Method for illuminating samples in microscopic imaging methods

Abstract

A method for illuminating samples in microscopic imaging methods, wherein a number m of different wavelengths λ.sub.i, with m>I and i=I, . . . , m, is selected for the illumination. For each of the wavelengths λ.sub.i a target phase function Δφ.sub.i(x, y, λ.sub.i) is predefined, wherein x and y denote spatial coordinates in a plane perpendicular to an optical axis z and each target phase function Δφ.sub.i(x, y, λ.sub.i) is effective only for the corresponding wavelength λ.sub.i. The target phase functions Δφ.sub.i are predefined depending on the structure of the sample and/or the beam shape and/or illumination light structure to be impressed on the light used for illumination. A total phase mask is then produced which realises all target phase functions Δφ.sub.i(x, y, λ.sub.i). This total phase mask is then illuminated simultaneously or successively with coherent light of wavelengths λ.sub.i such that the predefined structure of the illumination light is generated in the region of the sample.

Claims

1. A method for illuminating samples in microscopic imaging methods, comprising selecting a number m of different wavelengths λ.sub.i, with m>1 and i=1, . . . , m, for illumination purposes, specifying a target phase function Δφ.sub.i(x, y, λ.sub.i) for each of the wavelengths λ.sub.i, where x and y denote spatial coordinates in a plane perpendicular to an optical axis z and wherein each target phase function Δφ.sub.i(x, y, λ.sub.i) effectively only acts for the respective wavelength λ.sub.i, specifying the target phase functions Δφ.sub.i(x, y, λ.sub.i) based on a structure of the sample or an illumination light structure or beam shape to be impressed on light used for illumination purposes, wherein said target phase functions at least approximately satisfy a system of equations (G1) $\begin{array}{cc}\left(\begin{array}{c}\frac{{\mathrm{\Delta \phi }}_1\left(x,y,{\lambda }_1\right)}{2\pi }\\ \frac{{\mathrm{\Delta \phi }}_2\left(x,y,{\lambda }_2\right)}{2\pi }\\ \mathrm{.Math.}\\ \frac{{\mathrm{\Delta \phi }}_m\left(x,y,{\lambda }_m\right)}{2\pi }\end{array}\right)=\left(\begin{array}{cccc}\frac{\left(n_1\left({\lambda }_1\right)-1\right)}{{\lambda }_1}& \frac{\left(n_2\left({\lambda }_1\right)-1\right)}{{\lambda }_1}& \mathrm{.Math.}& \frac{\left(n_m\left({\lambda }_1\right)-1\right)}{{\lambda }_1}\\ \frac{\left(n_1\left({\lambda }_2\right)-1\right)}{{\lambda }_2}& \frac{\left(n_2\left({\lambda }_2\right)-1\right)}{{\lambda }_2}& \mathrm{.Math.}& \frac{\left(n_m\left({\lambda }_2\right)-1\right)}{{\lambda }_2}\\ \mathrm{.Math.}& \mathrm{.Math.}& \ddots & \mathrm{.Math.}\\ \frac{\left(n_1\left({\lambda }_m\right)-1\right)}{{\lambda }_m}& \frac{\left(n_2\left({\lambda }_m\right)-1\right)}{{\lambda }_m}& \mathrm{.Math.}& \frac{\left(n_m\left({\lambda }_m\right)-1\right)}{{\lambda }_m}\end{array}\right)\left(\begin{array}{c}{D}_1\\ D_2\\ \mathrm{.Math.}\\ D_m\end{array}\right),& \left(\mathrm{G1}\right)\end{array}$ where D.sub.i denotes a location-dependent or constant thickness of an optically effective material M.sub.i with a wavelength-dependent refractive index n.sub.i, wherein the refractive index n.sub.i is location-dependent if the thickness is constant and wherein an overall phase mask realizing the target phase functions Δφ.sub.i is generated based on a solution to the system of equations (G1); generating an overall phase mask, which realizes the m target phase functions Δφ.sub.i, and illuminating the sample simultaneously or successively with coherent light of the wavelengths λ.sub.i via the overall phase mask.

2. The method as claimed in claim 1, wherein that the same optically effective material M with a location-dependent thickness D(x, y) is used for all wavelengths λ.sub.i.

3. The method as claimed in claim 2, further comprising using a transmissive spatial light modulator or a liquid crystal layer arranged on a silicon substrate (LCOS) with a constant thickness as part of a spatial light modulator as optically effective material and a phase is set by way of a refractive index difference Δn that depends on an applied, location-dependent voltage U.

4. The method as claimed in claim 1, wherein an illumination light structure that increases resolution is impressed on the light used for illumination purposes.

5. The method as claimed in claim 4, wherein the illumination light structure comprises, for each of the wavelengths λ.sub.i, at least two diffraction maxima, differing from the 0th order, corresponding to two orders of diffraction which are arranged in a common pupil plane, wherein the diffraction maxima at the same wavelength λ.sub.i are respectively arranged along a straight line and the diffraction maxima of the zeroth order of diffraction lie congruently on one another for all wavelengths λ.sub.i and wherein the remaining diffraction maxima of the same orders either lie congruently on one another for all wavelengths λ.sub.i or lie on lines that are rotated with respect to one another by an offset angle that depends on the number m of wavelengths λ.sub.i.

6. The method as claimed in claim 1, wherein the illumination light structure comprises, for each of the wavelengths λ.sub.i, diffraction maxima corresponding to a Dammann grating arranged in a pupil plane, wherein the respective same orders of diffraction are congruent for all wavelengths λ.sub.i.

7. The method as claimed in claim 1, further comprising generating, for at least one of the wavelengths λ.sub.i, target phase function Δφ.sub.i(x, y, λ.sub.i) by means of a Gerchberg-Saxton algorithm on account of an intensity distribution specified for this wavelength λ.sub.i.

8. The method as claimed in claim 1, further comprising impressing the form of a multichromatic light sheet.

9. The method as claimed in claim 1, wherein a plurality of regions of interest are defined within the sample before the target phase functions Δφ.sub.i(x, y, λ.sub.i) are determined, the wavelengths λ.sub.i of the light used to illuminate a region of interest are defined for each region of interest, and an illumination light structure by means of which only the regions of interest are illuminated with light of the respectively selected wavelengths λ.sub.i is defined.

10. The method as claimed in claim 8, wherein said multichromatic light sheet is a multichromatic sinc.sup.3 beam.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is explained in even greater detail below for example with reference to the accompanying drawings, which also disclose features essential to the invention. In detail:

(2) FIG. 1 shows a first example for target phase functions for two wavelengths,

(3) FIG. 2 shows the overall phase mask belonging to the first example,

(4) FIG. 3 shows a second example for target phase functions for two wavelengths,

(5) FIG. 4 shows the overall phase mask belonging to the second example,

(6) FIG. 5 shows a third example for target phase functions for two wavelengths,

(7) FIG. 6 shows the overall phase mask belonging to the third example,

(8) FIG. 7 shows a fourth example for target phase functions for two wavelengths, and

(9) FIG. 8 shows the overall phase mask belonging to the fourth example.

DETAILED DESCRIPTION OF THE INVENTION

(10) Described below is a method for illuminating samples in the microscopic imaging method, within the scope of which a number of m wavelengths λ.sub.i, with m>1 and i=1, . . . , m are selected for illumination purposes, wherein for each one of the wavelengths λ.sub.i a target phase function Δφ.sub.i(x, y, λ.sub.i) that effectively only acts on this wavelength is specified. Here, x and y denote spatial coordinates in a plane perpendicular to an optical axis z. Here, the target phase functions Δφ.sub.i(x, y, λ.sub.i) are specified on the basis of a structure of the sample and/or a beam shape and/or illumination light structure to be impressed on the illumination light. The target phase functions are used to generate an overall phase mask, by means of which the sample is illuminated simultaneously or successively with coherent light of the wavelengths λ.sub.i. Here, the target phase functions approximately satisfy the system of equations G1, for example:

(11) $\begin{array}{cc}\left(\begin{array}{c}\frac{{\mathrm{\Delta \phi }}_1\left(x,y,{\lambda }_1\right)}{2\pi }\\ \frac{{\mathrm{\Delta \phi }}_2\left(x,y,{\lambda }_2\right)}{2\pi }\\ \mathrm{.Math.}\\ \frac{{\mathrm{\Delta \phi }}_m\left(x,y,{\lambda }_m\right)}{2\pi }\end{array}\right)=\left(\begin{array}{cccc}\frac{\left(n_1\left({\lambda }_1\right)-1\right)}{{\lambda }_1}& \frac{\left(n_2\left({\lambda }_1\right)-1\right)}{{\lambda }_1}& \mathrm{.Math.}& \frac{\left(n_m\left({\lambda }_1\right)-1\right)}{{\lambda }_1}\\ \frac{\left(n_1\left({\lambda }_2\right)-1\right)}{{\lambda }_2}& \frac{\left(n_2\left({\lambda }_2\right)-1\right)}{{\lambda }_2}& \mathrm{.Math.}& \frac{\left(n_m\left({\lambda }_2\right)-1\right)}{{\lambda }_2}\\ \mathrm{.Math.}& \mathrm{.Math.}& \ddots & \mathrm{.Math.}\\ \frac{\left(n_1\left({\lambda }_m\right)-1\right)}{{\lambda }_m}& \frac{\left(n_2\left({\lambda }_m\right)-1\right)}{{\lambda }_m}& \mathrm{.Math.}& \frac{\left(n_m\left({\lambda }_m\right)-1\right)}{{\lambda }_m}\end{array}\right)\left(\begin{array}{c}{D}_1\\ D_2\\ \mathrm{.Math.}\\ D_m\end{array}\right)& \left(\mathrm{G1}\right)\end{array}$

(12) D.sub.i is the location-dependent or constant thickness of an optically effective material n with a wavelength-dependent refractive index n.sub.i. The refractive index n.sub.i itself is only location-dependent if the thickness of the material n is constant. On the basis of the solution to this system of equations G1, an overall phase mask that realizes the target phase shifts Δφ.sub.i can be generated as a stack of the materials n with the location-dependent thicknesses D.sub.i(x, y). The sample is then illuminated through the overall phase mask; i.e., the overall phase mask is then illuminated simultaneously or successively by a coherent light with the various wavelengths λ.sub.i such that, for example, a specified structure of the illumination light is generated in the region of the sample. This method will be explained in more detail below on the basis of simplified embodiments, in which two wavelengths λ.sub.1 and λ.sub.2 are used. Moreover, the assumption is made that there is only one material M with the thickness D present; specifically, an LCOS-based spatial light modulator should be used as the latter can be set most flexibly. In this case, the thickness of the material is constant and the refractive index is set in location-dependent fashion in x and y by way of a voltage U that is specified in location-dependent fashion.

DETAILED DESCRIPTION OF THE DRAWINGS

(13) FIG. 1 initially shows, in FIG. 1a) on the left-hand side, a target phase function Δφ.sub.1 for a first wavelength λ.sub.1, for example 488 nm. Shown on the right-hand side is the target phase function Δφ.sub.2 for a second wavelength λ.sub.2, for example 561 nm. This relates to one-dimensional phase gratings. These phase gratings were specified in such a way that the orders of diffraction shown in FIG. 1b)—this relates to the −1st and 1st order of diffraction—are congruent for the two wavelengths λ.sub.1 and λ.sub.2, i.e., the diffraction angles are identical. Since the wavelengths are different, the periods of the phase gratings must likewise be different. The diffraction patterns shown in FIG. 1b) arise in the far field, i.e., at a certain distance from the phase grating. If the phase grating is respectively arranged in an intermediate image plane, the diffraction patterns are located in a pupil plane. A superposition of the two diffraction patterns shown in FIG. 1b) is shown in FIG. 1c). The masks can be calculated from specified diffraction patterns, for example with the aid of a Gerchberg-Saxton algorithm. The representation is implemented here in the xy-plane with arbitrary coordinates.

(14) Then, using the method described above, it is possible to generate a phase mask which combines the desired imaging behavior of the two individual phase masks from FIG. 1a) and which can be displayed on an LCOS-SLM. The resultant phase mask is shown in FIG. 2, with different hatching representing different phase shifts. Here, the phase shifts are different for each of the two wavelengths, even in the same region.

(15) A development of FIG. 1 is shown in FIG. 3. In contrast to FIG. 1, the orders of diffraction of the two wavelengths are arranged on lines that intersect at an angle of 90° in the masked 0th order of diffraction. FIG. 3a) shows the target phase masks for the two individual wavelengths, FIG. 3b) shows the resultant diffraction patterns in the far field or the diffraction patterns from which the target phase functions are calculated, and FIG. 3c) shows the superposition of the two diffraction patterns of FIG. 3b). Here, the direction of the effect of the phase element depends on the wavelength, which can be exploited during structured illumination microscopy by virtue of the direction of the arrangement of the orders of diffraction changing in the pupil in the case of small wavelength changes. If a corresponding overall phase mask is determined by means of the above-described method, said overall phase mask can be realized by means of an LCOS-SLM in an intermediate image plane and, by switching through the wavelengths, it is possible by way of the wavelength variation to switch through the minimum of three directions of the orders of diffraction required for such an illumination microscopy, which must include an angle of 120° with respect to one another in a pupil plane. The arising optimized overall phase mask is illustrated in FIG. 4; different hatching/filling corresponds to different phase shifts in this case too.

(16) The above-described method can also be used to determine an overall phase mask for generating a multichromatic light sheet, wherein the shape of a multichromatic light sheet, preferably the shape of a multichromatic sinc.sup.3 beam, is impressed on the illumination light, i.e., the light used for illumination purposes. This should be explained on the basis of FIG. 5 and FIG. 6. Once again, the phase plate should be realized or simulated by an LCOS-SLM. Once again, the two wavelengths λ.sub.1=488 nm and λ.sub.2=561 nm are used. The representations on the left-hand side of FIGS. 5a)-d) relate to the wavelength of 488 nm in this case; the representations on the right-hand side relate to the wavelength of 561 nm. FIG. 5a) initially shows the illumination of the spatial light modulator; the illumination is already implemented in light-sheet-type fashion but still is unstructured. Here, the different lines denote different intensities; the intensity is greatest in the center. The target phase functions for both wavelengths Δφ.sub.i are illustrated in FIG. 5b); the corresponding diffraction patterns in a pupil plane are shown in FIG. 5c). The resultant individual, structured light sheets sinc.sup.3 beams are illustrated in FIG. 5d). While the intensities are congruent to the pupil plane, the target phase functions, and hence also the light sheets, differ in the sample region.

(17) The resultant phase plate, which can be realized either from a material with a location-dependent, varying thickness or on an LCOS-SLM with a constant thickness but location-dependent varying voltage and hence location-dependently varying refractive index, is illustrated in FIG. 6. Here, in the regions filled in white, the phase deviation is 0π for both wavelengths; i.e., there is no phase shift. In the regions labeled by crosshatching, there is a phase shift of 1.1π for the wavelength of 488 nm and a phase shift of 0.93π for the wavelength of 561 nm. In the regions labeled by oblique hatching from bottom left to top right, there is a phase shift of 3.8π for the wavelength λ.sub.1 and a phase shift of 3.25π for the wavelength λ.sub.2. Finally, the regions filled in black denote a phase shift of 4.88π for the wavelength λ.sub.i and of 4.15π for the wavelength λ.sub.2.

(18) A further example is shown in FIGS. 7 and 8; here, a diffraction maximum distribution is specified as illumination light structure for each of the two wavelengths λ.sub.i, as arises in the case of a Dammann grating, which only generates odd diffraction maxima, for example, arranged in a pupil plane. The two Dammann gratings that should be arranged in pupil planes are illustrated in FIG. 7a); a multispot pattern, which is respectively illustrated in FIG. 7b) for both wavelengths, is respectively generated therewith in the region of the sample. Here, the periods of the two target phase functions in FIG. 7a) were chosen in such a way that the points are respectively at the same location in this case. The superposition of the two diffraction patterns is illustrated in FIG. 7c). One of the two phase shifts in accordance with the overall phase mask realizing target phase functions Δφ.sub.i is illustrated in FIG. 8; using the latter, it is possible to generate the superposed diffraction pattern illustrated in FIG. 7c). Regions filled differently again describe different phase shifts in this case too. In the white regions there is no phase shift, neither for the wavelength λ.sub.1=488 nm nor for the wavelength λ.sub.2=561 nm. In the crosshatched regions, the phase deviation is 1.1π for λ.sub.1 and 0.93π for λ.sub.2. In the regions hatched obliquely from bottom left to top right, the phase deviation is 3.82π for λ.sub.1 and 3.24π for λ.sub.2. In the regions filled in black, the phase shift is 4.90π for λ.sub.1 and 4.13π for the wavelength λ.sub.2.

(19) Using the method described above, it is possible—particularly when using LCOS-SLM—to generate multichromatic illumination patterns in a simple manner, in the case of which, for example, the relative positions of the orders of diffraction are identical for a plurality of different illumination wavelengths in a pupil plane, without having to resort to complex technology such as photonic integrated circuits, for example. This yields great cost savings, for example because there is no need to use photonic integrated circuits that are specifically matched to the respective illumination structure.

(20) While the invention has been illustrated and described in connection with currently preferred embodiments shown and described in detail, it is not intended to be limited to the details shown since various modifications and structural changes may be made without departing in any way from the spirit of the present invention. The embodiments were chosen and described in order to best explain the principles of the invention and practical application to thereby enable a person skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.