阅读说明：本技术 在至少两个波长范围之间进行辨别的高分辨率扫描显微术 (High resolution scanning microscopy for discriminating between at least two wavelength ranges ) 是由 I.克莱普 R.内茨 于 2018-06-21 设计创作，主要内容包括：出于高分辨率扫描显微术的目的,样品(2)由照明辐射(5)激发以发射荧光辐射,使得将照明辐射聚焦在样品(2)中或样品(2)上的点处,以形成衍射受限的照明光斑(14)。以衍射受限的方式将点成像在空间分辨二维检测器(19)上的衍射图像(23)中,其中二维检测器(19)具有对衍射图像(23)的结构进行分辨的空间分辨率。以小于照明光斑(14)的一半直径的增量,通过不同的扫描位置来扫描样品(2)。从二维检测器(19)的数据以及从向这些数据分配的扫描位置生成样品(2)的图像,该图像的分辨率增加以超过成像的分辨率极限。出于在样品(2)的荧光辐射中在至少两个预先确定的波长范围之间进行辨别的目的,对应于至少两个预先确定的波长范围的一定数目的衍射结构(30-37)生成在二维检测器(19)上,所述衍射结构区别于彼此但是具有共同的对称中心(40)。当生成样品(2)的图像时评估衍射结构(30-37)。(For the purpose of high resolution scanning microscopy, the sample (2) is excited by the illumination radiation (5) to emit fluorescence radiation such that the illumination radiation is focused at a point in or on the sample (2) to form a diffraction limited illumination spot (14). The spots are imaged in a diffraction-limited manner in a diffraction image (23) on a spatially resolved two-dimensional detector (19), wherein the two-dimensional detector (19) has a spatial resolution which resolves the structure of the diffraction image (23). The sample (2) is scanned by different scanning positions in increments smaller than half the diameter of the illumination spot (14). An image of the sample (2) is generated from the data of the two-dimensional detector (19) and from the scanning positions assigned to these data, the resolution of the image being increased beyond the resolution limit of the imaging. For the purpose of discriminating between at least two predetermined wavelength ranges in the fluorescence radiation of the sample (2), a number of diffractive structures (30-37) corresponding to the at least two predetermined wavelength ranges are generated on a two-dimensional detector (19), said diffractive structures being distinct from each other but having a common center of symmetry (40). The diffractive structures (30-37) are evaluated when generating an image of the sample (2).)

1. A method for high resolution scanning microscopy of a sample (2), wherein:

-the sample (2) is excited by illumination radiation (5) to emit fluorescence radiation such that the illumination radiation is focused at a point in or on the sample (2) to form a diffraction limited illumination spot (14),

-imaging the spots in a diffraction image (23) on a spatially resolved surface detector (19) in a diffraction limited manner, wherein the surface detector (19) has a spatial resolution resolving diffractive structures (30-37) of the diffraction image (23),

-shifting the spot in relation to the sample (2) into different scanning positions in increments smaller than half the diameter of the illumination spot (14),

-reading the surface detector (19) and generating an image of the sample (2) from data of the surface detector (19) and from scan positions assigned to the data, the resolution of the image increasing beyond a resolution limit of the imaging,

-for the purpose of distinguishing between at least two predetermined wavelength ranges by means of a spectral selection module (15), a diffraction image (23) having a plurality of diffraction structures (30-37) is generated on the surface detector (19), the number of diffraction structures corresponding to the number of wavelength ranges, the diffraction structures each being point-symmetric with respect to a center of symmetry and all lying on the surface detector (19),

-evaluating the diffractive structure (30-37) when generating an image of the sample (2),

it is characterized in that the preparation method is characterized in that,

-the point-symmetric diffractive structures (30-37) cover different areas in the image plane (18), but have a common center of symmetry (40).

2. The method of claim 1, wherein the at least two predetermined wavelength ranges are subjected to different phase manipulations.

3. The method according to claim 2, characterized in that the spectrum selection module (15) comprises phase influencing means (17, 17a, 17 b).

4. A method according to claim 3, characterized in that only radiation at one wavelength is influenced by the phase influencing means (17, 17a, 17b) and radiation at another wavelength is not influenced by the phase influencing means, so that the diffractive structure (30) of the radiation is an airy disc.

5. The method according to any of the preceding claims, wherein the diffractive structures (30-37) are coaxial.

6. Method according to any of claims 1 to 4, characterized in that the diffractive structures (32-37) have the same basic structure but are rotated relative to each other over an angle on the surface detector (19).

7. Method according to any of the preceding claims, characterized in that the spectral selection module (15) only affects the illumination, wherein the illumination spots (14) are constituted by illumination diffractive structures (38, 39) that are distinct but have a common center of symmetry (40).

8. Method according to any of the preceding claims, characterized in that the spectral selection module (15) only affects the imaging, in particular is arranged upstream of the surface detector (19).

9. A microscope for high resolution scanning microscopy comprising:

a sample space for receiving a sample (2) excitable for emitting fluorescent radiation,

an optical unit (11-13), the optical unit (11-13) having a focal plane (29) and a diffraction limit located in the sample space,

an illumination device (3), the illumination device (3) having an inlet (6) for supplying illumination radiation (5) and illuminating the sample space with the illumination radiation (5) via the optical unit (11-13) such that the optical unit (11-13) focuses the illumination radiation (5) to a point in the focal plane (29) to form a diffraction-limited illumination spot (14),

-an imaging device (4), which imaging device (4) is configured to diffractively image, via the optical unit (11-13), a point in the focal plane (29) in a diffraction image (23) on a spatially resolved surface detector (19), which spatially resolved surface detector (19) is located in an image plane (18) conjugate to the focal plane (29), wherein the surface detector (19) has a spatial resolution that resolves structures of the diffraction image (23),

-a scanning device (10), the scanning device (10) displacing the spot into different scanning positions in increments smaller than half the diameter of the illumination spot (14), and

-an evaluation device (C) reading the surface detector (19) for evaluating the structure of the diffraction image (23) from the data of the surface detector (19) and from the scan positions assigned to the data and generating an image of the sample (2) whose resolution is increased beyond the resolution limit,

-wherein the microscope (1) comprises a spectral selection module (15), the spectral selection module (15) distinguishing between at least two predetermined wavelength ranges, the selection module generating a diffraction image (23) on the surface detector (19) with a number of diffraction structures (30-37), the number of diffraction structures corresponding to the number of wavelength ranges,

-the surface detector (19) and the spectral selection module (15) are implemented such that the diffractive structures (30-37) are all located on the surface detector (19), and

-the evaluation device (C) evaluates the diffractive structure (30-37) when generating an image of the sample (2),

it is characterized in that the preparation method is characterized in that,

-the point-symmetric diffractive structures (30-37) cover different areas in the image plane (18), but have a common center of symmetry (40).

10. A microscope as claimed in claim 9 characterised in that the spectrum selection module (15) comprises phase influencing means (17, 17a, 17b) for influencing the diffractive structure, which phase influencing means subject the at least two predetermined wavelength ranges to different phase manipulations.

11. A microscope according to claim 10 characterised in that the phase influencing means comprises at least one phase mask (17, 17a, 17b) and/or at least one LCOS-SLM.

12. A microscope according to claim 10 or 11 characterised in that the phase affecting means (17) affects radiation in one wavelength range only and not in another so that its diffractive structure (30) is an airy disc.

13. A microscope as claimed in any one of claims 9 to 12 characterised in that the diffractive structures (30-37) are rotationally symmetric and coaxial.

14. A microscope according to any one of claims 9 to 13 characterised in that the diffractive structures (33-37) have the same basic structure but are rotated relative to each other by an angle on the surface detector (19).

15. A microscope according to any one of claims 9 to 14 characterised in that the spectrum selection module (15) is provided in the illumination device (3) but not in the optical unit (11-13), which is also effective for imaging, so that the illumination spot (14) is constituted by illumination diffractive structures (38, 39) which are distinct but have a common centre of symmetry (40).

16. A microscope according to any one of claims 9 to 14 characterised in that the spectrum selection module (15) is provided in the imaging device (4) rather than in the optical unit (11-13), which is also effective for illumination.