EP3019904A1 - Système de microscopie à feuille de lumière - Google Patents

Système de microscopie à feuille de lumière

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Publication number
EP3019904A1
EP3019904A1 EP14735991.3A EP14735991A EP3019904A1 EP 3019904 A1 EP3019904 A1 EP 3019904A1 EP 14735991 A EP14735991 A EP 14735991A EP 3019904 A1 EP3019904 A1 EP 3019904A1
Authority
EP
European Patent Office
Prior art keywords
illumination
detection
objective
sample
light
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14735991.3A
Other languages
German (de)
English (en)
Inventor
Jörg SIEBENMORGEN
Thomas Kalkbrenner
Helmut Lippert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Carl Zeiss Microscopy GmbH
Original Assignee
Carl Zeiss Microscopy GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Carl Zeiss Microscopy GmbH filed Critical Carl Zeiss Microscopy GmbH
Publication of EP3019904A1 publication Critical patent/EP3019904A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/0025—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/15—Preventing contamination of the components of the optical system or obstruction of the light path
    • 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
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/0088—Inverse microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/02—Objectives
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/33—Immersion oils, or microscope systems or objectives for use with immersion fluids
    • 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
    • G02B21/36—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
    • G02B27/0025—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration
    • G02B27/0068—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for optical correction, e.g. distorsion, aberration having means for controlling the degree of correction, e.g. using phase modulators, movable elements
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0389—Windows
    • G01N2021/0396—Oblique incidence

Definitions

  • the invention relates to an arrangement for light-sheet microscopy.
  • Such an arrangement comprises a sample vessel for receiving a sample contained in a medium, wherein the sample vessel is aligned with respect to a flat reference surface.
  • the assembly also includes an illumination optic having an illumination objective for illuminating the specimen with a light sheet, the optical axis of the illumination objective and the light sheet lying in a plane including a non-zero illumination angle ⁇ with the reference plane normal.
  • the arrangement finally comprises detection optics with a detection objective whose optical axis encloses a non-zero detection angle ⁇ with the normal of the reference surface.
  • the illumination objective and the detection objective can also be configured as a so-called double objective, as described for example in EP 0 866 993 B1. Both lenses are then combined in a common unit, the respective optics - i. Lenses with associated beam paths and optical elements arranged therein - then share some elements.
  • Such a device is used in particular in the examination of biological samples, in which the illumination of the samples with a light sheet whose plane intersects the optical axis of the detection at a non-zero angle occurs.
  • the light sheet encloses a right angle with the detection direction, which generally corresponds to the optical axis of the detection objective.
  • SPIM Selective Plane Illumination Microscopy
  • the SPIM technique is preferably used in fluorescence microscopy, where it is also referred to as LSFM (Light Sheet Fluorescence Microscopy).
  • LSFM Light Sheet Fluorescence Microscopy
  • the LSFM technique has several advantages: Since the detection in the far field can take place, larger sample areas can be detected. Although the resolution is somewhat lower than with confocal laser scanning microscopy, the LSFM technique allows thicker samples to be analyzed, since the penetration depth is higher. In addition, the light exposure of the samples in this method is the lowest, which among other things reduces the risk of bleaching a sample, since the sample is illuminated only by a thin light blue at a non-zero angle to the detection direction.
  • both a static light sheet which is generated for example by means of cylindrical lenses, can be used, as well as a quasi-static light sheet.
  • This can be generated by quickly scanning the sample with a light beam.
  • the light sheet-like illumination is created by the light beam is subjected to a very fast relative movement to the observed sample and this time successively multiple strung together.
  • the integration time of the camera, on the sensor of which the sample is finally imaged is selected such that the scanning is completed within the integration time.
  • a line sensor in combination with a rescan in the detection optics can also be used.
  • the detection can also be confocal.
  • One of the main applications of light sheet microscopy is in the imaging of medium-sized organisms with a size of several 100 microns to a few millimeters, usually these organisms are embedded in an agarose gel, which in turn is in a glass capillary.
  • the glass capillary is inserted from above or from below into a water-filled sample chamber and the sample is pushed out of the capillary a bit.
  • the sample in the agarose is illuminated with a light sheet and the fluorescence is imaged onto a camera with a detection objective that is perpendicular to the light sheet and thus perpendicular to the light sheet optics.
  • This method of light sheet microscopy has three major disadvantages. First, the samples to be examined are relatively large, they come from developmental biology.
  • the light sheet is relatively thick and thus limits the achievable axial resolution.
  • sample preparation is expensive and incompatible with standard sample preparations and standard sample holders, as is common in fluorescence microscopy for single cell examination.
  • a SPIM structure has been implemented in which the illumination objective and the detection objective are perpendicular to one another and are directed at an angle of 45 ° from above onto the sample. If, for example, the plane of a table on which the sample holder is mounted, or another horizontal plane, is used as reference surface, the illumination angle ⁇ and the detection angle ⁇ are each 45 °.
  • WO 2012/110488 A2 is described, for example, in WO 2012/110488 A2 and in WO 2012/122027 A2.
  • the sample is in such structures, for example, on the bottom of a Petri dish.
  • the Petri dish is filled with water, the lighting lens and detection lens are immersed in the liquid, the water also performs the function of an immersion liquid.
  • This approach offers the advantage of higher resolution in the axial direction because a thinner sheet of light can be generated. Due to the higher resolution then smaller samples can be examined. Also, the sample preparation has become much easier. Nevertheless, the sample preparation and the sample holder still do not conform to the standard currently valid in single-cell fluorescence microscopy. Thus, the Petri dish must be relatively large, so that the two lenses can be immersed in the shell, without hitting the edge of the shell.
  • a further disadvantage is that with this structure an analysis of a large number of samples in a short time (high-throughput screening) is not readily possible since the objectives must be cleaned when changing the sample in order to avoid contamination of the various samples ,
  • the object of the invention is to further develop an arrangement for light sheet microscopy of the type described above in that the analysis in particular of a plurality of samples is simplified by cross-contamination is effectively prevented when changing between two samples.
  • this object is achieved in an arrangement for light sheet microscopy of the type described above in that this arrangement is a separation layer system with one or more layers with predetermined thicknesses and of predetermined materials for the spatial separation of the medium in which the sample is located from the illumination objective and the Detection lens includes.
  • the separating layer system with an aligned parallel to the reference surface interface at least in the area that is responsible for the The illumination objective and the detection objective for illuminating the sample and detecting light coming from the sample are in contact with the medium - completely or at least almost completely.
  • the illumination angle ⁇ and the detection angle ⁇ are predefined on the basis of the numerical apertures NA D of the detection objective and NA B of the illumination objective.
  • the specification is made in the sense that the components are arranged to each other so that the existing aberrations are minimal without further action. Of course, you can also adjust other angles if you accept larger aberrations, but the image quality then decreases.
  • the separation layer system consists only of a single layer, this layer can also be an air layer, wherein the illumination and detection lens are designed as dry lenses.
  • the separating layer system may, however, also comprise a plurality of layers, for example a glass or plastic layer which, as a foil or plate, covers the sample vessel with respect to both objectives. Between this glass or plastic layer and the lenses is then an air layer or a layer with an immersion liquid, with which the two lenses are in contact.
  • the separation layer system can also consist of a single liquid layer, if it is ensured that this liquid layer does not mix with the medium in which the sample is located. This liquid can then also serve as immersion medium.
  • the introduction of a separating layer system can effectively prevent contamination, extreme light aberrations occur even at low numerical apertures such as 0.3 due to the passage of illumination and detection light through the interfaces of the separating layer system to the medium in which the sample is located like spherical aberrations and coma.
  • the oblique passage adds further, asymmetrical aberrations, or the others are amplified.
  • the illumination angle ⁇ and the detection angle ⁇ are predetermined on the basis of the numerical apertures NA D , NA B of the detection objective or of the illumination objective.
  • the objective with the lower numerical aperture which is usually the illumination objective, is arranged at a larger angle than the detection objective.
  • the detection lens may also have a larger numerical aperture than the illumination objective.
  • symmetrical configurations are also used in which the illumination objective and the detection objective are the same and both lenses include the same angle with the normal.
  • the sum of the illumination angle ⁇ and the detection angle ⁇ is ideally 90 ° in all cases. Is deviated from, for example, because both lenses can be arranged at a more acute angle, so that the sum is smaller than 90 ° is, then, since the object plane is now wrong with respect to the optical axis of the detection lens, to ensure that the Scheimpflug condition is met - the image sensor of the camera must then also be aligned obliquely. Arrangements are also conceivable in which the illumination and detection objectives are combined in an optical module such as the double objective mentioned above.
  • the illumination optics and / or the detection optics therefore comprise correction means for reducing the aberrations, in particular those aberrations which arise through the oblique passage of illumination light and / or light to be detected through interfaces of the separation layer system.
  • the correction means therefore comprise correction lenses and / or correction elements in the illumination objective or in the detection objective.
  • the correction lenses can be configured, for example, as cylindrical lenses, as lenses tilted against the respective optical axis or as non-axially arranged lenses whose axis of symmetry therefore does not lie on the optical axis of the illumination or detection objective.
  • the correction elements can be designed, for example, as elements with aspherical surfaces or free-form surfaces. Various corrective lenses / correction elements of one type or different types can also be combined in one lens.
  • a separate set of illumination and detection objectives can then be generated for each separating layer system, which however involves a high cost, since several sets have to be purchased, as well as an increased amount of work, which is associated with the change of lenses when changing the separation layer system is connected.
  • the correction means therefore comprise adaptive optical elements arranged in the illumination beam path and / or in the detection beam path for manipulating the phase fronts of the illumination or detection sound light.
  • These can be designed, for example, as deformable mirrors, phase plates or spatial light modulators.
  • These elements can preferably be configured controllable, so that an adaptation to a variety of possible Separation layer systems with one and the same arrangement of illumination and detection lens is possible.
  • the separating layer system preferably comprises, as already mentioned, a plate-shaped or foil-shaped cover which terminates the sample vessel and consists of a predetermined material and of a predetermined thickness.
  • a first large area of this plate- or film-shaped cover is in this case almost completely in contact with the medium in which the sample is located, at least in the area accessible to the illumination objective and the detection objective for illumination and detection.
  • a second large area of the cover is preferably in contact with a gas, for example air, or an immersion medium as further component of the separating layer system, at least in the area accessible to the illumination objective and the detection objective for illumination and detection.
  • Alternatively or in addition to the said correction means in the lenses or in the beam path and the separation layer system can be adjusted accordingly to reduce the aberrations. With a corresponding adaptation of the interface materials may also be a further correction of the lenses may be waived, or these corrections must not be so strong.
  • the material for the cover has a refractive index which differs from the refractive index of the medium in which the sample is located by less than 5%. If both materials have the same refractive index, aberrations at the interface between the medium and the cover can be completely avoided.
  • This material is an amorphous polymer, here the glass transition temperature can be adjusted so that the polymer in the cooled state has the refractive index of the medium in which the sample is located.
  • the glass transition temperature can be adjusted so that the polymer in the cooled state has the refractive index of the medium in which the sample is located.
  • other amorphous polymers with adjustable glass transition temperature can be used.
  • the separation layer / cover should be as thin as possible and should not be thicker than a few hundred ⁇ m. If the cover serves at the same time as the bottom of the sample vessel, as is the case with an inverse arrangement, or as a side wall in a horizontal observation arrangement, then of course sufficient stability against the pressure exerted by the medium in which the sample is located must be ensured become. This is not required for a cover as a lid of the sample vessel for an upright observation, here, the material can be made significantly thinner with thicknesses of less than 100 microns.
  • the material for the cover is a nanostructured material composed of a first and a second component, wherein the refractive index of the first component is smaller and the refractive index of the second component is greater than the refractive index of the medium for receiving the sample is.
  • the average structure sizes of the regions of material of the first component have a diameter which is smaller than the wavelengths of light used for illumination and the light to be detected, since only then an effective refractive index in a range of 5 % to the refractive index of the medium, for example water, can be adjusted.
  • different polymers can be used whose mixing behavior or demixing behavior, if the materials do not mix, is exploited, or nanoporous silica.
  • the first component is the air and the second component is silicon dioxide.
  • Such nanostructured materials are described, for example, in the article "Optical thin-film materials with low refractive index for broadband elimination of Fresnel reflection" by J. Q.
  • the separation layer system including the cover may comprise, for example, a vessel lid for conventional microtiter plates, then the known upright construction of a light sheet microscope can be used, with appropriate positioning means for positioning the sample in the upper quarter of the sample vessel, based on its depth, or for positioning in the vessel lid it is ensured that the sample is accessible to the microscope setup.
  • the arrangement for light-sheet microscopy can also include an inverted light-sheet microscope in which the illumination and detection objective are arranged below the sample vessel.
  • the cover forms part of the separation layer system, the bottom of the vessel sample, so it must be kept special sample containers or standard multi-well plates with transparent bottom of the vessel.
  • the sample vessel suitably comprises means for positioning the sample in a lateral or upper region of the sample vessel within the working distance of the illumination objective and the detection objective.
  • FIG. 3 shows a further arrangement for light-sheet microscopy with correction means arranged in the beam path
  • FIG. 5 shows an example of a nanostructured cover.
  • FIG. 1 shows first an arrangement suitable for upright observation for light sheet microscopy.
  • This comprises a sample vessel 1 for receiving a medium in a second
  • the sample vessel 1 is aligned with respect to a flat reference surface, which is defined here by a sample stage 4.
  • the arrangement comprises an illumination optical unit with a light source 5 and an illumination objective 6 for illuminating the sample 3 with a light sheet, wherein the optical axis 7 of the illumination objective 6 and the light sheet lie in a plane which corresponds to the normal of the reference surface a non-zero illumination angle ⁇ includes.
  • the arrangement also comprises detection optics with a detection objective 8 whose optical axis 9 encloses a non-zero detection angle ⁇ with the reference plane normal. Light coming from the sample 3 is directed to a detector 10, registered there; the registered signals are made available for further processing and / or presentation on a screen.
  • the arrangement also comprises a separating layer system with one or more layers of predetermined thicknesses and of predetermined materials for the spatial separation of the medium 2, in which the sample 3 is located, from the illumination objective 6 and the detection objective 8.
  • the separating layer system has an alignment parallel to the reference surface Interface 11, with which it is completely or at least almost completely in contact with the medium 2 at least in the area accessible for the illumination objective 6 and the detection objective 8 for illumination and detection.
  • Beieuchtungswinkel ß and detection angle ⁇ are given by numerical apertures NA D and NA B of the detection lens 8 and the illumination objective 6, respectively.
  • the medium 2 for example, water can be used, but also the use of other liquids or even gels is possible.
  • a first, not necessarily necessary, measure may be to specify the illumination angle ⁇ and the detection angle ⁇ based on the numerical apertures of the illumination objective 6 and the detection objective 8. This is also shown in FIG.
  • the numerical aperture NA B of the illumination objective 6 is smaller than the numerical aperture NA D of the detection objective 8. Since the aberrations in the case of objectives with a large NA To make more noticeable, for such lenses positioning their optical axis as close to the normal of the reference surface, so with a particularly small angle to this advantageous, since the aberrations are also greater, the more oblique the light is, the greater than that Angle, which includes the optical axis of the lens with the normal of the reference surface.
  • a lens with a smaller numerical aperture can be used, since numerical apertures of less than 0.5 are generally sufficient for the generation of a light sheet, while the highest possible numerical aperture of 1.0 or higher for detection due to the high resolution necessary is.
  • the numerical aperture NA B of the illumination objective 6 is smaller than the numerical aperture NA D of the detection objective 8.
  • the illumination angle ⁇ can therefore be selected larger than the detection angle ⁇ . It is preferred, as shown in Figure 1, the sum of illumination angle ß and detection angle ⁇ 90 °. In the case of deviating angles, the detector 10 must be tilted accordingly, so that the so-called Scheimpflug condition is fulfilled.
  • the separating layer system here has a plate-shaped cover 12 terminating the sample vessel and made of a predetermined material and of a predetermined thickness.
  • a first large area of the plate-shaped cover 12, which here coincides with the boundary surface 11, is almost completely in contact with the medium 2 at least in the area accessible to the illumination objective 6 and the detection objective 8 for illumination and detection.
  • a second Crufikiee 13 of the cover 12 is here with a gas, such as air, in contact and forms another interface.
  • a gas such as air
  • the second large area 13 also acts as an interface and is sometimes referred to as such in the following.
  • the illumination optics and / or the detection optics therefore comprise correction means for reducing such aberrations, which result from the oblique passage of illumination light and / or to light to be detected through boundary surfaces 11, 13 of the separation layer system.
  • correction means may include, for example, corrective lenses and / or correction elements in the illumination objective 6 and / or in the detection objective.
  • the correction lenses may be formed as cylindrical lenses, as tilted against the optical axis and / or not axially arranged lenses and / or as correction elements with aspherical surfaces or free-form surfaces.
  • 1 shows, as an example, a non-axially arranged lens 14 in the illumination objective 6 and an off-axis lens 15 in the detection objective 8.
  • FIG. 2 shows an arrangement for light-sheet microscopy, the components of which are constructed and arranged similarly to FIG. 1, with the difference that here the illumination objective 6 and the detection objective 8 are arranged below the sample vessel 1, that is to say an arrangement for inverse light sheet microscopy.
  • the cover 12 is formed in this case by the bottom of the sample vessel 1.
  • Such an inverse arrangement is particularly suitable for the analysis of samples in microtiter plates, since the samples are usually located by gravity on the vessel bottom, so that they are more accessible for an inverse structure than for an upright structure, since the wells very closely conceived and difficult to access from the top.
  • the illumination objective 6 and the detection objective 8 are arranged above the sample vessel 1, it will therefore be expedient to use sample vessels 1 which have means for positioning the sample 3 in the upper region of the sample vessel 1, so that the sample 3 is also accessible from above.
  • the arrangement comprises correction means arranged in the illumination beam path and / or in the detection beam path and which are adaptive optical elements for manipulating the phase fronts of the illumination system. or the detection law.
  • correction means can be combined with corrected objectives, as also shown in FIGS. 1 and 2.
  • Figures 3 and 4 only the inverse structure of the arrangement for light sheet microscopy is shown, in an equivalent way to Figures 1 and 2, a structure can be readily constructed, are arranged at the illumination objective 6 and detection lens 8 above the sample vessel 1.
  • FIG 3 an arrangement is shown, which is initially similar to the structure in Figure 2.
  • a sample 3 is stored in a sample vessel 1, which is arranged on a sample table 4.
  • the sample is in a medium 2, for example water.
  • the illumination objective 6 and the detection objective 8 are identical in this case, they can therefore be arranged at an angle of 45 ° to the normal of the reference surface.
  • a deformable mirror 16 is arranged, in the detection beam path a deformable mirror 17, which is hit by the light to be detected, before it is imaged onto the detector 10 via a lens 18.
  • deformable mirrors 16 and 17 which can also be replaced by spatial light modulators or phase plates at these positions, are controllable and thus adaptable to different illumination angles ⁇ and detection angle 5 as well as to different lens configurations and different separating layer systems, in particular different covers 12. In this way, the aberrations can be almost completely corrected.
  • Deformable mirrors and spatial light modulators can also be used in addition to correct for aberrations caused by the sample.
  • FIG. 1 A somewhat simplified construction, in which the illumination objective 6 and the detection objective 8 are constructed identically and the illumination angle .beta. And the detection angle .delta. Are also identical, but in which it is sufficient to use only one deformable mirror 19, is illustrated in FIG.
  • a beam splitter 20 is arranged in front of the light source 5, which is permeable to the illumination wavelength range and is designed to be reflective for the wavelengths of the fluorescent light to be detected.
  • a beam splitter 21 is arranged, which in turn is permeable to the detection wavelength range to be detected, but is designed to be reflective for the illumination wavelength range.
  • Another possibility for reducing or avoiding aberrations, which can be combined with the already mentioned possibilities of the correction means in the beam paths or in the objectives, is to select a material for the cover 12 which has a refractive index which is of the refractive index of the medium 2 in which the sample 3 is embedded differs by less than 5%.
  • the aberrations are thereby already greatly reduced, the correction means must then no longer engage as strongly in the beam path, as without such a measure, which simplifies the Hersannon and makes cheaper, for example, in that aspherical lenses can be used instead of free-form surfaces.
  • the medium 2 in which the sample 3 is located can be used as the material for the cover 12, for example PTFE, CYTOP ®, FEP, Teflon ® AF or a perfluordioxolanes polymer.
  • amorphous polymer such as Teflon® AF
  • its glass transition temperature is preferably adjusted so that the polymer when cold has the refractive index of the medium 2 in which the sample 3 is located.
  • water is used as the immersion medium on the other side of the cover 12, if the refractive indices are identical or differ only in the per thousand range, the occurrence of aberrations in the passage of light through the interfaces can be completely avoided.
  • a nanostructured material comprising a first component 22 and a second component 23 as the material for the cover 12.
  • the refractive index of the first component 22 is smaller than the refractive index of the medium 2 for receiving the sample
  • the refractive index of the second component 23 is greater than the refractive index of the medium 2 for receiving the sample 3.
  • From these two components 22 and 23 can be a nanostructured Produce material which has an effective refractive index, which differs from the refractive index of the medium 2 by less than 5%.
  • the average structure sizes or mean diameters of regions of the first component 22 are smaller than the light wavelength of the light used for illumination and of the light to be detected.
  • the effective refractive index results from the volume ratio of the two components.
  • nanostructured silica An example of such a nanostructured material is shown in Figure 5, nanostructured silica.
  • a cover 12 which may form, for example, the vessel bottom or the vessel lid shown.
  • silicon dioxide can be selected as the second component 23, and air can be used as the first component 22 in this case.
  • the refractive index of water is between the refractive indices of the two components.
  • the drawing serves only to illustrate, in fact, the openings may also have more random, for example, generated by etching, forms.
  • Essential is the volume ratio and that it is ensured that the average opening diameter are smaller than the wavelengths of light used or to be detected.
  • a two-component mixed material or a segregated material may also be used.
  • the thickness of the cover 12 as small as possible in each case, here suffices a thickness of a few hundred microns for a designed as a vessel bottom cover 12 and a few microns for a designed as a film cover 12, which serves as a lid for the sample vessel 1.

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  • Optical Elements Other Than Lenses (AREA)

Abstract

L'invention concerne un système de microscopie à feuille de lumière. Ce système comprend un récipient à échantillon (1), destiné à recevoir un échantillon (3) contenu dans un milieu (2), qui est aligné par rapport à une surface de référence plane. Le système comprend en outre une optique d'éclairage équipée d'un objectif d'éclairage (6) qui sert à éclairer l'échantillon (3) avec une feuille de lumière, l'axe optique (7) de l'objectif d'éclairage (6) et la feuille de lumière se situant dans un plan qui fait un angle d'éclairage β différent de zéro avec la normale à la surface de référence. Le système comprend également une optique de détection équipée d'un objectif de détection (8) dont l'axe optique (9) fait un angle de détection δ différent de zéro avec la normale à la surface de référence. Un tel système comporte un ensemble de couches de séparation comprenant une ou plusieurs couches d'épaisseurs prédéfinies, constituées de matériaux prédéfinis, servant à séparer dans l'espace le milieu (2) dans lequel l'échantillon (3) se trouve de l'objectif d'éclairage (6) et de l'objectif de détection (8). Cet ensemble de couches de séparation est en contact avec le milieu (2), par une surface limite (11) parallèle à la surface de référence, au moins dans la zone accessible à l'objectif d'éclairage (6) et à l'objectif de détection (8) pour l'éclairage et pour la détection. L'angle d'éclairage (β) et l'angle de détection (δ) sont prédéfinis au moyen d'ouvertures numériques (NAD, NAB) de l'objectif de détection (8) ou de l'objectif d'éclairage (6).
EP14735991.3A 2013-07-10 2014-07-08 Système de microscopie à feuille de lumière Withdrawn EP3019904A1 (fr)

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DE201310107297 DE102013107297A1 (de) 2013-07-10 2013-07-10 Anordnung zur Lichtblattmikroskopie
PCT/EP2014/064551 WO2015004108A1 (fr) 2013-07-10 2014-07-08 Système de microscopie à feuille de lumière

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WO (1) WO2015004108A1 (fr)

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WO2015004108A1 (fr) 2015-01-15
JP6492073B2 (ja) 2019-03-27
CN105359025A (zh) 2016-02-24
US10712553B2 (en) 2020-07-14
US20160154236A1 (en) 2016-06-02
JP2016525229A (ja) 2016-08-22
CN105359025B (zh) 2019-01-25

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