Classifications

• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0036—Scanning details, e.g. scanning stages
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/0004—Microscopes specially adapted for specific applications
  • G02B21/002—Scanning microscopes
  • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
  • G02B21/0052—Optical details of the image generation
  • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/24—Base structure
  • G02B21/26—Stages; Adjusting means therefor
• • 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
• • 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

Definitions

• the invention relates to a method for operating a sample chamber for microscopic imaging and a device for performing the method.
• the invention further relates to a sample chamber which is used in particular in the method according to the invention.
• these can be treated chemically in such a way that structures deep in the tissue or organ are also visible.
• the tissues or organs - hereinafter generally referred to as a sample - are subjected to a treatment with certain chemicals, which is also referred to as "clearing".
• the action of the chemicals removes or replaces existing lipids, for example, while supporting structures remain unchanged.
• the structures that have been preserved can be made visible and imaged using fluorescent markings.
• the effective penetration depth of microscopy methods can be significantly increased, for example, because of a reduced scatter of illuminating or detection radiation due to lipids removed in this way.
• Methods for "clarifying" samples, for example of brains are for example from Silvestri, L. et al. (2016): Clearing of fixed tissue: a review from a microscopist's perspective. Journal of biomedical optics, 21 (8): 081205 or from Richardson, DS & Lichtman, JW (2015): Clarifying tissue clearing, Cell, 162 (2): 246-257.
• Specimen clarification is used in particular for brain examinations.
• Typical samples are cleared brains from mice. Their size of 8 - 10 mm requires an optical penetration depth of at least about 10 mm.
• the chemicals used for clarification are sometimes very aggressive or also toxic and often expensive. If the immersion arrangements customary in the prior art are used, undesirable imaging errors can occur. If, for example, a clearing medium used for clarification ultimately forms the immersion as a sample medium, then aberrations can be caused by unknown or non-fully compensated optical properties of the clearing medium (refractive index, Abbe number). In addition, the lens comes into contact with the aggressive and / or toxic chemicals, which can damage the lens and mean increased effort in ensuring occupational safety and health protection and a reduction in the service life of the optical components.
• the object of the invention is to propose a possibility both on the process side and on the device side with which the disadvantages occurring in the prior art are reduced.
• the object is achieved by a method for operating a sample chamber for microscopic imaging according to claim 1, by a sample chamber according to claim 10 and a device for acquiring image data according to claim 14.
• the invention also relates to a sample holder for receiving at least one sample chamber according to the invention.
• the method for operating a sample chamber for microscopic imaging comprises the steps of providing a sample chamber in an illumination beam path and detecting it a detection radiation.
• the sample chamber used encompasses a sample space in which a sample can or is positioned.
• the sample chamber has at least one wall delimiting the sample space with an outside facing away from the sample space.
• the illumination beam path is directed through the outside into the sample space.
• the section of the illumination beam path directed into the sample space through the outside is also referred to and understood as the illumination axis.
• a detection radiation caused by an illumination radiation directed into the sample space along the illumination axis is detected along a detection axis running through the wall of the sample chamber from the sample space.
• the detection axis is part of a detection beam path.
• the method is characterized in that a sample chamber is used, the outside of which has the shape of a spherical section with a circular disk as the base, which is also referred to below as a spherical shape.
• the sample chamber and the detection axis are rotated and / or pivoted relative to one another about the center of the circular disk, so that different angular relative positions of the sample chamber and detection axis are set. With different angular relative positions of the sample chamber and the detection axis, image data are acquired.
• the detection axis always runs through the spherical outside.
• the essence of the invention is the use of a sample chamber with a spherical outside, as will be described in more detail below.
• the design of at least one outside of the sample chamber as a sphere (spherical section) and the movement of the sample chamber around the center of the circular disk advantageously enables the setting of a large number of different angular relative positions.
• an angle of the detection axis relative to the outside is kept constant.
• the detection axis always coincides with a normal of the outside, regardless of which angular relative position is set.
• This constancy of the orientation of the detection axis is only limited by the actual shape of the sample chamber, in particular by the covered angular range on the outside and the arc length on the outside.
• Equivalent are other shapes of the base of the sample chamber than that of a circular disk, for example as a polygon or free form.
• sample chamber advantageously allows a lens used for the detection of the detection radiation to be separated from the sample medium.
• image data acquisitions are carried out in at least two different z relative positions in a respective angular relative position.
• a respective Z relative position is set in that the sample chamber and / or a focal plane of an imaging optical system are displaced relative to one another along the detection axis.
• the direction of the detection axis is referred to as the z-direction (z-axis).
• Az relative position denotes a position of the focal plane along the detection axis.
• a z stack of image data is acquired under an angular relative position and a number of z relative positions.
• an image of the focal plane (Field of View; FoV) is acquired and imaged. If image data is recorded in two different z-relative positions under the same angular relative position, a sample volume is imaged if the two focal planes in the z-direction do not completely overlap or adjoin one another (Volume of View; VoV). If a stack (z-stack) of image data of mutually adjacent or overlapping focal planes is recorded in this way, a (volume) section of the sample can be completely recorded along the detection axis and under the relevant relative angle position.
• FoV Field of View
• the illuminating radiation is advantageously formed into a light sheet and irradiated into the sample space. Illuminating the sample with a light sheet is particularly advantageous for large and scattering samples.
• the illuminating radiation is advantageously used in the form of a non-diffractive beam.
• a non-diffractive beam is, for example, a beam limited in terms of its frequency spectrum by means of a sinc filter, as described in DE 10 2012 013 163 A1 (also referred to as a Sinc 3 beam).
• the Sinc 3 beam does not have to be scanned laterally to generate a light sheet.
• lateral scanning is required for other non-diffractive beams that can be used, such as a Mathieu beam or a Bessel beam.
• a Sinc 3 beam in particular, light sheets with a small thickness in the Z direction can potentially be generated.
• Non-diffractive rays that can be used to generate the light sheet are, in particular, Airy rays, circular Airy rays [circular airy beam], coherent Bessel rays (coherent Bessel beam; Gao, L. et al. (2012), Noninvasive Imaging beyond the Diffraction Limit of 3D Dynamics in Thickly Fluorescent Specimens; Cell 151: 1370 - 1385] and structured light sheets [lattice light sheet; Chen, Bi-Chang, et al. (2014), Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution; Science 346: 1257998]
• a light sheet with a known beam shape for example a scanned or Gaussian beam shaped by means of cylinder optics.
• a known beam shape for example a scanned or Gaussian beam shaped by means of cylinder optics.
• the advantages of non-diffractive beam shapes cannot be used.
• the refraction of the light sheet on the wall of the spherical sample chamber must be taken into account. This is particularly true when image data are acquired in different z-relative positions. It doesn't matter whether the sample chamber is moved or the lens.
• the angle of incidence of the light sheet is changed as a function of the z relative position (z position). The angle of incidence is understood to be the angle between the illuminating beam and a normal of the point of incidence of the illuminating beam on the outside.
• a portion of the volume of the sample can be imaged using the process design described above.
• image data from adjacent or overlapping partial areas of the sample are acquired as z-stacks at different angular relative positions of the sample chamber and detection axis.
• the angular relative position is changed in such a way that, for example, after a completed detection of a z-stack, a further angular relative position is set and a further z-stack is recorded.
• parameters such as focus position, angle of incidence and light sheet position can be stored as lookup tables.
• a light sheet can advantageously be combined with a plenoptic detection or light field detection [e.g. B. Prevedel, R. et al. (2014): Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nature methods, 11: 727]
• plenoptic detection a thicker and longer light sheet can be generated and irradiated.
• the light field detection can be used to reconstruct part of the sample in the z direction along the thickness of the light sheet from the additionally available angle information with an exposure. In this way, the required total exposure time can be significantly reduced, since in addition to the lateral parallelization (wide field), parallelization is also carried out along the detection axis (Plenoptics).
• the problem of non-focal background fluorescence, which complicates the plenoptic reconstruction, is advantageously reduced by the light sheet illumination.
• the illumination can take place using a point or multipoint scanner.
• the image data can be detected confocal, in the wide field or by means of plenoptic detection (see further below).
• the sample chamber is designed for use in the field of microscopy. It encompasses a sample space for positioning a sample and has at least one wall delimiting the sample space with an outside facing away from the sample space.
• the sample chamber is characterized in that the outside of the wall has the shape of a spherical section with a circular disk as the base. There is also a sample holder for holding the sample in the plane of the circular disk.
• the spherical section covers an angular range of at least 90 °, but preferably at least 120 °.
• Sample chambers with a covered angular range of at least 150 °, in particular 180 °, are well suited for the microscopic examination of clarified samples.
• the angular range covered by the spherical section can also be more than 180 °, for example 210 °, 240 ° or 270 °.
• the sample chamber advantageously allows the positioning of a sample with an extension in each spatial direction of at least 3 mm, for example at least 5 or 10 mm.
• the sample chamber can advantageously be sealed with a further wall, so that, for example, a sample medium is held in the sample chamber and cannot accidentally emerge from it.
• This further wall can be a separate wall and also serve as a sample holder.
• an inside of the further wall extends in the plane of the circular area.
• the sample holder in the form of a further wall can also be a sample carrier with at least one carrier surface.
• a parking space can have, for example, a structure such as an elevation and / or an annular depression. The sample chamber can be put over the elevation and is held by this at the parking space.
• a depression can also be a lateral guide for the sample chamber.
• the depression can also contain or accommodate a seal so that the sample chamber is closed in a liquid-tight manner.
• a sealing effect can be achieved in further embodiments by the surfaces of the areas of the sample chamber placed in the depression and / or the surface of the depression having a high accuracy of fit to one another and being sealed, for example, by adhesive forces or capillary forces of the surfaces or a liquid film located between them.
• a sample holder can have at least two sample chambers arranged on the sample holder.
• Such a sample carrier with sample chambers can be handled in the sense of a multiwell plate, as is known from laboratory operations.
• the latter has a plurality of carrier surfaces which are arranged at an angle to one another.
• the offset can be in one plane or spatial.
• support surfaces can be arranged around an axis. The individual carrier surfaces can be moved into a detection position in a controlled manner while the other carrier surfaces are in a waiting position.
• sample chambers which are closed, for example, with a separate wall, or sample carriers can have channels for supplying and / or discharging media into or out of the sample space.
• Different media such as chemicals for clarifying and / or rinsing the sample and sample media can be fed in and out via the channels.
• the channels are advantageously in fluid communication with pumps and media reservoirs.
• sensors and a control unit are advantageously present.
• the sample carrier has at least one carrier surface.
• a circumferential wall stands on this, which encloses a parking space as a lateral boundary and, together with the sample holder, forms a hollow cylinder which is open on one of its end faces.
• the sample chamber is arranged on the position, the hollow cylinder can be filled with an immersion medium into which a lens can be immersed.
• This additional chamber also called “dipping chamber” for simplicity, can also have a different cross-section in other versions, for example rectangular or polygonal with, for example, 5, 8, 10 or 20 corners.
• the sample chamber is arranged in a vessel, for example placed on the floor.
• Such a design of a sample carrier with a sample chamber enables a complete separation of the optics, in particular the objective, from the aggressive media within the sample chamber despite the use of an immersion medium.
• the immersion medium present in the hollow cylinder higher numerical apertures can be realized than would be the case with an air gap. It is advantageous if the immersion medium in the dipping chamber has the same refractive index as the sample medium. A further improvement in the imaging quality can be achieved if the immersion medium and sample medium have the same Abbe number and even the same partial dispersion.
• the refractive index, Abbe number and / or partial dispersion are often from New or further developed media for clarification not known or only roughly known.
• the refractive index of the immersion medium can be dynamically adjusted by mixing two or more suitable starting media. When working with a dynamically mixed immersion medium, it is advantageous if the refractive index of the wall material of the sample chamber is close to the refractive index of the sample medium and / or immersion medium.
• the method according to the invention can be carried out by means of a device for acquiring image data with a sample chamber or with a combination of sample carrier and sample chamber.
• the device has a light source for providing illuminating radiation along an illuminating beam path.
• beam-shaping optics for shaping the illuminating radiation into a light sheet are provided, which cooperate with a first scanner, which is arranged in or near a pupil of the illuminating beam path and serves to deflect the illuminating radiation.
• the device is characterized in that a second scanner that is movable about a pivot point is arranged near an intermediate image in the illumination beam path.
• the scanning optics map the pivot point of the second scanner to an entry point of the light sheet on the outside of the sample chamber.
• the functions of the first scanner and the second scanner consist in tracking the angle of incidence of the light sheet by the first scanner and a displacement of the light sheet by means of the second scanner.
• the angle tracking is implemented by the first scanner, which can be a conventional galvanometric scanning mirror. Since the movement of the angle tracking does not have to be fast in the sense of a scanning movement of a laser scanning microscope (LSM), other actuators are also possible. For example, piezo actuators, DC motors or stepper motors with e.g. Spindle drive can be used.
• LSM laser scanning microscope
• a displacement of the light sheet is necessary in real operation of the device, for example, in order to e.g. correct aberration-related focus deviations.
• the second scanner is used for this. In contrast to a typical scanner arrangement in an LSM, not both scanners are in one pupil of the illuminating beam path.
• the optical path lengths in the sample medium also change. It may therefore be necessary to correct the focus along the light sheet propagation direction. Such a correction can be done, for example, by moving the illumination lens or a tube lens or by other focusing optics.
• the first and the second scanner move in one plane, while in a classic scanner arrangement in an LSM the scan planes are orthogonal to one another.
• a beam shape should be selected to generate the light sheet that requires lateral “smearing” by a scanning movement, this can be done by a third scanner, which - comparable to the classic LSM arrangement - is close to the first scanner.
• Such beam shapes are, for example Sinc 3 beam or the radially symmetrical Bessel beam.
• the first and the third scanner are therefore in such an arrangement close to the pupil.
• the third scanner causes a rapid movement of the light sheet orthogonal to the plane of movement of the first scanner.
• Adaptive optical elements or units can advantageously be used to correct them. Suitable adaptive Optical units are, for example, deformable mirrors, spatial light modulators (SLM) and their combinations. The use of optical auxiliary structures, for example a so-called Guide Star, is possible.
• the adaptive optical elements can be arranged in the illumination beam path and / or in the detection beam path.
• the invention is particularly suitable for displaying clarified and large samples such as brains of mice.
• imaging methods that are suitable for thick, dense and scattering samples, i.e. in particular multiphoton methods (point scanner, temporal focusing)
• lenses or imaging systems with rather moderate magnifications are advantageously used.
• These are advantageously combined with cameras with very large numbers of pixels (“macroscope”) in order to take into account a mapping of the entire brain, for example, with as few individual images as possible.
• the advantages of the invention lie in particular in the separation of sample medium and objective. If the sample carrier is equipped with microfluidics, i.e. channels, clearing protocols can be carried out automatically and reproducibly. It is not necessary to change the sample carrier or the sample chamber between the clarification and imaging steps.
• the sample chamber according to the invention can be integrated in a “lab on the chip” environment. In addition to the automated execution of clarification protocols and image acquisition, storage of the sample with controlled media exchange is also possible.
• Figure 1 is a schematic representation of an embodiment of a sample chamber according to the prior art.
• FIG. 2 shows a schematic illustration of a first exemplary embodiment of a sample chamber according to the invention in a sectional illustration (FIG. 2a) and a top view from below (FIG. 2b);
• FIG. 3 shows a schematic illustration of an exemplary embodiment of a sample chamber according to the invention and a sample carrier
• FIG. 4 shows a schematic illustration of a first embodiment of the method according to the invention, with FIG. 4a capturing image data in a first angular relative position and a first Z relative position;
• FIG. 4b shows the acquisition of image data in a second angular relative position and a second Z relative position and
• FIG. 4c shows the acquisition of image data in the second angular relative position and a third Z relative position;
• 5a to 5c show a schematic illustration of a sequential detection of z-stacks at different angular relative positions and a resulting covering of the sample with a number of z-stacks; 6 shows a schematic illustration of a coordinate transformation of a z stack into a horizontally oriented coordinate system;
• FIG. 7 shows a schematic illustration of a coordinate transformation of a z stack into a spherical coordinate system
• FIG. 8 shows a schematic illustration of an exemplary embodiment of an imaging system with the possibility of selecting different imaging methods as a function of the sample
• FIG. 9 shows a schematic illustration of an exemplary embodiment of an optical arrangement for location and angle tracking in the case of light sheet illumination on a sample chamber according to the invention, and a schematic illustration of the angle tracking (insert figure);
• FIG. 10 shows a schematic illustration of a first exemplary embodiment of a sample carrier with a number of sample chambers according to the invention.
• FIG. 11 shows a schematic illustration of a second exemplary embodiment of a sample carrier with a number of sample chambers and channels according to the invention for supplying and / or discharging media.
• the sample chamber 1 shows a sample chamber 1 known from the prior art, which encloses a sample space 2, in which a sample 3 can be positioned.
• the sample space 2 is filled with a sample medium 4, into which a lens 5 of an imaging optics of an optical device, not shown, can be immersed.
• the sample medium 4, which also functions as an immersion medium 14, has a first refractive index n1 and is in direct contact with the objective 5. If the sample medium 4 is a chemical or a mixture of chemical components, such as those used for clarifying the sample 3, there is a risk of damage to the objective 5 due to their often aggressive properties.
• the sample space 2 is enclosed on three sides by a wall 6 which in the prior art is essentially formed from flat partial surfaces, the partial surfaces not having to be optically effective, for example not transparent.
• the sample 3 can be illuminated with an illuminating radiation along an illuminating beam path 7 coinciding with an optical axis OA of the objective 5.
• a detection radiation caused in the sample 3 is detected with the objective 5 along a detection axis DA detection beam path 8, the optical axis OA and the detection axis DA coinciding.
• a first exemplary embodiment of a sample chamber 1 according to the invention is shown in a side sectional view in FIG. 2a.
• the sample space 2 is delimited by a wall 6, an outer side 6.1 of the wall 6 having the shape of a spherical section (FIG. 2a).
• the spherical section covers a circular surface 9 with a center point 10 (see also FIG. 2b).
• the sample space 2 is delimited by a further wall 11, which extends in the plane of the circular surface 9.
• the sample chamber 1 is provided so that when it is used as intended, the Illumination beam path 7 through the outside 6.1 into the sample space 2 and the detection beam path 8 likewise through the outside 6.1. Illumination beam path 7 and detection beam path 8 can coincide or, as symbolized in FIG. 2a, be separated from one another.
• the spherical sample chamber 1 encloses the sample 3, for example a brain to be imaged, as closely as possible.
• the sample medium 4 used for clarification is located in the sample chamber 1. Detection of detection radiation can take place using an objective 5 designed as an air objective (see, for example, FIGS. 4a to 4c).
• the sample chamber 1 is placed on a carrier surface 12 of a sample carrier 13 and surrounded by a wall 15 in the form of a hollow cylinder standing on the carrier surface 12 (FIG. 3).
• Sample carriers 13 and carrier surface 12 can also form an open vessel together in further versions.
• the wall 15 or cylinder wall can have a different, for example an angular or oval, shape in plan view.
• An existing space 16 above the sample chamber 1 is filled with air or with an immersion medium 14, in which the objective 5 is immersed or can be immersed.
• the immersion medium 14 has a second refractive index n2 and serves to increase the numerical aperture (NA) of the objective 5.
• NA numerical aperture
• a non-aggressive chemical compound or such a mixture of substances can be selected as the immersion medium 14, which is also usually inexpensive.
• the first and second refractive indices nl, n2 and possibly also their Abbe number vl or v2 can be chosen to be approximate or identical.
• the thickness of the wall 6 is then as small as possible and / or the wall 6 consists of a material whose refractive index is close to the refractive index n1 or n2.
• the immersion medium 14 can also be mixed from two or more suitable components and so the refractive indices n1 and n2 can be matched to one another as best as possible.
• Such an adaptation of the composition of the immersion medium 14 can also take place dynamically in further embodiments of the invention, in that components of the immersion mixture 14 or an already mixed immersion medium 14 are supplied to the room 16 in a controlled manner via supply and discharge lines (not shown). In this way, such a “dipping chamber” can be flexibly adapted to different operating conditions.
• a sample chamber 1 is brought into a first angular relative position. This means that it is inclined by a certain angle in relation to a reference coordinate system, for example the Cartesian coordinate system shown with the axes x, y and z orthogonal to one another.
• a reference coordinate system for example the Cartesian coordinate system shown with the axes x, y and z orthogonal to one another.
• the circular surface 9 runs parallel to a plane spanned by the axes x and y.
• illuminating radiation is irradiated into the sample space 2 through the outside 6.1 at an angle of incidence a.
• the illuminating radiation is refracted when it passes through the wall 6 and then runs approximately horizontally through the sample space 2 and the sample 3.
• the positioning of the refracted section of the illuminating beam path 7 along the Z axis z gives one first z relative position.
• the objective 5 is focused on the broken section of the illuminating beam path 7, which can in particular be designed as a light sheet 17.
• An optical axis OA of the objective 5 used for detecting detection radiation is directed perpendicular to the broken section of the illuminating beam path 7 and through the center 10. Using the lens 5, image data of a part of the illuminated areas, in particular an illuminated plane, of the sample 3 can now be acquired.
• a so-called z-stack 18 (see FIG. 5) of object planes in different z-relative positions is recorded (symbolized by the vertical double arrow) and subsequently assembled (see also FIG. 5). Any necessary corrections to the angle of incidence a of the illuminating beam path 7 are explained with reference to FIG. 9.
• the sample chamber 1 is therefore pivoted and / or rotated around the center 10, as shown in FIGS. 4b and 4c.
• the sample chamber 1 in FIG. 4b is brought into a further angular relative position and a further z relative position.
• a z-stack 18 can again be acquired (FIG. 4c), stored and finally assembled into a representation of the sample 3.
• corresponding relative angular positions can be freely selected and z-stacks 18 can be acquired.
• a first z stack 18 is detected along a detection beam path 8 in the direction of the Z axis z and stored together with data on the respective angular relative position and z relative position (FIG. 5a).
• the sample chamber 1 is then pivoted and / or rotated about the center point 10 by a certain angle.
• a z-stack 18 of image data is again acquired from the sample 3 thus brought into a second or a further angular relative position (FIG. 5b).
• Occurring overlaps of the individual z-stacks 18 can be used to improve the data quality, for example by carrying out an evaluation using a multiview fusion algorithm. Remaining aberrations can be corrected using adaptive optical elements (see, for example, FIG. 8).
• the image data obtained by means of the method according to the invention can be represented in different coordinates. 6 and 7, a transformation of a tilted z-stack 18 into a horizontal orientation (FIG. 6) or a tilted z-stack into spherical coordinates (FIG. 7) is shown only as a representative.
• a weighted interpolation can be used to transform the coordinates.
• detected intensity values l x, y, z of four points of a z-stack 18 are shown, which lead to a transformed intensity value l X ' y , z - a point (coordinate) of the horizontal (FIG. 6) or the spherical coordinate system (FIG. 7) be interpolated.
• a schematic representation of an exemplary embodiment of an imaging system with the possibility of selecting different imaging methods depending on the sample 3 comprises a beam shaping unit 19 for providing and shaping an illuminating radiation to form a light sheet 17 and / or a laser scanning microscope LSM (hereinafter also briefly : LSM).
• An adaptive optical unit 20 can optionally be present in the illumination beam path 7 of the beam shaping unit 19 and / or the LSM.
• at least one optical lens 23 and a beam splitter 21 are arranged in the illumination beam path 7 of the LSM.
• the illuminating radiation is directed to the objective 5 by the action of the beam splitter 21 and is radiated into the sample space 2 of the sample chamber 1.
• a detection radiation produced in the sample 3 passes through the objective 5 and the beam splitter 21 which is transparent to the detection radiation along the detection beam path 8 to a tube lens 22 and further to a detector 25, for example a camera.
• the beam splitter 21 can optionally be inserted or swiveled into the illumination beam path 7 and / or the detection beam path 8.
• the illumination of the sample 3 can optionally take place by means of a light sheet 17, by means of a point scanner of an LSM or by means of a multipoint scanner of an LSM.
• the LSM or the beam shaping unit 19 serve as a light source.
• the detection radiation can be recorded confocal, in the wide field or plenoptically.
• the latter variant can be carried out using a microlens array 26.
• a correspondingly controllable adaptive optical unit 20 can be arranged in the detection beam path 8.
• the microlens array 26 can optionally be inserted or swiveled into the detection beam path 8 using appropriate adjusting devices (not shown).
• the sample chamber 1 can be pivoted about each of the axes X, Y and Z by means of a sample table 27 which can be driven and displaced along these axes. Rotation around the Z axis is possible.
• the sample chamber 1 is pivoted about the center 10 (see FIGS. 4a to 5c) and optionally rotated about an axis running through the center 10 and in the direction of the Z axis Z (see, for example, FIG. 4a to 5).
• the objective 5 is designed to be positioned in the direction of the Z-axis z by means of a controllable drive 30.
• the sample chamber 1 can be shifted in a controlled manner along the axes x, y and in particular in the direction of the Z axis z by means of the sample table 27, in order to position the sample chamber 1 relative to the illumination beam path 7, for example.
• the possibility of movement along the Z axis z represents an option for the detection of the z stack 18.
• the angle relative positions and / or the z relative positions can be set by a corresponding movement of the objective 5. It is also possible that, for example, the settings of the angular relative positions and / or the z relative positions are set by combinations of the movements of objective 5 and sample table 27.
• a control unit 28 for example a computer or a correspondingly configured part of a computer, is provided to control the sample table 27 and the various actuating devices or at least one drive 30, and with the sample table 27, the drives 30, in a manner suitable for transmitting data and control commands (only one shown by way of example) and optionally connected to the camera 25 (shown only indicated).
• the z-stacks 18 can be moved as a z-relative position by moving the sample 3 and / or by moving the lens 5 and changing the respective focal plane be included.
• the rotation of the sample chamber 1 described above has no optical effect if the pivot point and center point 10 coincide (see, for example, FIG. 5).
• a relocation may be necessary to correct aberration-related focus shifts that occur in practice.
• angular tracking and relocation are possible with a device shown in FIG. 9.
• FIG. 9 various angles of a sample chamber 1, an illumination beam path 7 and a light sheet 17 are shown schematically.
• the sample chamber 1 and the illumination beam path 7 are shown with continuous solid lines in a first Z-relative position and with interrupted solid lines in a second Z-relative position.
• the illuminating radiation passes through a medium with the first refractive index na.
• the sample chamber 1 with the sample medium 4 has a second refractive index nb with nb> na.
• the respective angle of incidence a n is measured between the respective illumination beam path 7 and a normal on the outside 6.1.
• the wall 6, in particular its outside 6.1, is designed as a spherical section with a radius R, an extension of the normal extends through the center 10 and closes an angle with another wall 11 functioning as the bottom of the sample chamber 1 or with the plane of the circular surface 9 gh a.
• the illuminating radiation is directed along the illuminating beam path 7 at a first angle of incidence ai onto the outside 6.1 of the spherically shaped wall 6 of the sample chamber 1.
• the extension of the normal occurs at an angle y1 through the center 10.
• the illuminating beam path 7 is refracted in accordance with the differences in the refractive indices na and nb.
• the angle of incidence al is selected such that the broken section of the illuminating beam path 7 runs parallel to the plane of the circular surface 9 and image data can be acquired, for example, by means of a lens 5 along the detection axis DA.
• the broken section of the illuminating beam path 7 forms an angle ⁇ 1 with the extension of the normal.
• image data can be acquired with the focus position of the objective 5 remaining the same if an angle of incidence a2 is set.
• the extension of the normal at an angle g2 now occurs through the center 10.
• the broken section of the illuminating beam path 7 forms an angle ⁇ 2 with the extension of the normal, where ⁇ 1> 32.
• the optical arrangement for tracking the position and angle in the case of light sheet illumination on a sample chamber according to the invention in accordance with the illustrated exemplary embodiment has the beam shaping unit 19, in which an illuminating radiation provided by a light source (not shown in more detail) is shaped into a light sheet 17.
• a light source not shown in more detail
• the first scanner S1 is located in or near a pupil P of the illumination beam path 7 and is used for relocation. It is designed, for example, in the form of a galvanometric scanning mirror. An angle in the pupil P corresponds to a location in the sample 3, which is why a change in the angle of the first scanner S1 leads to a displacement of the light sheet 17 in the direction of the z-axis z.
• a second scanner S2 is arranged between the beam shaping unit 19 and the first scanner S1. This is located near an intermediate image ZB. The two scanners S1 and S2 deflect the illumination beam path 7 in the same movement planes.
• the pivot point of the second scanner S2 is imaged onto the outside 6.1 by means of the scanning optics 24.
• the second scanner S2 changes an angle in the intermediate image ZB, which corresponds to a change in location in the pupil P and on the first scanner S1 and an angle in the sample 3. Since the angle is to be set shortly before sample 3, an angular deviation can be corrected accordingly by axially displacing the optical lens 23.
• a necessary correction of the focusing along the direction of propagation of the light sheet 17 can take place, for example, by moving the objective 5, the tube lens 22 or by other focus optics.
• a correction of the focusing may be necessary since the optical path length in the sample medium 4 is different at different Z-relative positions and therefore changes during the acquisition of a z-stack 18.
• a third scanner S3 (not shown) can be arranged close to the pupil and, for example, close to the first scanner S1.
• the action of the third scanner S3 causes the light sheet 17 to move rapidly perpendicular to the plane of the drawing.
• a beam shape that requires lateral “smearing” e.g. Bessel beam
• a plurality of sample chambers 1 can be arranged on a sample carrier 13.
• the sample holder 13, which has a plurality of storage spaces for sample chambers 1, can be of flat design (FIG. 10).
• a sample chamber 1 to be observed in each case is delivered to the objective 5 by a corresponding control of the sample table 27 and / or the objective 5 is delivered to the respective sample chamber 1.
• a first z stack 18 can be detected in a first angular relative position.
• the objective 5 can be pivoted and / or the sample carrier 13 can be pivoted around the center 10 of the sample chamber 1 currently to be detected.
• a sample carrier 13 for a number of sample chambers 1 according to the invention, the latter has carrier surfaces 12 offset from one another, on each of which a sample chamber 1 is arranged or can be arranged.
• the carrier surfaces 12 can be circumferential surfaces of a carousel or revolver, for example. Such an embodiment increases the accessible angular range compared to an embodiment according to FIG. 10, under which angular relative positions can be set.
• a sample carrier 13 can have at least one channel 29 which opens at one of the parking spaces (FIG. 11).
• the channel 29 serves for the supply and / or discharge of sample medium 4 into and from the sample space 2.
• the sample chamber 1 is delimited in the plane of the circular surface 9 by the support surface 12 or in a further wall 11 there are openings corresponding to some or all of the channels (indicated indicated). In further versions, these openings can be equipped with a valve or a Closure may be provided to enable the sample chamber 1 to be removed from the carrier surface 12.
• sample chamber 1 can have the wall 6 and for a closure in the plane of the circular surface 9 to be formed by a surface of the support surface 12 as a further wall 11.
• the sample space 2 can be supplied with a sample medium 4 via the channel 29 or the channels 29.
• the sample medium 4 can be, for example, a compound for clarifying the sample 3, a nutrient solution, a buffer or a compound for supporting the storage of the sample 3.
• FIG. 11 shows carrier surfaces 12 offset from one another, on each of which a sample chamber 1 is placed on a parking space.
• a support surface 12 with the sample chamber 1 provided thereon can be brought into a detection position, which in the example shown is the middle position in which the support surface 12 located in the detection position is oriented orthogonally to the optical axis OA of the objective 5.
• the objective 5 can be pivoted relative to the sample chamber 1 and different focus planes can be captured, so that the sample chamber 1 and objective 5 can be brought into different angular relative positions and / or z relative positions with respect to one another .
• the mutually angularly offset and angled arrangement of the support surfaces 12 enables better access to the respective sample chamber 1 to be recorded, since the respectively adjacent sample chambers 1 are swung out of the plane of the sample chamber 1 to be recorded.
• the current alignment of the sample carrier 13 and the supply and / or discharge of media via the channels 29 are controlled by means of the control unit 28 and by means of drives 30 and / or pumps 30.
• sample carriers 14 immersion medium

Landscapes

• Physics & Mathematics (AREA)
• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Optics & Photonics (AREA)
• Engineering & Computer Science (AREA)
• Multimedia (AREA)
• Computer Vision & Pattern Recognition (AREA)
• Microscoopes, Condenser (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Priority Applications (2)

Applications Claiming Priority (2)

Publications (1)

Family

ID=68766759

Family Applications (1)

Country Status (4)

Families Citing this family (3)

Citations (6)

Family Cites Families (7)

• 2018
  • 2018-12-19 DE DE102018222271.1A patent/DE102018222271A1/de active Pending
• 2019
  • 2019-12-02 JP JP2021535872A patent/JP7399168B2/ja active Active
  • 2019-12-02 WO PCT/EP2019/083222 patent/WO2020126419A1/de not_active Ceased
  • 2019-12-02 US US17/309,793 patent/US12259538B2/en active Active

Patent Citations (6)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events