Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/361—Optical details, e.g. image relay to the camera or image sensor
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
  • G02B21/365—Control or image processing arrangements for digital or video microscopes
  • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
  • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/061—Sources
  • G01N2201/06113—Coherent sources; lasers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/12—Circuits of general importance; Signal processing

Definitions

• the invention relates to a method for high-resolution 3D fluorescence microscopy, wherein in a sample repeatedly fluorescence emitter excited to emit fluorescence radiation and the sample with a microscope having an imaging beam path with an optical resolution and a focal plane, individual images are generated, the Fluorescence emitter are excited to emit fluorescence radiation such that at least a subset of the fluorescence emitter is isolated in each frame so that the images of these fluorescence emitters are separable in the individual images within the frame of the optical resolution, in the generated individual images from the images of the isolated fluorescence emitter Layers of these fluorescence emitters are localized with a spatial resolution going beyond an optical resolution and from this a high-resolution overall image is generated, each individual image being divided into a first and a second partial image by means of a divider element wherein the first partial image images a first focal plane in the sample and the second partial image images a second focal plane in the sample, both partial images are imaged side by side on a camera.
• the invention further relates to a fluorescence microscope for three-dimensional imaging of a sample with a spatial resolution increased via an optical resolution, comprising: an illumination device which is designed to repeatedly excite fluorescence emitters in the sample to emit fluorescence radiation, an imaging beam path with the optical path A resolution-comprehensive imaging device, which is adapted to generate from the sample with the optical resolution frames, a control device which is adapted to drive the illumination device and the imaging device so that from the sample a plurality of individual images are generated, the fluorescence emitter such Emission of fluorescence radiation are excited, that at least a subset of the fluorescence emitter is isolated in each frame such that the images of these fluorescence emitters are separable in the individual images within the optical resolution , wherein the control device is adapted to, in the generated individual images, the layers of the isolated fluorescent fluorescence emitter with a via a Locating optical resolution going to localization accuracy and to produce a high-resolution overall image, the imaging beam path has a splitter element dividing each frame into
• PALM abbreviated method photo-activated light microscopy
• a marking substance for imaging a sample which z. B. can be activated by means of optical radiation. Only when activated can the marker release certain fluorescence radiation.
• Non-activated molecules of the labeling substance emit no or at least no appreciable fluorescence radiation even after irradiation of excitation radiation. Therefore, the activation radiation is generally referred to as a switching signal.
• the switching signal is now applied so that at least a certain proportion of the activated marker molecules of adjacent activated molecules is so spaced that these marker molecules are separated from the optical resolution of the microscopy separately or subsequently separated by image processing methods. It is said that a subset of the fluorescence emitters is isolated. After recording the fluorescence radiation, the center of its resolution-limited radiation distribution is then determined for these isolated emitters. From this one can mathematically determine the position of the molecules with higher accuracy than the optical resolution actually allows. This process is called localization.
• the PALM principle exploits statistical effects.
• a marker molecule which can be excited to fluoresce radiation after receiving the switching signal of a given intensity it can be ensured by adjusting the intensity of the switching signal that the probability of activating marker molecules present in a given area of the sample is so small that there are sufficient partial regions in which only distinguishable marking molecules emit fluorescence radiation within the optical resolution.
• the PALM principle was further developed with regard to the activation of the molecules to be detected. For example, for molecules that have a long-lived non-fluorescent and a short-lived fluorescent state, separate activation with spectrally different from the excitation radiation activation radiation is not required.
• the sample is first activated with high-intensity illumination radiation in such a way that the vast majority of the molecules are brought into the non-fluorescent, long-lived state (eg a triplet state). The remaining, then still fluorescent molecules are thereby isolated in terms of optical resolution.
• PALM PALM principle in the literature has now received other abbreviations, such as STORM etc.
• the abbreviation PALM is used for all microscopic images that reach a spatial resolution beyond the optical resolution of the equipment used by first isolating and then localizing fluorescent molecules.
• the PALM method has the advantage that no high spatial resolution is required for the illumination. A simple wide field illumination is possible.
• the PALM principle requires that many frames of the sample be taken, each containing subsets of isolated molecules. In order to fully model the sample, the amount of all frames must ensure that as many molecules as possible were contained at least once in a subset.
• the PALM method therefore regularly requires a large number of individual images, which requires a certain amount of time for recording an overall image. This involves a considerable amount of computation, since a large number of molecules must be located in each individual image. There are a lot of data.
• depth direction is meant the direction along the incidence of light, ie along the optical axis.
• the image of the molecule on the camera is elliptically distorted as soon as the molecule is above or below the focal plane, ie the point of symmetry of the point image ashing function.
• the information about the depth of the luminescent marker molecule can be obtained.
• a disadvantage of this method is that even the local environment and the orientation of a molecular dipole can lead to a distortion of the image of the luminescent marker molecule, which has nothing to do with the depth. Such luminescent marker molecules are then assigned a wrong depth value, depending on their orientation.
• the depth position of marker molecules that lie between these two planes is now obtained by analyzing the two partial images of the same marker molecule (eg, the width of the point wash image, which can be subtractive) or by fitting a three-dimensional point spread image function.
• the method requires two high-resolution fields and a precise adjustment of the beam paths and calibration measurements in order to achieve a sub-pixel-precise superimposition of the two partial images.
• the two sub-images of a marker molecule are usually of different shape, since the lateral extent of the point-image spreading function of an imaging system changes as a function of the position of the considered object plane.
• the invention has for its object to improve such a method in that the structure is simplified and Justieranjoen are easy to meet.
• the object is achieved by a method for high-resolution 3D fluorescence microscopy, in which a sample repeatedly excited fluorescence emitter for emitting fluorescence radiation and the sample with a microscope, which has an imaging beam path with an optical resolution and a focal plane, individual images are generated, the Fluorescence emitter are excited to emit fluorescence radiation such that at least a subset of the fluorescence emitter is isolated in each frame so that the images of these fluorescence emitters are separable in the individual images within the frame of the optical resolution, in the generated individual images from the images of the isolated fluorescence emitter Layers of these fluorescence emitters are localized with a spatial resolution going beyond an optical resolution and from this a high-resolution overall image is generated, each individual image being divided into a first and a second partial image by means of a divider element wherein the first partial image images a first focal plane in the sample and the second partial image images a second focal plane in the sample, both partial images are imaged onto at least one camera, in which an adaptive mirror is
• a fluorescence microscope for three-dimensional imaging of a sample with a resolution increased by an optical resolution, comprising: a lighting device which is adapted to repeatedly excite fluorescence emitter in the sample to emit fluorescence radiation, an imaging beam path with the optical Resolution comprehensive imaging device, which is adapted to generate from the sample with the optical resolution frames, a Control device which is designed to control the illumination device and the imaging device in such a way that a plurality of individual images are generated by the sample, the fluorescence emitters being excited to emit fluorescence radiation in such a way that at least a subset of the fluorescence emitters in each individual image is isolated such that the Images of these fluorescence emitters are separable in the individual images as part of the optical resolution, wherein the control device is designed to localize the positions of the isolated fluorescent fluorescence emitters in the generated individual images with a spatial accuracy that goes beyond an optical resolution and to produce a high-resolution overall image therefrom, the imaging beam path has a divider element which divides each individual image
• an adaptive mirror is used as the beam-dividing element. It has a dual function, as it spatially separates the partial images as well as assigns them to different focal planes. This reduces the adjustment effort which in the prior art was characteristic of the principle used by the invention. This is to generate for each frame at least two fields associated with different focal planes in the sample. In the partial images then appear the dot images of the isolated fluorophores with a size which depends on the depth of the fluorophore. For example, if the fluorophore is in the focal plane of a field, the dot image of the fluorescence emitter is as small as the diffraction limit allows. As the distance to the focal plane increases, the dot image grows with otherwise the same geometry.
• the size of the dot image of a fluorescence emitter encodes the distance to the focal plane.
• the fluorescence emitter is above or below the focal plane. Therefore, in the principle pursued by the invention, at least two partial images are generated for two different focal planes in the sample in order to be able to determine not only the distance from the focal plane but also the absolute position relative to the focal plane.
• the two partial images can be imaged simultaneously on two image acquisition areas or one after the other on a camera.
• the adaptive mirror further has the advantage of high light efficiency over the concepts used in the prior art. Losses, as occur at beam splitters, etc. in the prior art, does not know the solution according to the invention.
• the distance of the focal planes of the two partial images can be adjusted over a wide range, without the need for a complex mechanical actuation of beam path deflecting elements, etc.
• the mirror also allows the partial images to be reproduced in rapid succession on a camera, since a quick change of focal length can be realized.
• parts of the mirror surface of the adaptive mirror are set with a slightly different focal length and an angle tilted with respect to the remaining elements.
• the latter leads to the desired lateral offset and the adjacent image of the two partial images on the camera.
• the mirror surface portions it can be set with which intensity ratio the two partial images are generated.
• the two sub-images must be adjusted sub-pixel-precise to each other, so that the desired high resolution with depth indication can be achieved.
• a particularly simple procedure for adjusting the partial images is to first produce them with the same focal length. There are then two identical sub-images on the camera side by side, d. H. The single image is displayed side by side twice on the camera. In this state, it is easily possible to obtain the reference coordinates for the two fields.
• the coordinates of the high-resolution fluorescence emitter simply provide the relative positions of the two partial images on the camera. After the adjustment has been carried out in such a way, the mirror is controlled so that the different focal lengths are realized. The adjustment position of the partial images does not change, so that the previously determined relative indication of the coordinates in the two partial images is still valid.
• Positional adjustment is understood here to mean that a coordinate specification is determined which makes it possible to find a point in the first partial image in the second partial image as well, In the simplest case it can be a relative coordinate.
• the use of the adaptive mirror makes it easy to change the difference in the focus lengths, making the depth resolution or the detected depth range easily adjustable.
• the mirror surface components of the adaptive mirror which cause the image of the first partial image are simply reduced in size and, by this measure, the mirror surface components which cause the imaging of the second partial image are enlarged.
• the adaptive mirror makes it particularly easy to correct aberrations of the imaging beam path. For this purpose, it is expedient to detect radiation reflected at the mirror (also) with a wavefront sensor.
• Particularly suitable as an adaptive mirror are mirrors with a segmented surface or continuous, so-called membrane mirrors, as are known to the person skilled in the art, for example from the publication www.bostonmicromachines.com/light-modulator.htm or www.imagine-optic.com. An overview of adaptive mirrors can be found in http://en.wikipedia.org/wiki/Deformable_mirror.
• the adaptive mirror is combined with a wavefront sensor which detects the wavefront of the radiation which has been reflected at the mirror.
• a wavefront sensor which detects the wavefront of the radiation which has been reflected at the mirror.
• This will optionally correct aberrations of the microscope or aberrations caused by the sample.
• a shift of the focal plane within certain limits is possible without problems, without the microscope objective would have to be adjusted. Mechanical disturbances of the sample by movements of the microscope objective can be avoided.
• the image of a fluorescence emitter means its diffraction-limited dot image as a rule.
• FIG. 1 is a schematic representation of a microscope for depth-resolved and high-resolution fluorescence microscopy
• FIG. 4 is a schematic representation of the operation of an adaptive mirror in the imaging beam path of the microscope of FIG. 1.
• FIG. 1 schematically shows a fluorescence microscope 1 whose operation is controlled by a control unit C. It is connected via connections, not shown, with the elements or components of the microscope 1.
• the microscope 1 is adapted to perform fluorescence microscopy according to the PALM principle, etc. It comprises an illumination beam path 3, as well as an imaging beam path 4, which illuminate a sample 2 via a common objective 5 and image the fluorescent sample.
• the illumination beam path 3 is combined with the imaging beam path 4 via a beam splitter 6, which is generally dichroic, so that both the illumination radiation from the illumination beam path 3 passes through the objective 5 to the specimen and the image of the specimen through the objective 5 takes place.
• the illumination beam path 3 can have a plurality of spectral channels, in the representation of FIG. 1 only one laser source L1 is shown by way of example.
• the illumination beam path illuminates the sample so that fluorescence radiation in the sample 2 is excited.
• an excitation radiation source can additionally be coupled into the illumination beam path 3.
• the sample 2 emits fluorescence radiation, and in the imaging beam path 4, the image of the fluorescent sample 2 is directed onto a high-resolution camera K.
• the resolution of objective 5, imaging beam path 4 and camera K is selected so that a diffraction-limited point image of a single fluorescence emitter falls on several pixels. This enables the localization required for the PALM principle described above a fluorescence emitter with a lateral position accuracy, which goes beyond the optical resolution of the microscope 5 and imaging beam path 4.
• the microscope 1 can also be configured with multiple color channels. Then several cameras are provided in the imaging beam path 4, which are coupled via suitable beam splitter in the beam path.
• the imaging beam path 4 comprises, in addition to unspecified optical elements which are not further characteristic of the microscope 1 and otherwise customary in the art, an adaptive mirror 7 whose mirror surface is curved and which is part of the imaging beam path 4. It focuses the radiation from the fluorescent sample 2 in the direction of the camera K. Its function will be explained later.
• the adaptive mirror is controlled by the control unit C, which adjusts the geometry of the mirror surface.
• a wavefront sensor 9 connected via a beam splitter to the imaging beam path 4 serves the control device C to be able to detect the current mirror effect as precisely as possible. It increases accuracy, but is not mandatory.
• the controller C controls the microscope 1 so that the PALM principle is executed.
• the sample 2 is thus illuminated by the illumination beam path 2 so that fluorescence emitters are isolated in the sample 2, d. H. can be separated within the optical resolution.
• a multiplicity of individual images is recorded, each of which contains different subsets of the fluorescence emitters isolated in the sample 2.
• the position is then determined with high precision by the control device C by means of known mathematical algorithms for each isolated fluorescence emitter, so that a positional accuracy exceeding the optical resolution of the image is achieved.
• This is referred to in the literature as so-called high resolution or super resolution.
• the PALM principle allows highly accurate lateral localization of fluorescence emitters, i. H. the detection of the position perpendicular to the optical axis.
• the extent to which a fluorescence emitter is located in a focal plane, in front of or behind a focal plane, ie the depth position of the fluorescence emitter, is not specified with higher accuracy by the PALM principle than with other wide field microscopes.
• the microscope 1 is operated under the control of the control unit C so that each individual image in which the lateral localization is divided into two sub-images, which are assigned to different focal planes in the sample 2.
• FIGS. 2 and 3 show these partial images 12 and 13.
• the partial image 12 is a single image which images a focal plane that is higher than the partial image 13 with respect to the light incidence direction, ie the optical axis.
• Figures 2 and 3 show different states a, b, c for the partial images 12 and 13, and contain by way of example only a single fluorescence emitter.
• the partial images 12a and 13a show a state in which a fluorescence emitter is located so that it is in the focal plane, which is associated with the partial image 13. Consequently, the image 11a of the fluorescence emitter in the partial image 13a is a diffraction-limited spot whose size is predetermined by the diffraction limit.
• the same spot can also be found in sub-image 12a, but the image 1 1 a is extended here larger, since the imaged fluorescence emitter is not in the focal plane of the sub-image 12. This leads to a defocusing, which enlarges the picture 1 1 a.
• the comparison of the images 11a in the partial image 12a and 13a clearly shows the depth of the fluorescence emitter, namely in the focal plane of the partial image 13.
• the control unit C can therefore derive a corresponding depth specification for the fluorescence emitters from the size of the dot images of the fluorescence emitters and the comparison of the partial images.
• the lateral localization is obtained as known from the dot image 1 1 a.
• the partial images 12b and 13b show a state in which a fluorescence emitter is located between the focal planes of the partial images. This can be seen from the fact that the dot image 1 1 b of this fluorescence emitter has a size that is above the diffraction-limited minimum, but also below the extent that would result if the fluorescence emitter were arranged in the focal plane of the sub-image 13.
• the partial images 12c and 13c finally show a state that is inverted to the state a.
• the fluorescence microscope 1 reaches the partial images 12 and 13 by using the adaptive mirror 7.
• This causes both the division of the image of the sample 2 in the two partial images 12, 13, as well as the assignment to different focal positions by different focal lengths for the partial images 12, 13th
• FIG. It shows the adaptive mirror 7, on which the radiation from the sample 2 along an optical axis OA is incident.
• the reflecting surface of the mirror 7 is divided into segments 15 and 16 or is divided by the control by the control unit 10 in such segments.
• the adaptive mirror acts as a beam splitter, which divides the diffractive image of the sample 2 in the imaging beam 4 in the two fields 12 and 13.
• Other imaging elements such as lenses etc. can be used, but are not shown in the illustration of Figure 4 for the sake of clarity.
• the adaptive mirror 7 is adjusted to have mirror surface segments 15 which deflect the optical axis OA in a first direction. Interleaved mirror surface segments 16 direct the optical axis OA in a second direction.
• the first direction is an optical axis OA1, which is assigned to the camera image area Ka
• the second direction corresponds to an optical axis OA2, which is assigned to the camera image area Kb.
• the corresponding marginal rays for the imaging by the first mirror surface segments 15 are like the optical axis OA1 dotted registered in Figure 4, the marginal rays and the optical axis OA2, which are passed through the mirror surface segments 16 on the camera image area Kb are drawn in phantom.
• the mirror surface segments 15 cause a focal length difference in the generated partial image 12.
• the adaptive mirror 7 is thus adjusted so that it not only generates an image division, but also assigns both partial images 12 and 13 to different focal planes in the sample 2. This can be seen from the fact that the marginal rays for the partial image 12 meet on the optical axis OA in the camera image area Ka, whereas the dot-dashed marginal rays for the partial image 13 on the camera image area Kb are still widened.
• the camera image areas Ka and Kb are preferably but not necessarily image areas of the same camera K. This has the advantage that a sub-pixel-precise adjustment of the partial images 12 and 13 is easily possible.
• the following options are available: 1. In an adjustment step upstream of the actual measuring method, the adaptive mirror 7 is adjusted such that the partial images 12 and 13 are produced along diverging optical axes OA1 and OA2, but with the same focal length of the mirror 7. The partial images 12 and 13 then originate during the adjustment step thereof focal plane. The high-resolution lateral localization of one or more structures in the partial images 12 and 13 then makes it easy to obtain a reference with which structures in the partial image 12 are converted to the coordinate system of the partial image 13 in the later measuring operation and vice versa. 2.
• the partial images 12 and 13 can first be superpositioned, ie displayed in overlay.
• the mirror 7 is adjusted so that the optical axes OA1 and OA2 coincide, the angular difference between the mirror segments 15 and 16 is therefore no longer given.
• this overlay can be easily ensured that despite different focal lengths of the segments 15 and 16, the images are superimposed. Also, a referencing of the images to each other can be easily performed.
• the mirror is changed so that the angular differences between the mirror segments 15 and the mirror segments 16 are present.
• the angular divergence between the optical axes OA1 and OA2 occurs, and the sub-images 12 and 13 are juxtaposed.
• the thus caused difference in the coordinates between the sub-images 12 and 13 is easy to derive from the mirror parameters, so that a highly accurate relative referencing between the sub-images 12 and 13 is ensured by the previously achieved adjustment in the superpositioned state.

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