 30. In the exemplary embodiment, this may be a planar, transmissive amplitude mask 17 which is orientated perpendicular to the direction of propagation 24a of the illuminating light 27a in the illuminating beam path 24. Mention was made in the introduction that instead of a transmissive mask, other technical solutions for the illumination modulator 17 are possible. In accordance with the invention, the illumination modulator 17 is configured to produce an illumination pattern 17a of the illuminating light 27a in the sample 32 (see
Furthermore, a line generator 28 is located in the illuminating beam path 24. The line generator 28 has optics which generate a light strip 3 of the illuminating light 27a at the site of the illumination modulator 17. As an example, the line generator 28 may be a Powell lens or a diffractive optical element (DOE).
Furthermore, a scanning unit 29 is disposed in the illuminating beam path 24 between the line generator 28 and the illumination modulator 17, which may, for example, comprise a reflecting Galvo scanner. The scanning unit 29 is operated (for example by rotation of a mirror, see the arrow) in a manner such that the light strip 3 moves in the scanning direction 4 at the location of the illumination modulator 17.
The illumination modulator 17 is mounted on a displacement unit 33 or is carried by it. The displacement unit 33 is configured to displace the illumination modulator 17 in one or more directions in space 34, for example by means of one or more servomotors.
A controller 35 (for example a computer or microprocessor) is connected to the light source 27, the scanning unit 29, the displacement unit 33 and the surface detector 31 via signal lines and/or data lines 36. The controller 35 is configured and programmed to control, acquire and/or synchronise these components in a suitable manner. As an example, the controller 35 may control the light source 27 for pulsed operation of the light source 27, the scanning unit 29 for scanning the light strip 3 in the scanning direction 4, the displacement unit 33 for displacing the illumination modulator 17 and the surface detector 31 in order to select and acquire a region of interest 12. This enables a sequence in which initially, one or more (possibly digital) acquisitions are obtained from the region of the sample 32 to be examined, while the light strip 3 is scanned over the illumination modulator 17. After one complete scan of the light strip 3, a displacement of the illumination modulator 17 is carried out in one or two directions in space 34 by means of the displacement unit 33, before a further scan of the light strip 3 is carried out and one (or more) further acquisitions are made. These acquisitions are stored in a memory 37 which may, for example, be located in the controller 35. If a two-dimensional or three-dimensional area of the sample 32 is detected by sufficient acquisitions, the acquisitions are compiled in an evaluation unit 38 into a complete image. The evaluation unit 38 may also be part of the controller 35.
In a first insert, above the displacement unit 33,
In a second insert in
In a further embodiment, the illumination modulator 17 comprises only the cylindrical lenses 16, without an amplitude mask with holes. At the same time, however, the operation of the light source 27 may be pulsed. When the microscopy apparatus 100 is operating, pulsing of the light source 27 with simultaneous scanning of the light strip 3 along the scanning direction 4 ensures that an illumination pattern 17a in the form of an asymmetrical 2D Bravais lattice G4, G5, G6 is generated with the precisely similar structure that is shown in
A specific example will now be described with a 40× 1.25 water immersion objective 20. An amplitude lattice 17 is illuminated with a scanned laser line 3 and focussed into the sample 32 with a tubular lens 23 and the objective 20. The fluorescent light 25a is collected from the sample 32, transmitted by the dichroic mirror 21 and focussed with a tubular lens 22 onto a RS camera 31 with a continuously moved ROI 12 synchronised to the movement of the light line or laser line 3 with a height “k” of 16 μm-20 μm. In the example, a transmitting amplitude lattice 17 with rectangularly (2D-asymmetrical) disposed holes 2, analogous to the pattern shown in
In the sample 32, for the 40× 1.25 objective 22, this corresponds to an illumination pattern 17a with a=5 μm, b=1 μm, D=0.5 μm. The resolution of the objective is ca. 200 nm FHMM (FWHM), and resolution-related dispersion of the pattern 17a is negligible for this broad estimation. If the lattice 17 is displaced in a manner such that there is a point of illumination 8 in the sample 32 every 330 nm (which results in a relatively homogeneous illumination), then 14×3=42 raw images are required. This corresponds to a displacement of exactly three points in direction “a” and exactly one point in direction “b”, corresponding to the displacement vector shown in
If an image with a comparable confocality and with a comparable SNR were to be acquired with a symmetrical 2D pattern (square G2 or hexagonal G3), for the square G2 (a=“b”=5 μm), a total of 14*14=196 raw images would be required. For the hexagonal pattern G3, then, (5/0.33−1)×(5*sin 60°/0.33)=14*13=182 raw images would be required. Compared with this, the method in accordance with the invention is four to five times faster, because it needs correspondingly fewer acquisitions. By using cylindrical lenses 16 orientated onto the 2D holed masks 17, in addition with this example, the required power for the laser line 3 can be reduced by up to a factor of 5 μm/0.5 μm=10.
In this example, a rectangular lattice structure G4 was used. One advantage of this rectangular lattice structure G4 is that it can be efficiently combined with cylindrical lenses 16. In principle, however, all three asymmetrical 2D Bravais structures G4, G5, G6 may be envisaged (see
Starting from the exemplary embodiments shown, the method in accordance with the invention and the microscopy apparatus 100 in accordance with the invention may be varied in many ways. As already discussed, examples which may be envisaged are different technical solutions for the illumination modulator 17. It is also possible to envisage a plurality of different asymmetrical 2D Bravais lattice structures G4, G5, G6 for the illumination modulator 17a.
While various details have been described in conjunction with the exemplary implementations outlined above, various alternatives, modifications, variations, improvements, and/or substantial equivalents, whether known or that are or may be presently unforeseen, may become apparent upon reviewing the foregoing disclosure. Accordingly, the exemplary implementations set forth above, are intended to be illustrative, not limiting.
| Number | Date | Country | Kind |
|---|---|---|---|
| EP23173519.2 | May 2023 | EU | regional |
