Generating a field of light with multiple localized zeros

Description

[0001] The invention relates to a method for generating a light field which has defined zero points at predetermined positions in a defined working area. State of the art

[0002] Fluorophores or other point emitters can be localized in a working area, for example on a sample under investigation, by using their secondary light (e.g. luminescence). When a point emitter is irradiated with excitation light of a suitable wavelength, it in turn emits secondary light (scattered light of the same wavelength or luminescence light of a different wavelength). However, if the light field of the excitation light has a zero point that lies exactly at the position of the point emitter, the point emitter emits no or minimal secondary light. A zero point here is a clear local intensity minimum. By sequentially scanning an extended sample while monitoring the intensity of the secondary light, a point emitter can be localized. A corresponding method is known from (F. Balzarotti et al., "Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes", Science 355, 606-612 (2017)). A modification of this method is known from DE 10 2016 119 262 A1.

[0003] DE 10 2011 055 367 A1 discloses a method and a device for tracking the movement of a particle, in particular a single molecule, in a sample. A minimum of an intensity distribution with which the sample is irradiated is tracked to the particle moving within the sample in such a way that the rate of photons emitted by the particle remains minimal.

[0004] WO 2013 / 167 479 A1 discloses a special microscope for the high-resolution imaging of a structure of interest in a sample comprising a substance having a first state with first spectral properties and a second state with second spectral properties.

[0005] US 2014 / 042 340 A1 discloses another method for tracking a particle, in which a minimum of an intensity distribution with which the sample is irradiated is tracked to the particle moving within the sample in such a way that the rate of photons emitted by the particle remains minimal. The position of the minimum is considered the position of the particle within the sample. Task and solution

[0006] An object of the present invention is to provide a method by which a work area can be exposed to a light field having multiple zero points that can be positioned independently of one another within the work area. A further object of the invention is to provide a method by which multiple point emitters can be located and / or tracked simultaneously within an extended work area.

[0007] These objects are achieved by a method for generating a light field according to the main claim and by a method for locating and / or tracking point emitters according to the secondary claim. Further advantageous embodiments emerge from the dependent claims. Disclosure of the invention

[0008] Within the scope of the invention, a method was developed for generating a light field in a predetermined working area by means of an at least partially coherent light source. The coherent light source can in particular be a laser, for example. However, partially coherent light, such as that from a lamp with a color filter or from light-emitting diodes (LEDs), is also suitable. Light from the light source is fed to at least one modulator so that the modulator imposes different changes in at least one property of the light on different components of the light. Examples of such properties are the phase, the polarization, and the intensity of the light. The light is then guided into the working area. The working area can, for example, be the sample plane or image plane in a microscope.The modulator is controlled with a zero-point control pattern such that the sum of the amplitude distributions of the light components guided through the modulator has zero points at at least two independently predeterminable positions within the operating range. These zero points each border on areas where the said sum of the amplitude distributions is non-zero. A zero point is defined as a local intensity minimum. This zero point can be defined in one spatial direction or in two or three spatial directions and is then referred to as a 1D zero point, 2D zero point, or 3D zero point.

[0009] A zero point can be understood, for example, as a location in the light field where the lowest possible intensity close to zero is generated, even if an actual zero is practically impossible to achieve. A sharply defined 1D / 2D / 3D zero point can thus, for example, describe an intensity curve with a local minimum that exhibits a strong curvature in one, two, or three spatial directions.

[0010] A strong curvature can be understood, for example, as a significant increase in illumination intensity at a predetermined distance from the zero point, such as λ / 5 (or less), which is detectable or usable for measurements. A significant increase can be understood, for example, as a significant increase at the predetermined distance from the zero point, which has increased by at least 3% of the highest maximum closest to the zero point (on either side of the zero point) compared to the zero point.

[0011] Through the various changes, such as phase changes, experienced by different components of the light, constructive or destructive interference can be established between these components. Since the light is at least partially coherent, this phase relationship, and therefore also the interference, is maintained on the path between the modulator and the working area. If any number of zeros are desired at a specific location in the working area, a zero control pattern that achieves the desired result can be calculated using a parameterized approach, for example. The modulator can be placed in any optical plane relative to the working area. These optical planes are, for example, planes conjugate to the working area, which are referred to below as the image plane, or planes that have a Fourier relationship (Fourier transform) to the image plane, which are referred to below as the Fourier plane.The Fourier planes are the conjugate planes of the back focal plane (considered for the emitted light, i.e., the front focal plane of the imaging illumination system) of an optical imaging system. However, any other position of the modulator outside of these planes is also conceivable. The decisive factor is the correct selection of the zero-point control pattern according to the selected optical plane.

[0012] It is precisely the possibility of generating zeros at several independently predeterminable positions that distinguishes the method proposed here from other methods that, for example, generate a spatial pattern of zeros with a fixed periodicity, as is the case with the structured illumination method or SIMFlux.

[0013] The accuracy with which the zeros can be positioned in the working area is not limited by the spatial resolution with which the zero control pattern can be generated in the modulator. The reason for this is that, according to claim 1, in addition to a spatial distribution of regions that change the phase of the light incident on the modulator, the zero control pattern also has the respective local amount of this phase change as an additional degree of freedom. The phase resolution with which the phase can be changed is also technically limited in most modulators, but is sufficient to increase the positioning accuracy significantly beyond the extent that the pixelation of the modulator allows on its own and that results for the distribution of the zeros through pixel-precise displacement of a zero control pattern on a modulator divided into pixels.

[0014] The light field generated by this method is particularly advantageous for working with fluorophores or other point emitters on an extended sample forming the working area. With multiple independently positionable zero points, several of the point emitters located in their vicinity can be independently excited, localized, and / or tracked. This saves considerable measurement time and enables the simultaneous measurement of the dynamics of several moving point emitters, compared to a measurement using only one zero point, for example, by scanning a single zero point across the sample. Excitation is understood to mean exposing the point emitter to light from the light source that causes it to emit scattered light, luminescence light, or other secondary light.By exploiting the special properties of some types of point emitters, for example fluorophores such as Alexa Fluor ®< 647 or P AmCherry, it is possible to ensure that even with a high point emitter density, the point emitters only emit secondary light in isolated or optically isolated quantities, making localization and / or position tracking particularly easy. In this case, this optical isolation of the densely packed point emitters is made possible by the prior activation of isolated point emitters in a stochastic manner on the surface of the extended sample. Only activated point emitters can then be excited to emit secondary light. Isolated or optically isolated point emitters means that no other excitable, similar point emitters are present within a minimum distance to the nearest point emitter.This minimum separation defines the minimum distance between two point emitters at which their emitted secondary light can be detected separately, often referred to as the resolution limit. This minimum separation is maintained if the separation is at least equal to the diffraction limit at the wavelength of the secondary light from the point emitters.

[0015] When scanning a single zero point surrounded by areas with an intensity other than zero, there is a comparatively high probability that several point emitters in these areas will be activated simultaneously and must be removed from the measurement series, or the activation process must be separated from the actual localization process.

[0016] Furthermore, the modulator's standard control electronics are sufficient to generate the zeros. However, commercially available optical elements such as lenses, masks, aperture stops, and mirrors can be deliberately positioned after the modulated light field up to the working area in such a way that they still exert a calculated influence on the resulting zeros or on the light field in general within the working area. Based on the desired positions of the zeros, the appropriate zero control pattern can be calculated using a standard computer.

[0017] Not only does a single modulator have to be used, but the modulation can also be distributed across multiple modulators, or one or more modulators and additional optical elements. For example, with modulators, there may be a trade-off between achieving the best possible phase resolution on the one hand and a fast response time on the other.

[0018] For example, a first modulator can be used to place zeros slowly but with high accuracy onto the work area. A second optical element, which could also be a modulator, can then be used to quickly change the position of the zeros by small amounts to improve the accuracy of the position estimation by measuring only a few photons of secondary light emitted from the work area. This rapid position change can advantageously be achieved using technical means separate from those used for the slow translation. For example, resonant or non-resonant galvanometer mirrors or digital micromirror devices can be used, but also other means suitable for shifting an image simultaneously. Examples include a vibrating rotating glass plate, a vibrating displacement of the modulator, or similar.

[0019] To quickly change the zero position without changing the zero control pattern, mechanically interchangeable glass plates of different thicknesses but with the same alignment angle (e.g., in a wheel similar to a filter wheel) can be positioned behind the modulator (or in a conjugate plane) in such a way that the light is offset in a different parallel direction. By varying the glass thickness appropriately, this parallel offset can be precisely selected so that the zeros in the working area (sample plane) are shifted by a selectable distance, e.g., in the range of a few 10 nm (or less).

[0020] Instead of interchangeable glass plates, an optical device can be used, for example, in which a flat glass plate, for example, can be adjusted by a definable angle to the optical axis (see the implementation, for example, in the "ApoTome" product from Zeiss). In a particularly advantageous embodiment, a flat glass plate rotates at a constant angle α to the optical axis around the optical axis at an angle φ(t). This allows a global offset of all zeros previously generated via the modulator to be generated. This can be used, for example, by positioning several zeros as precisely as possible on the fluorophores in a first localization step, and then offsetting all zeros by the same amount.In addition to fast localization without complex feedback, another advantage is that the same zero-point curve can be used for later position determination for each particle.

[0021] A first modulator can also be used to generate intense, locally limited light patterns on a second modulator, with the second modulator then imposing the zero point and the precise shift on this light. This allows the available light to be efficiently distributed across several such zero points.

[0022] In a particularly advantageous embodiment, a modulator with a planar active surface is selected. This modulator is designed to impart phase changes to light components that pass through the active surface and / or are reflected by the active surface. The modulator can, for example, be a phase spatial light modulator (phase SLM). This phase SLM can only change the phases of the light field, but not its amplitudes (or intensities). The desired positions of the zero points in the working range can then be translated into locations on the active surface at which the phase of the light is to be changed, and into amounts by which the phase is to be changed.

[0023] In a particularly advantageous embodiment, the zero control pattern consists of any number of sub-zero control patterns, each of which can generate one or more (approximately two) independent zeros by influencing the light field in different ways. The sub-zero control patterns, in turn, can consist of sub-sub-zero control patterns, which are considered parts of the sub-control patterns. The sub-control patterns can—but do not have to—be based on the same basic patterns. In this particularly simple embodiment, for example, certain parameters of the sub-sub-zero control patterns can be changed in order to control the respective zeros independently of one another. For the sake of clarity, the term "sub-control pattern" is used below as a synonym for the term "sub-zero control pattern."Furthermore, the term "sub-sub-control pattern" is used as a synonym for the term "sub-sub-zero-control pattern".

[0024] In a particularly advantageous embodiment, at least two adjacent regions of the above-described phase-changing active surface, which can be located, for example, in the image plane, are controlled such that an average phase shift of π is set for the light leaving these regions between light components on which these regions respectively act. These adjacent regions can, for example, each correspond to sub-sub-zero control patterns. In the case of inhomogeneous illumination, the average of the phase shift can be weighted, in particular, by the amplitude strength of the illumination. Light components from the adjacent regions can then destructively interfere with each other, resulting in a zero crossing of the amplitude of the light in the sample, and thus a narrowly defined zero point (local minimum).The desired positions of the zeros in the working area can be easily translated using ray optics into the positions of the adjacent regions on the active surface that are to be targeted to generate these zeros. It should be noted that the sizes of the zeros generally do not correspond to the size of the zero-generating regions. The design of the zero-generating regions is more crucial, which influences, for example, the spatial directions in which the zero is confined.

[0025] The active area of ​​the modulator can, for example, be divided into pixels that can be controlled separately. These pixels can then serve as basic building blocks for generating any desired zero-point control pattern.

[0026] For example, if adjacent, rectangular, areas on an active area divided into pixels are controlled in such a way that a phase shift of π occurs between the light components influenced by these areas, both rectangles advantageously form another rectangle whose side lengths correspond to an even number of pixels, for example, 6 × 10 pixels. This special zero-subcontrol pattern with its two rectangles as a sub-sub-zero control pattern will be referred to as a "phase box" in the following. The algorithms for calculating the positions of the rectangular areas from the desired positions of the zeros then become particularly simple. The same applies if the areas are not rectangular, but round (e.g., circular) with point symmetry around a center. For example, circular areas can be inscribed in a square of 11 × 11 pixels.Due to the address structure often found on phase SLMs with a spacing of two pixels, other box sizes can also be particularly useful. It may also be useful to choose an odd number of pixels as the edge length and, for example, intentionally leave the center unmodulated. It may also be useful to combine even and odd edge lengths.

[0027] In a particularly advantageous embodiment, the positions of the zeros can be specified with greater accuracy than defined by the zero shift that would result from a translation of the zero control pattern on the active area of ​​the modulator by one pixel in at least one coordinate direction. As previously explained, other degrees of freedom (such as the introduction of gratings with phase shift adjustable by phase change, which lead to an amplitude change of the light directly reflected in the zeroth order from the box) can be used for this purpose, which are not directly linked to the resolution of the pixels on the active area.

[0028] In a particularly advantageous embodiment, the modulator is controlled at a first time with a first zero control pattern and at a second, later time with at least one second zero control pattern. At least two zero control patterns differ in that the position of at least one zero in the operating range changes by a smaller amount than would result from the translation of the first zero control pattern on the active area of ​​the modulator by one pixel.

[0029] The zeros can therefore be shifted in the working area by sub-pixel amounts, measured by the shift of the zero, which is obtained by shifting the relevant modulator pixels by exactly one pixel. Accordingly, the accuracy with which, for example, point emitters can be tracked in the working area can be significantly increased. As feedback for the tracking, the intensity of secondary light emitted by the point emitters as a result of excitation by the light field generated by the modulator can be monitored. A point emitter is only not excited at all (or is only minimally excited) to emit secondary light if it is exactly covered by a zero of the light field in the working area. Even with a relative shift between the zero and the point emitter of, for example, 1 nm, significantly more measurable secondary light is emitted, from which the distance to the zero can be determined.

[0030] In a further particularly advantageous embodiment, at least one sub-zero control pattern of the zero control pattern is superimposed or otherwise composed of at least two sub-sub-zero control patterns. The light influenced by each of these sub-sub-zero control patterns generates a complex amplitude at the specified location of at least one zero. The sub-sub control patterns are then selected such that the superposition of these complex amplitudes results in zero or a minimum magnitude close to zero at the specified location of the zero.

[0031] In this way, starting with the desire to generate a zero at a specific, predefined location, it is particularly easy to obtain a sub-zero control pattern that delivers a zero at precisely this location. In particular, by changing the complex amplitudes of the light field using the sub-sub-zero control pattern, the position of the generated zero can be shifted in a controlled manner.

[0032] In another particularly advantageous embodiment, at least one sub-zero control pattern is superimposed or otherwise composed of at least three sub-sub control patterns. The light influenced by each of these sub-sub control patterns generates a complex amplitude at the specified location of at least one zero. The sub-sub control patterns are selected such that the superposition of these complex amplitudes generates at least two zeros in the working area. With such an arrangement, the two zeros can be shifted independently of one another by sub-pixel amounts and also brought very close to one another.

[0033] To enable easy shifting of zero points, at least one sub-sub-control pattern advantageously contains an additional control element in the plane of the active surface, which is suitable for influencing the amplitude strength generated by this sub-sub-control pattern at the specified zero point location. This control element can, for example, be a periodic portion of the sub-sub-control pattern or a Fresnel zone plate with an adjustable focal length. The latter can be implemented, for example, in a phase-changing modulator by adding a Fresnel zone plate ("Fresnel lens") to at least one sub-sub-zero-control pattern. By adjusting the focal length of the Fresnel lens, an additional focus or defocus term is impressed on the light field leaving this sub-sub-control pattern, which means that the generated amplitude strength can be influenced at the specified zero point location.This influencing of the generated amplitude strength at the specified zero position can also be achieved by adding a suitable periodic pattern, such as a grating, from which only a portion of the diffracted light (e.g., only the zeroth order) reaches the plane of the zeros to be generated. Changing the amplitude strength of the light from one (or more) of the sub-sub-control patterns leads to a sub-pixel shift of the zero. If necessary, phases of the light from the sub-sub-control patterns that do not precisely match the desired zero position can be compensated for by additionally imposing offset phases in the sub-sub-control patterns.

[0034] In a particularly advantageous embodiment, a phase SLM is used as the phase-changing light field modulator, which can only change the phases of the light field. However, to also allow the intensities (or amplitudes) to be changed with the phase SLM—which is not readily possible from a constructional perspective—a low-pass filter is integrated into the beam path, or the low-pass filter properties of an existing optical component (e.g., the microscope objective) are utilized for this purpose. Examples of low-pass filters that can be used are an aperture stop, a pupil stop, or a mask in the Fourier plane of an optical system. An additional grating is then generated, for example, in both halves of the phase box.Since the additional grating has been chosen to be so high-frequency that its diffracted light is blocked by the low-pass filter and thus the amplitude removed from the image by the grating does not contribute to the image of this part of the box in the sample, the amplitude strength of the image of each part of the phase box in the image plane can be controlled independently, for example by a symmetrically designed phase of the grating (+ / - phase value P), via the choice of the phase value P.

[0035] In practice, this means that a larger relative difference in the phase values ​​+ / - P (modulation depth) of the additional grating applied causes more light to be blocked from this area of ​​the phase box, all other conditions remaining unchanged.

[0036] To improve zero-point quality during sequential 2D localization, e.g., by rotating the zero-point control pattern in the image plane by 90°, it is advantageous to also rotate the polarization of the light in front of the lens to maintain its azimuthal polarization. Active (e.g., liquid crystal modulators, electro-optical modulators, or motorized λ / 2 plates) and passive optical elements (e.g., segmented λ / 2 plates in the Fourier plane) are suitable for this purpose.

[0037] By changing the relative overall phase and the described grating modulation depth between the two halves of the "phase box," it is possible to shift the zero from the original position with sub-pixel precision while still qualitatively maintaining a deep (close to zero) and "sharp" (with high maxima nearby) zero. In practice, however, this often means that when the zero is shifted by sub-pixels, an exact 0-π phase difference between the two halves of the box is no longer maintained. This 0-π phase difference then results at the desired sub-pixel position, and at the same time, the respective amplitudes of the contributions from the respective halves are adjusted in such a way that they interfere destructively there, thus resulting in a zero.

[0038] To keep the positioning algorithm simple, it may be useful to always provide one half of the phase box with a fixed grid around a fixed center phase, while only the other half of the box varies the grid modulation depth (+ / - P) and relative phase F compared to the center phase of the other "phase box".

[0039] Since phase SLM often has technical limitations due to its pixelation and the achievable phase gradation (e.g., 8 bits), it can also be particularly useful to vary both P and F on both sides of the phase box. Through simulations and / or calibration solutions, optimal values ​​for subpixel shifts can be determined while maintaining the best possible zero point, which can then also be used in a "look-up" table.

[0040] By additional modulation with the grating, the positions of zeros can be shifted, particularly in a linear direction. To control the positions of zeros two-dimensionally, a new pair of zero control patterns can be generated for the second direction, which is rotated by 90° relative to the zero control patterns used for the first direction.

[0041] However, subzero control patterns can also be used, for example, consisting of approximately four parts and generating a local minimum that is tightly confined in two dimensions. Analogous to the previously described approach, the individual parts of this pattern can be modulated with a grating or other periodic patterns to control the position of the zero in two dimensions with subpixel precision.

[0042] Even when two patterns are used, the subzero control patterns can have many different identical shapes (e.g., semicircles, triangles) or even different shapes. Different shapes or sizes can also be useful for influencing the respective amplitude strengths and thus for controlling the zero position.

[0043] To obtain a tightly confined local minimum along the optical axis, the sub-zero control pattern, for example, the phase box described above, can be constructed with an inner and an outer region. These regions can interact to create a sharp minimum along the optical axis. The previously described principle for adjusting the amplitude strength at the specified zero location can be applied equally to these regions to control the positions of the zeros with sub-pixel precision. This principle can be generalized to create zeros that are sharply confined in two or three dimensions.

[0044] For example, one can create sub-zero control patterns consisting of four sub-sub-zero control patterns, producing a zero that is tightly confined in two dimensions. Using a principle analogous to that described with rectangular "phase boxes" for creating one-dimensional zeros, the two-dimensional position perpendicular to the optical axis can then also be controlled with subpixel precision.

[0045] By combining the described sub-zero control patterns, the generation of zeros in all three spatial directions (X, Y, Z) can be implemented analogously. This allows control over the zero in all spatial directions individually, sequentially combined, or simultaneously in all possible combinations (XY, XZ, YZ, or XYZ). The spatial direction "Z" is parallel to the optical axis. The spatial directions "X," "Y," and "Z" are all perpendicular to each other. These spatial directions can be expressed by Cartesian unit vectors, which are perpendicular to each other and thus linearly independent.

[0046] Furthermore, subzero control patterns can be created with multiple sub-subzero control patterns, each generating more than one zero in the working area. These zeros can then also be located within the minimum distance at which the point emitters are considered optically isolated. For example, to generate two 1D zeros, a subzero control pattern consisting of three sub-sub control patterns can be selected in a phase-changing modulator. These three sub-sub control patterns are composed, for example, of rectangular areas, each with an edge length of 6x10 pixels, so that the entire rectangular subzero control pattern has an edge length of 18x10 pixels.The sub-control patterns are selected so that adjacent rectangular areas are controlled in such a way that a phase shift of π occurs between the light components influenced by these areas. Analogous to the "phase box," other edge lengths or different sizes of the sub-sub-control patterns can be advantageous. Similarly to the "phase box," the positions of the zeros can be positioned with sub-pixel precision by changing the amplitude strength using a corresponding element in at least one of the sub-sub-zero control patterns. If, in the example given, the zero position is to be independently changeable, one could, for example, add independently controllable elements for changing the amplitude strength, such as a Fresnel lens or a periodic pattern, in the two non-adjacent areas of the sub-zero control pattern.All further considerations for generating and optimizing the generation of a zero apply analogously to the generation of multiple zeros with a sub-zero control pattern.

[0047] It is also possible to determine the position of multiple emitters from multiple measurements using shifted and relatively rotated 1D zero patterns in 2D, analogous to tomographic methods (such as computed tomography). The same applies to 3D position determinations, for example, using 2D or 1D zeros.

[0048] In another particularly advantageous embodiment, a zero point can also be shifted with sub-pixel precision as follows: A phase box A with a phase difference of π between adjacent rectangular regions is positioned with pixel precision at a location using the modulator. If this phase box A is shifted in the plane by one pixel along or against the normal of the phase boundary, a second phase box B is created, in which two rectangular regions with a phase difference of π also adjoin each other. Thus, the zero point will also shift accordingly (neglecting the influence of other phase boxes), taking the image scale into account.

[0049] In order to generate a zero-point control pattern whose zero-point position lies between the "extremes" specified by the phase boxes A and B, an average of the phase boxes A and B can now be formed by weighting these two phase boxes A and B with a constant W relative to each other.

[0050] In the areas where both phase boxes A and B have phase 0 or π, this new zero-point control pattern also has the corresponding phase 0 or π. In the one-pixel-wide overlap region in the middle, the phase shifts by a value between 0 and π due to the weighted averaging according to the value of the constant W. In the two edge regions, each one-pixel wide, there is a mixture of the respective control pattern of the phase box and the background, which may, for example, be occupied by the blocking control pattern.

[0051] The overall resulting pattern initially leads to a deterioration in the zero point quality. However, for a specifically specified W, the zero point quality can be restored. To do this, the area of ​​the pattern in which the phase changes by 0 is provided with a first additional phase. Analogously, the area of ​​the pattern in which the phase changes by π is provided with a second additional phase. The two additional phases can now be optimized so that the illuminance in the zero point, whose position is specified by W, reaches the smallest possible minimum. In particular, the second additional phase can be obtained by negating the first additional phase. However, the second additional phase can also be optimized independently of the first additional phase, which further improves the zero point quality.

[0052] The dependence of the first additional phase, or the second additional phase, on W can be determined numerically using an algorithm, tabulated, and approximated if necessary. It can also be calculated theoretically. Once the additional phases are determined, the position of the zero point, which depends on W, can also be determined using an algorithm (or theory), tabulated, and approximated. These dependencies can be inverted, so that, starting from a desired sub-pixel zero point position, the value of W and the values ​​of the additional phases can be determined. This then clearly describes and defines the weighted phase box control pattern corrected for the additional phases.

[0053] In a particularly advantageous embodiment, a parameterized approach is established for the zero-point control pattern. The parameters of this approach are optimized with the goal of ensuring that the sum of the amplitude distributions has zeros at at least two specified positions. In this way, the search, in particular for zero-point control patterns that do not merely consist of prefabricated "phase boxes," can be significantly accelerated. The parameters can, for example, be the phase values ​​to be found within an available control region on a light modulator. However, they can also refer to other values ​​(e.g., significantly fewer values, such as offset phase, blaze angle, and modulation depth), from which the corresponding control patterns can then be determined.

[0054] In another particularly advantageous embodiment, the modulator is controlled with a superposition of a blocking control pattern and the zero-point control pattern. The blocking control pattern is used to essentially keep the light guided through the modulator away from the working area. The light field then only has a significant intensity in the immediate vicinity of the zero points. In this way, the light dose to which the working area is exposed overall can be limited. In this way, for example, the overall phototoxic load of a sensitive sample forming the working area can be reduced.

[0055] This blocking control pattern can, for example, be a high-frequency grating, which is used to essentially keep the light guided through the modulator away from the working area. The mode of operation is analogous to the example described when overlaying the phase box with an additional grating. Since the applied blocking pattern has been selected to be so high-frequency in the sense of a schlieren optics, it can be blocked by a corresponding low-pass filter. The low-pass filtering can, for example, be implemented by an aperture stop or a Fourier mask in a Fourier plane between the modulator and the working area. High-frequency components of the light field are thus suppressed. In this embodiment, the light field exhibits high frequencies essentially in the respective vicinity of the zero points and in the areas of the blocking control pattern.

[0056] It may be advantageous to design the edges of at least one zero-point control pattern, sub-zero-point control pattern, or sub-sub-zero-point control pattern as a smooth transition to the adjacent blocking pattern. This minimizes unwanted edge effects that could lead to deterioration of the zero point.

[0057] A ring aperture (bandpass filter) can be advantageously selected as the Fourier mask. This allows reflections, such as unmodulated reflections from the modulator, to be blocked. This contributes to achieving higher zero quality.

[0058] The modulator can also be illuminated and controlled at an oblique angle, for example, so that light components whose phase is not altered by a sub-zero control pattern are essentially kept out of the working area (e.g., by a diaphragm or the aperture limiter). This can also reduce the phototoxicity of the working area. The phase boxes then contain a grating (e.g., with blaze) designed to allow light to enter the working area from there.

[0059] In another particularly advantageous embodiment, the light is modulated in several different optical planes. For this purpose, several modulators can be used, for example. It is also possible, for example, to use at least two parts of a modulator in different optical planes in combination with at least one additional optical element. The modulators or parts of modulators can then cooperate in a division of labor, as already explained above.

[0060] In another particularly advantageous embodiment, an optical plane in which the modulator is arranged is imaged in a reduced size onto the working area or onto another optical plane using an optical system. Given a given spatial resolution of the pixels controllable on the active surface of the modulator, positioning of zero points in the working area with greater precision is then possible. The imaging optical system can furthermore, for example, guide the light field into the working area in such a way that secondary light emitted from the working area as a result of excitation by the light field is not shadowed by the modulator but can be guided to a detector.

[0061] In a further particularly advantageous embodiment, the working area and the modulator are arranged at optically conjugated positions and / or in optically conjugated planes. "Conjugated" in this context means, in particular, that an optical system provides sharp images between the respective (conjugated) positions / planes.

[0062] In this way, the relationship between the positions of the zeros in the working range on the one hand and the phase changes required by the modulator to generate these zeros on the other hand is easier to manage.

[0063] However, the modulator can also be placed in a Fourier plane, for example. The position of the Fourier plane is determined by the specific optical system used. By placing the modulator in the Fourier plane, phase changes can be introduced by the modulator in the spatial frequency domain. These changes can be determined, for example, by Fourier transforming the desired spatial distribution of zeros in the operating range, or using iterative algorithms (such as the Gerchberg-Saxton algorithm or algorithms that find an optimum based on gradients).

[0064] The placement in the Fourier plane has the advantage that the total available intensity can be better utilized.

[0065] A modulator located neither in the image plane nor in the Fourier plane can also be advantageous. For example, with a phase-changing modulator, the available intensity of the light field can be more effectively utilized by virtually shifting the modulator back into the image plane by incorporating additional focus terms. This only slightly increases the computational effort for the zero-point control patterns, as no time-consuming iterative algorithms are required to generate the zero-point control patterns, especially during pattern application.

[0066] The use of a static phase mask in combination with zero-point control patterns can be useful for generating a "generalized phase contrast." A "generalized phase contrast" is created by applying a phase mask in the Fourier plane.

[0067] Particularly advantageously, two modulators, or even two parts of the same modulator, can be arranged in different optical planes with respect to the imaging optical system. For example, one modulator, or part of the modulator, can then take on the task of creating local intensity maxima near the desired positions of the zeros. The other modulator can then take on the task of moving the zeros to the desired positions with sub-pixel accuracy and optimizing the quality of the zeros. One advantage of this arrangement is very high light efficiency and the ability to individually control the curvature of the intensity distribution near the zero, which is ultimately related to the intensity near the zero.

[0068] A further embodiment involves using two or more modulators, or two or more parts of one or more modulators, arranged in different optical planes with respect to the imaging optical system, such that a first number of modulators (or parts) prefocuses the light from the light source, for example, into different partial beams. The remaining number of modulators (or parts) can then use the prefocused light to generate the nulls, which are then bordered by stronger intensity maxima.

[0069] As previously explained, a primary application of an arbitrarily positionable arrangement of multiple zeros of a light field in a work area is working with point emitters in that work area. Therefore, the invention also relates to a method for applying a light field to a work area containing point emitters, which enables excitation, localization, tracking, or other work with these point emitters.

[0070] Within the scope of this method, at least two positions at which the point emitters are located in the working area are at least approximately acquired. This can be achieved, for example, by prior activation and localization of point emitters, for example, via a model-based fit of the respective emitted intensity distribution. The working area is then exposed to a light field using the method described above, which has zeros at the previously acquired positions. In a particularly advantageous embodiment, the point emitters are separated – i.e., optically isolated. If the optical isolation of the point emitters is not present, the positions of the point emitters can still be obtained, for example, using sub-zero control patterns, which can be used to create multiple zeros within the minimum distance required for optical isolation of the point emitters.A sufficient number of measurements with offset zeros can also provide the necessary information. It can be particularly useful to also use image information.

[0071] As previously explained, the initially only approximately determined positions of the point emitters can be subsequently optimized by shifting the positions of the zeros within the workspace with sub-pixel accuracy. Once the exact positions of the point emitters have been determined, the point emitters can be tracked independently of each other on their path through the workspace. Iterative optimization is particularly simple if one can assume that the emitters are not moving or are moving very little during the optimization process. However, optimization can also incorporate the possible movement of the emitters during the optimization process into the model.

[0072] Therefore, in a particularly advantageous embodiment, the intensity (and, if applicable, its spatial distribution) of secondary light emitted by the point emitters in response to excitation with the light field is measured. This allows, for example, the study of how well the point emitters can be excited by the light field at any given time. The secondary light can also be used to locate the point emitters and track their movement.

[0073] In a further particularly advantageous embodiment, the positions at which the zero points of the light field are located can be actively controlled so that the intensity of the secondary light assumes a minimum or the minimum is intentionally located in its immediate vicinity, so that very precise distance information to the zero point can be obtained from the emitted light.

[0074] For this purpose, the positions of the zeros can be shifted with subpixel precision, as described above. In general, the rate of detected photons per unit of time can be determined depending on the shift. The closer the zero is to the emitter, the more precisely each point emitter can ultimately be located for a given illumination dose.

[0075] Another possibility is to initially position at least two zeros with subpixel precision at a reference point (assumed position of the point emitter) and to shift these zeros at at least three points by a fixed distance from the reference point with a relative angular separation that is as isotropic as possible (e.g., three steps with a relative rotation of 120° around the center of rotation). The position of the point emitter can then be fitted based on the number of photons registered at the individual measurement points.

[0076] The choice of circular polarization for illuminating the working area can be useful in order to minimize the dependence on the orientation of the molecule or, more precisely, the transition dipole moment of the molecules.

[0077] A combination of the two methods is also useful. For example, the amounts by which the zero point is shifted from the reference point can be progressively reduced. The position of the point's center can then be fitted, for example, using a maximum likelihood estimation.

[0078] The method for determining the zero points can also be chosen such that in each step the zero points are iteratively repositioned so that the result of the measurement (number or image of the emitted photons) always provides maximum information ("information capacity") with regard to the determination of the emitter position, which leads to a particularly efficient determination of the emitter position.

[0079] In another particularly advantageous embodiment, the working area is illuminated by a plurality of light fields, each of which has zero points at different positions. For each of these light fields, the intensity of the secondary light and / or the spatial distribution of this intensity is measured. The position of at least one point emitter in the working area is evaluated from the measurements. If, for example, at least three light fields are selected and the zero points are shifted in a plane from one light field to the next, a two-dimensional position of at least one point emitter can be evaluated from the measurements. The evaluation can, in particular, include a fit, for example, using the maximum likelihood method.

[0080] In another particularly advantageous embodiment, at least four light fields are selected. The union of the positions of zeros from all light fields extends across the spatial directions. The evaluation, which may include a fit, for example, can then provide the three-dimensional position of at least one point emitter.

[0081] In a further particularly advantageous embodiment, the working area is first exposed to a first light field, and the intensity of the secondary light and / or the spatial distribution of this intensity is measured.

[0082] After this measurement, a second light field is determined, which promises maximum information gain with regard to a given target. This given target can, for example, include the precise localization of at least one point emitter. The first light field and the second light field differ primarily with regard to the positions of the zeros they contain. The working area is then exposed to the second light field, and the intensity, or rather the spatial distribution of the intensity, is measured again. Special description section

[0083] The subject matter of the invention is explained below with reference to figures, without limiting the subject matter of the invention. It is shown: Figure 1 : Embodiment of the method 100; Figure 2 : Generation of zeros 7a, 7b by phase changes Δϕ a , Δϕ b ; Figure 3: Superposition of a blocking control pattern 6e with zero control pattern 6 from several sub-zero control patterns 60; Figure 4 : Exemplary zero control patterns 6a and 6b with sub-zero control patterns 60a, 60b, which in turn contain sub-sub control patterns 6a1, 6a2 and 6b1, 6b2, respectively; Figure 5 : Illustration of the shifting of a zero point 7a by changing a sub-sub control pattern 6b1, 6b2; Figure 6 : Illustration of the shifting of two zeros by changing sub-sub control patterns 6b2, 6b3; Figure 7 : Embodiment of the method 200; Figure 8 : Exemplary device for carrying out the method 200.

[0084] Figure 1is a schematic flow diagram of an embodiment of the method 100 for generating a light field 2 in a predetermined working area 1. In step 110, light 4 from a light source 3 is supplied to at least one modulator 5. According to block 111, this modulator 5 can have a controllable active surface 51, which imposes phase changes Δϕ a , Δϕ b on light components 4a, 4b of the light 4. The effect of the modulator 5 is described in detail in Figure 2 The entire optical structure is shown in Figure 8 shown in more detail.

[0085] In step 120, the modulator 5 is controlled with a zero-point control pattern 6, 6a, 6b such that the sum of the amplitude distributions of the light components 4a, 4b guided via the modulator 5 in the working area 1 has zero points 7a, 7b at at least two predetermined positions. These zero points each border on areas 8a, 8b in which this sum of the amplitude distributions is different from zero. This is also shown in Figure 2 shown in more detail.

[0086] In step 130, the light 4 is guided by the modulator 5 into the working area 1 and forms the light field 2 there.

[0087] Within box 120, several exemplary embodiments for controlling the modulator 5 are given.

[0088] According to block 121, adjacent regions 51a, 51b of the active area 51 of the modulator 5, which may correspond to sub-sub-control patterns 6a1, 6a2; 6b1, 6b2, can be controlled such that an average phase shift of π is set between light components 4a, 4b, on which these regions 51a, 51b respectively act, for the light leaving these regions 51a, 51b. The sub-zero control patterns, composed of sub-sub-control patterns, are referred to as "phase boxes" in a special embodiment. Their effect is described in Figure 2 explained in more detail.

[0089] According to block 122, the modulator 5 is controlled at a first time with a first zero control pattern 6a, for example, consisting of at least two (possibly differently configured) sub-zero control patterns 60, and at a second, later time with at least one second zero control pattern 6b, for example, consisting of at least two (possibly differently configured) sub-zero control patterns 60. At least two zero control patterns 6a, 6b differ in that the position of at least one zero 7a, 7b in the working area 1 changes by a smaller amount than would result from the translation of the first zero control pattern 6a by one pixel 52 on an active area 51 of the modulator 5, which is divided into pixels 52. The zero 7a, 7b can therefore be shifted by sub-pixel amounts in the working area 1.An exemplary subdivision of the active area 51 into pixels 52 is shown in . Figure 2 shown.

[0090] According to block 123, a parameterized approach 6d can be established for the zero-point control pattern 6. According to block 124, the parameters 6d' of approach 6d can be optimized with the goal of ensuring that the zeros 7a, 7b are located exactly at the desired positions.

[0091] According to block 125, the modulator can be controlled with a superposition of the zero-point control pattern 6, 6a, 6b on the one hand and a blocking control pattern 6e on the other. The blocking control pattern 6e alone would essentially keep all of the light 4 guided via the modulator 5 away from the working area 1; this blockage is broken at the points where the zero-point control pattern 6, 6a, 6b acts. The superposition according to block 125 is shown in Figure 3 outlined.

[0092] According to block 126, at least one sub-zero control pattern 60 can be superimposed or composed in some other way of at least two sub-sub-control patterns 6a1, 6a2 or 6b1, 6b2. The light influenced by each of these sub-sub-control patterns 6a1, 6a2 or 6b1, 6b2 then generates, according to block 127, a complex amplitude A 1 , A 2 at the predetermined location of at least one zero 7a, 7b. The sub-sub-control patterns 6a1, 6a2 or 6b1, 6b2 are selected according to block 128 such that the superposition A* of these complex amplitudes A 1 , A 2 results in zero (or almost zero) at the predetermined location of the zero 7a, 7b. This is described in detail in Figure 5 shown in more detail. The reference symbol A* should not be misunderstood here as a "complex conjugation" in the mathematical sense.

[0093] Within box 130, various embodiments are specified as to how the light 4 can be guided in detail from the modulator 5 into the working area 1 in order to form the light field 2 there.

[0094] According to block 131, the light between the modulator 5 and the working area 1 can be low-pass filtered in the spatial frequency domain.

[0095] According to block 132, the modulator 5 can be illuminated by the light source 3 at an oblique angle and controlled such that light components 4 whose phase is not changed by a zero-point control pattern 6, 6a, 6b are essentially kept away from the working area 1.

[0096] According to block 133, an optical plane in which the modulator is arranged can be imaged in a reduced manner onto the working area 1 or onto another optical plane using an optical system 9.

[0097] According to block 134, the working area 1 and the modulator 5 can be arranged at optically conjugated positions and / or in optically conjugated planes.

[0098] According to block 135, the modulator 5 can be arranged in a Fourier plane.

[0099] According to block 136, the modulator 5 can be arranged in any optical plane.

[0100] According to block 137, additional modulators (5) and / or at least two parts of a modulator in different optical planes may be used.

[0101] According to block 138, one or more modulators and / or at least two parts of a modulator in different optical planes may be used in combination with at least one additional optical element.

[0102] Figure 2illustrates the generation of a zero point 7a, 7b using a sub-zero control pattern 60 with the modulator 5 using a simplified sketch of the structure. For clarity, the structure is drawn in transmission, although commercially available modulators 5 that operate in reflection typically deliver better results.

[0103] A light source 3 radiates light 4 onto the modulator 5. The active area 51 of the modulator 5 is divided into pixels 52, which can be individually controlled and, depending on the control, impose a specific phase change Δϕ on the light incident on the respective pixel 52. In the Figure 2 In the state shown, the modulator 5 is controlled with the sub-zero control pattern 60, which creates two adjacent regions 51a and 51b on the active area 51. For illustration, Figure 2a sub-zero control pattern 60 is shown which generates only a single zero 7a, 7b.

[0104] The light component 4a, which falls on the area 51a, is subjected to a phase change Δϕ a. The light component 4b, which falls on the area 51b, is subjected to a phase change Δϕ b. The phase changes Δϕ a and Δϕ b differ by π. For the sake of clarity, Figure 2 only an arrangement of areas 51a and 51b is shown.

[0105] The light components 4a and 4b are guided by the modulator 5 through an optical imaging system 9 into the working area 1, where they form the light field 2. The Figure 2The symbol used should not be misunderstood to mean that a single lens is generally sufficient as an optical imaging system 9. Each region 51a and 51b individually generates an amplitude distribution A 1 and A 2 , with a value different from zero at the desired zero point, but there with the same magnitude and different sign, so that the total there is a zero point. In the light field 2, for each arrangement of regions 51a and 51b on the active surface 51, the zero point 7a is formed, which is surrounded by a region 8a, 8b with an intensity different from zero. The preferred use of these zero points is the localization and / or tracking of point emitters 11 in the working area 1.

[0106] As mentioned before, Figure 3a superposition of a blocking control pattern 6e (here a regular grid of horizontal lines) and a zero control pattern 6. The zero control pattern 6 comprises several sub-zero control patterns 60 according to the type of Figure 2 used sub-zero control pattern 60. This overlay can be switched to the active area 51 of the modulator 5.

[0107] Figure 4 shows two exemplary sub-zero control patterns 60a and 60b, which belong to two different zero control patterns 6a and 6b, respectively. Both sub-zero control patterns 60a and 60b each contain two sub-sub-control patterns 6a1 and 6a2, or 6b1 and 6b2, which can be switched, for example, to adjacent regions 51a and 51b on the active area 51 of the modulator 5.

[0108] In the sub-zero control pattern 6a, the second sub-sub control pattern 6a2 causes a phase change of the affected light by π compared to the first sub-sub control pattern 6a1. The light modulated according to the two sub-sub control patterns 6a1 and 6a2 superimposes at the location of the zero 7a to form a complex amplitude of zero.

[0109] The sub-zero control pattern 60b is modified compared to the sub-zero control pattern 60a in that the two sub-sub-control patterns 6b1 and 6b2 now each contain a periodic component 6c1, 6c2 in the plane of the active area 51. This influences the amplitude strengths generated by the sub-sub-control patterns 6b1 and 6b2 at the specified location of the zero. Figure 5 It is explained in more detail how the position of a zero point 7a can be shifted in this way.

[0110] Figure 5schematically illustrates how the position of a zero point 7a can be shifted by changing the amplitude strength using sub-sub control patterns 6a1, 6a2; 6b1, 6b2. For simplicity, only a shift along one coordinate direction x is shown here.

[0111] Under the already Figure 4 For the known sub-zero control pattern 6a, the complex amplitudes A 1 and A 2 , which the light in the working area 1 generated by the sub-sub control patterns 6a1 and 6a2, respectively, are plotted as a function of the coordinate x. The superposition A* of these complex amplitudes A 1 and A 2 has a zero 7a on the A-axis.

[0112] Under the also already Figure 4 Corresponding diagrams are drawn for the known sub-zero control pattern 6b. In the Figure 5In the example shown, by adding the periodic component 6c2 of the second sub-sub-zero control pattern 6b2, the complex amplitude A 2 of the light influenced by the second sub-sub-zero control pattern 6b2 is changed to a new curve A' 2 , which essentially only differs in strength from the previous curve A 2 . Accordingly, the complex amplitude A* of the superposition also changes to a new curve A* ,1 =A 1 +A 2 ', which no longer has the zero 7a on the A-axis, but at the new position x 2 .

[0113] However, it is also possible, for example, to add the periodic component 6c1 of the first sub-sub-zero control pattern 6b1 instead of the periodic component 6c2 of the second sub-sub-zero control pattern 6b2. When this happens, the complex amplitude A1 of the light influenced by the first sub-sub-zero control pattern 6b1 is modified to a new curve A1', which essentially differs only in strength from the previous curve A1. This shifts the zero 7a of the new curve A*,2 =A1'+A2 from the A axis to the left to the new position x1.

[0114] Figure 6 schematically illustrates how two zeros 7a, 7b can be generated with a sub-zero control pattern 60a, 60b. In addition, Figure 6schematically shows how the positions of two zeros 7a, 7b, which were generated with a sub-zero control pattern 60a, 60b, can be shifted by changing the amplitude strength using the sub-sub-control patterns 6a1, 6a2, 6a3; 6b1, 6b2, 6b3. For simplicity, only a shift along one coordinate direction x is shown here.

[0115] The Figure 6 The sub-zero control patterns 60a, 60b shown differ from those shown in Figure 5 shown sub-zero control patterns 60a, 60b by adding a third sub-sub-zero control pattern 6a3 or 6b3. Accordingly, there are three complex amplitudes A 1 , A 2 , and A 3 , which superimpose to form a resulting complex amplitude A**.

[0116] For the sub-zero control pattern 60a, the first zero 7a is located at position x 1,a , where A 1 superimposed with A 3 results in zero or almost zero. The second zero 7b is located at position x 2,a , where A 1 superimposed with A 2 results in zero or almost zero.

[0117] The sub-zero control pattern 60b differs from the sub-zero control pattern 60a in that the sub-sub-control patterns 6b2 and 6b3 have been given equal periodic components 6c2 and 6c3. As a result, the amplitude A 2 is modified to a curve A 2 ', and the amplitude A 3 is modified to a curve A 3 '. The amplitudes A 1 , A 2 ', and A 3 ' are thus superimposed to form the resulting complex amplitude A***. This complex amplitude A*** has a first zero 7a at position x 1,b , where A 1 superimposed with A 3 ' results in zero or almost zero, and a second zero 7b at position x 2,b , where A 1 superimposed with A 2 ' results in zero or almost zero.

[0118] Figure 7 is a schematic flow diagram of an embodiment of the method 200 for applying a light field 2 to a work area 1 containing point emitters 11. In step 210, positions 11a, 11b at which the point emitters 11 are located are at least approximately acquired using any method. In step 220, the work area 1 is exposed to a light field 2 using the previously described method 100, which has zero points 7a, 7b at the acquired positions 11a, 11b.

[0119] Light field 2 excites point emitters 11 to emit secondary light 12. This secondary light is measured in step 230. In step 240, the positions at which the zero points 7a, 7b of light field 2 are located are actively controlled so that the intensity of secondary light 12 assumes a minimum or the minimum is intentionally located in its immediate vicinity. The positions of the zero points 7a, 7b then serve as point emitter positions.

[0120] According to block 221, the work area 1 can be exposed to a plurality of light fields 2a-2d, each of which has zeros at different positions. For each of these light fields 2a-2d, the intensity of the secondary light and / or the spatial distribution of this intensity can then be measured according to block 231. From these measurements, the position 11a, 11b of at least one point emitter 11 in the work area 1 can be evaluated in step 250. If at least four light fields 2a-2d are selected according to block 221a and the union of the positions of zeros 7a, 7b from all light fields extends over three spatial directions X, Y, Z, the position 11a, 11b of the at least one point emitter 11 can also be determined three-dimensionally. This allows, in particular, the approximate value of this position 11a, 11b originally introduced in step 210 to be refined.The determined position 11a, 11b can also be fed back into the method 200 as a new approximate value in order to continuously track the at least one point emitter 11.

[0121] According to block 222, the work area 1 can initially be exposed to a first light field 2a. After measuring 230 the intensity of the secondary light 12 and / or the spatial distribution of this intensity, a second light field 2b can be determined in step 260, which promises maximum information gain with regard to a predetermined target. The work area can then also be exposed to this second light field 2b according to block 223.

[0122] Figure 8 shows an exemplary setup for implementing the method 200. In contrast to Figure 2The modulator 5 is operated in reflection mode, and the imaging optical system 9 between the modulator 5 and the working area 1 is more complex. The optical system 9 comprises a lens 9a, a semi-transparent dichroic lens 9b, a Fourier mask 9c, and an objective lens 9d. The light field 2 thus generated in the working area 1 is of the type shown in Figure 2 outlined.

[0123] The Figure 8 The structure shown makes it possible to guide secondary light 12, which point emitters 11 in the working area 1, in a straight line downwards through an emission filter 13 and a tube lens 14 into a detector 15. List of reference symbols

[0124] 1Working area 2, 2a-2dLight field in working area 1 3Light source 4Light 4a, 4bModulated components of light 4 5Modulator 51Active area of ​​modulator 5 51a, 51bAdjacent areas on active area 51 52Pixels of active area 51 6, 6a, 6bZero control pattern 60Sub-zero control pattern as a building block of pattern 6 60aSub-zero control pattern belonging to pattern 6a 60bSub-zero control pattern belonging to pattern 6b 6a1-6a3Sub-sub-zero control pattern of pattern 6a 6b1-6b3Sub-sub-zero control pattern of pattern 6b 6c1-6c3Periodic components of the sub-sub-control patterns 6a1-6a3; 6b1-6b3 6dparameterized approach for zero-point control pattern 6 6d'Parameters of approach 6d 6eBlocking control pattern 7a, 7bZeros 8a, 8bAreas with non-zero intensity around zeros 7a,7b 9Imaging optical system between modulator 5 and region 1 9aLens 9bDichroid 9cFourier mask 9dObjective 11Point emitters 11a-11bPositions of point emitters 11 12Secondary light emitted by point emitters 11 13Emission filter 14Tube lens 15Detector 100Method for generating the light field 2 in region 1 110Supplying the light 4 to the modulator 5 111Selection of a modulator 5 with active area 51 120Controlling the modulator 5 with the zero control pattern 6 121Controlling regions 51a, 51b with phase difference π 122Switching between sub-zero control patterns 6a and 6b 123Setting up the parameterized Approach 6d 124Optimizing the parameters 6d' 125Control with blocking control pattern 6e 126Forming the pattern 60 from sub-sub-control patterns 6a1, 6a2; 6b1, 6b2 127Generating complex amplitudes A 1 , A 2 128Selection of the patterns 6a1, 6a2; 6b1,6b2 for positioning the zero points 130 Guiding the light 4 from the modulator 5 into the area 1 131 Low-pass filtering of the light 4 132 Oblique illumination of the modulator 5 133 Reduced imaging with optical system 9 134 Arranging area 1 and modulator 5 in conjugate planes 135 Arranging the modulator in a Fourier plane 136 Arranging the modulator in any optical plane 137 Arranging modulators in different optical planes 138 Arranging 137 in conjunction with optical elements 200 Method for exciting and / or tracking point emitters 11 210 Determining approximate positions 11a of the point emitters 11 220 Exposing the area 1 to light field 2 221 Selection of several light fields 2a-2d 221a Selection of at least four light fields 2a-2d 222Selection of a first light field 2a 223Selection of the new light field 2b 230Measurement of the secondary light 12 231Separate measurement 230 for each light field 2a-2d 240Regulation of the positions of the zeros 7a, 7b 250Evaluation of the position 11a,11b of the point emitter 11 260 Determination of a new light field 2b after information gain A complex amplitude A 1 complex amplitude of the light influenced by pattern 6a1, 6b1 A 2 complex amplitude of the light influenced by pattern 6a2, 6b2 A 3 complex amplitude of the light influenced by pattern 6a3, 6b3 A 2 'change in the complex amplitude A 2 due to periodic component 6c2 A 3 'change in the complex amplitude A 3 due to periodic component 6c3 A*superposition of the complex amplitudes A 1 , A 2 A**superposition of the complex amplitudes A 1 , A 2 , A 3 A***superposition of the complex amplitudes A 1 , A 2 ', A 3 ' A* ,1 change in the superposition A* due to periodic component 6c1 A* ,2 Change of the superposition A* by periodic component 6c2 Δϕ a , Δϕ b Phase changes caused by areas 51a, 51b xCoordinate direction x 1 , x 2 Positions of a single zero 7a x 1,a , x 2,a Positions of two zeros 7a, 8a, caused by pattern 60a x 1,3 , x 2,b Positions of two zeros 7b, 7b, caused by pattern 60b,

Claims

1. A method (100) for generating a light field (2) in a specified work region (1) by means of an at least partly coherent light source (3), wherein • light (4) from the light source (3) is fed (110) to at least one modulator (5), with this modulator (5) impressing different changes in at least one property of the light on different components (4a, 4b) of the light (4), • the modulator (5) is controlled (120) by a zero control pattern (6, 6a, 6b) in such a way that the summation of the amplitude distributions of the light components (4a, 4b) guided via the modulator (5) has zeros (7a, 7b) at at least two positions that are specifiable independently of one another within the work region (1), with these zeros (7a, 7b) each adjoining regions (8a, 8b) in which this summation of the amplitude distributions differs from zero, wherein the zero control pattern (6, 6a, 6b) denotes a spatial distribution of regions which change the phase of the light incident on the modulator (5), as well as the respective local absolute value of this phase change, and • the light (4) is guided (130) from the modulator (5) into the work region (1).

2. The method (100) of claim 1, wherein a modulator (5) with a plane active surface (51) is chosen (111), said modulator being configured to impress phase changes (Δϕa, Δϕb) on light components (4a, 4b) which pass through the active surface (51) and / or are reflected by the active surface (51).

3. The method (100) of claim 2, wherein at least two adjoining regions (51a, 51b) of the active surface (51) are controlled (121) such that, between the light components (4a, 4b) on which these regions (51a, 51b) respectively act, a mean phase shift of π is set for the light leaving these regions (51a, 51b).

4. The method (100) of any one of claims 2 to 3, wherein the active surface (51) of the modulator is subdivided into pixels (52) which are controllable separately from one another, wherein the modulator (5) is controlled (122) using a first zero control pattern (6a) at a first time and using a second zero control pattern (6b) at a second, later time, wherein at least two zero control patterns (6a, 6b) differ to the effect that the position of at least one zero (7a, 7b) in the work region (1) changes by a smaller absolute value than the change that would arise from the translation of the first zero control pattern (6a) on the active surface (51) of the modulator (5) by one pixel (52).

5. The method of any one of claims 1 to 4, wherein the zero control pattern (6) comprises a plurality of zero control sub-patterns (60) that each generate an independent zero (7a, 7b) by virtue of the light respectively influenced by the zero control sub-patterns (60) being influenced in different ways.

6. The method (100) of claim 5, wherein • at least one zero control sub-pattern (60) is superimposed or otherwise composed (126) from at least two control sub-sub-patterns (6a1, 6a2; 6b1, 6b2) and • the light influenced by each of these control sub-sub patterns (6a1, 6a2; 6b1, 6b2) in each case generates (127) a complex amplitude (A1, A2) at the specified location of at least one zero (7a, 7b), • wherein the control sub-sub-patterns (6a1, 6a2) are chosen such that the superposition (A*) of these complex amplitudes (A1, A2) at the specified location of the zero (7a, 7b) yields (128) zero or a minimum in terms of absolute value that is near zero.

7. The method (100) of claim 6, wherein • at least one zero control sub-pattern (60) is superimposed or otherwise composed (126a) from at least three control sub-sub-patterns (6a1, 6a2, 6a3; 6b1, 6b2, 6b3) and • the light influenced by each of these control sub-sub-patterns (6a1, 6a2, 6a3; 6b1, 6b2, 6b3) in each case generates (127a) a complex amplitude (A1, A2, A3) at the specified location of at least one zero (7a, 7b), • wherein the control sub-sub-patterns (6a1, 6a2, 6a3; 6b1, 6b2, 6b3) are chosen such that the superposition (A*) of these complex amplitudes (A1, A2, A3) generates (128a) at least two zeros (7a, 7b) in the work region (1).

8. The method (100) of any one of claims 6 to 7, wherein at least one control sub-sub-pattern (6a1, 6a2, 6a3; 6b1, 6b2, 6b3) comprises an additional control element in the plane of the active surface (51), said additional control element being suitable for influencing the amplitude strength generated by this control sub-sub-pattern (6a1, 6a2, 6a3; 6b1, 6b2, 6b3) at the specified location of the zero (7a, 7b).

9. The method (100) of any one of claims 1 to 8, wherein • a parametrized approach (6d) for the zero control pattern (6, 6a, 6b) is set up (123), and wherein the parameters (6d') of this approach (6d) are optimized (124) with the goal that the summation of the amplitude distributions of the light components (4a, 4b) guided via the modulator (5) has zeros (7a, 7b) at the at least two specified positions, and / or • the modulator (5) is controlled (125) with a superposition of a blocking control pattern (6e) and a zero control pattern (6, 6a, 6b), wherein the blocking control pattern (6e) is configured to the effect that the light (4) guided via the modulator (5) is kept away from the work region (1), and / or • the light between the modulator (5) and the work area (1) is low-pass-filtered (131) in the spatial frequency domain, and / or • the modulator (5) is illuminated by the light source (3) at an oblique angle and is controlled (132) in a manner that is configured such that light components (4) whose phase is not changed by a zero control pattern (6, 6a, 6b) are kept away from the work region (1).

10. The method (100) of any one of claims 1 to 9, wherein • the light (4) is modulated in several different optical planes, and / or • an optical plane in which the modulator (5) is arranged is imaged (133) onto the work region (1) or onto another optical plane in a size-reducing manner with an optical system (9), and / or • the work region (1) and the modulator (5) are arranged (134) at optically conjugate positions, and / or in optically conjugate planes, and / or • the modulator (5) is arranged (135) in a Fourier plane.

11. A method (200) for subjecting a work region (1) containing point emitters (11) to a light field (2), comprising the steps of: • at least approximately obtaining (210) at least two positions (11a, 11b) at which point emitters (11) are located in the work region (1); • subjecting (220), by the method (100) according to any one of claims 1 to 10, the work region (1) to a light field (2) having zeros (7a, 7b) at the obtained positions (11a, 11b).

12. The method (200) of claim 11, wherein the intensity of secondary light (12) emitted by the point emitters (11) in response to an excitation with the light field (2) is measured (230), wherein the positions at which the zeros (7a, 7b) of the light field (2) are located are actively controlled (240) such that the intensity of the secondary light (12) assumes a minimum, or the minimum is deliberately located in their immediate vicinity.

13. The method (200) of claim 12, wherein • the work region (1) is subjected (221) to several light fields (2a-2d) that each have zeros (7a, 7b) at different positions; • the intensity of the secondary light (12), and / or the spatial distribution of this intensity, is measured (231) for each of these light fields (2a-2d); and • from the measurements, the position (11a, 11b) of at least one point emitter (11) in the work region (1) is evaluated (250).

14. The method (200) of claim 13, wherein at least four light fields (2a-2d) are chosen (221a), and wherein the union set of the positions of zeros (7a, 7b) from all light fields (2a-2d) extends over three spatial directions (X, Y, Z).

15. The method (200) according to any one of claims 12 to 14, wherein • the work region is subjected (222) to a first light field (2a); • after measuring (230) the intensity of the secondary light (12), and / or the spatial distribution of this intensity, a second light field (2b) is determined (260) which promises a maximum information gain in view of a predetermined goal; and • the work region is also subjected (223) to this second light field (2b).