EP3494429A1 - Microscope, en particulier microscope à feuillet de lumière ou microscope confocal, et équipement complémentaire pour microscope - Google Patents

Microscope, en particulier microscope à feuillet de lumière ou microscope confocal, et équipement complémentaire pour microscope

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Publication number
EP3494429A1
EP3494429A1 EP17749427.5A EP17749427A EP3494429A1 EP 3494429 A1 EP3494429 A1 EP 3494429A1 EP 17749427 A EP17749427 A EP 17749427A EP 3494429 A1 EP3494429 A1 EP 3494429A1
Authority
EP
European Patent Office
Prior art keywords
microscope
color
optical
illumination
wavelengths
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.)
Pending
Application number
EP17749427.5A
Other languages
German (de)
English (en)
Inventor
Florian Fahrbach
Werner Knebel
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.)
Leica Microsystems CMS GmbH
Original Assignee
Leica Microsystems CMS 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 Leica Microsystems CMS GmbH filed Critical Leica Microsystems CMS GmbH
Publication of EP3494429A1 publication Critical patent/EP3494429A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • G02B21/0004—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
    • 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
    • G02B21/36—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
    • G02B27/0031—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 for scanning purposes
    • 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
    • G02B27/0037—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 with diffracting elements
    • 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

Definitions

  • Microscope in particular a disc or confocal microscope
  • the invention relates to a microscope, in particular a lens or confocal microscope, with an illumination optical system for transmitting light of at least two wavelengths of at least one light source along each wavelength-dependent beam path from a lighting side of the illumination optical system to a sample side of the illumination optical system.
  • the invention further relates to an optical Nachrustsatz for a microscope, in particular for a lens or confocal microscope having an illumination optical system for transmitting light at least two wavelengths of at least one light source along each wavelength-dependent beam path from a lighting side of the illumination optical system to a sample side of the illumination optical system.
  • Microscopes such as a lense microscope or a confocal microscope
  • a lense microscope or a confocal microscope have in common that an illumination optical system does not illuminate the complete sample to be examined, but only a limited area, which in the case of a light-disk microscope is limited in one direction along an optical axis of an observation optical unit.
  • the illuminated area of a sample examined by means of a light-disk microscope thus has a substantially two-dimensional structure.
  • the illuminated areas in the confocal microscopy are essentially zero-dimensional and their size is of the order of the diffraction-limited minimum focus diameter of the illumination wavelength. In the model these are assumed to be punctiform.
  • An illuminated region which is limited at least along the optical axis of the observation optics has the advantage that the image information of the sample made available via the observation optics essentially consists of the image information of the illuminated, restricted volume region of the sample. In other words, only those pixels or that plane contribute to the image structure, which are illuminated by the illumination optics.
  • the aforementioned microscope and the above-mentioned optical retrofit set solve this task in each case by the microscope or the retrofit kit has a color cross-correction device with at least one optical color cross-correction element, wherein at the sample-side output of the color cross correction element, the beam paths of the at least two different Wavelengths have an offset parallel to one another and / or a tilt relative to the illumination side, which results on the sample side of the illumination optical system in an offset of the foci of at least two wavelengths transversely to an optical axis of the illumination optics.
  • An offset can be transformed into a tilt by a lens, as well as tilt by a lens can be transformed into an offset.
  • the microscope according to the invention and the optical retrofit kit according to the invention have the advantage that the offset of the foci on the sample side of the illumination optical system produced by the color cross-correction device improves both the quality of illumination and the imaging quality of the microscope. This advantage is also achieved when the illumination takes place through edge regions of the illumination optics.
  • the dispersion of dielectric materials i. their wavelength-dependent refractive index, causes coaxial light beams of different wavelengths at the entrance (and / or exit) and an angle different from 90 degrees to the surface are refracted to different degrees in the dielectric material, which in refractive optical elements, such as Lenses, leading to chromatic aberration.
  • a chromatic aberration includes a lateral chromatic aberration, the so-called lateral chromatic aberration, and an axial chromatic aberration, the so-called longitudinal chromatic aberration. Both express themselves in particular in the focal point or focus of the lens.
  • the axial chromatic aberration causes the foci of different wavelengths to be offset from each other along a direction parallel to the optical axis.
  • the lateral chromatic aberration causes the foci of different wavelengths to be offset from one another in a direction perpendicular to the optical axis.
  • the foci of different wavelengths are thus, with coaxial entry into the lens, at a different distance from the optical axis of the lens. This form of chromatic aberration becomes particularly apparent when an optical element is obliquely penetrated or in its peripheral areas by multispectral light beams.
  • the lateral chromatic aberration can significantly reduce the imaging quality in a light-optical microscope, since, when illuminated with two different wavelengths, two disc-shaped regions offset from one another along the optical axis of the observation optics are illuminated.
  • the observation optics are focused only on a single plane, so that the second disc-shaped illuminated plane is blurred.
  • the microscope according to the invention or the optical retrofit kit according to the invention in each case counteract a reduction of the imaging quality caused by chromatic aberration.
  • the offset of the foci of the at least two wavelengths transversely to the optical axis of the illumination optical system caused by the illumination optical system is directed counter to the optical axis of the illumination optical system caused by the illumination optical system on the sample side of the illumination optical system .
  • the color cross-correction device can be rotatable about the optical axis or about the propagation direction.
  • the illumination optics may comprise a plurality of transmitting optical elements and / or a beam path deflecting elements.
  • the illumination side of the illumination optics can have an optical input, to which an optical waveguide, via which the light of a light source is transmitted, can be connected.
  • a light source can be provided on the illumination side of the illumination optics.
  • the lighting side of an element, an optic or a device is to be understood as that side which points in the direction of the light source. Accordingly, the sample side of an element, optics or device is to be understood as the side pointing in the direction of the sample to be illuminated. When viewing directions in a folded optical setup, it is considered unfolded such that one side of an element always faces either the sample or the illumination side.
  • the color cross-correction device can be arranged on the sample side or on the illumination side of the illumination optics, or else in the illumination optics, i. For example, between two optical elements of the illumination optics, be positioned.
  • the color cross-correction device can be designed as a separate unit which can be built as a unit with an illumination-side input and a sample-side output, the color cross-correction device offset the beam paths of the at least two different wavelengths on the sample-side output of the color cross correction device from one another and / or tilted relative to one another ,
  • the offset and / or tilt is a relative offset and / or relative tilt of the optical paths of the at least two different wavelengths, i.
  • no absolute offset and / or tilt is generated between the optical paths of the at least two wavelengths.
  • the relative salting and / or the relative tilting can be applied to a possibly already existing offset and / or tilting of the beam paths of the at least two wavelengths.
  • the color cross-correction device may further comprise lenses which make a tilting of an offset of two beam paths to one another or an offset from a tilting of two beam paths relative to each other. Consequently, the offset of the beam paths of the at least two different wavelengths resulting at the sample-side output of the color cross-correction device can be generated by a tilting of the beam paths relative to one another and a subsequent lens.
  • An offset or tilting of two beam paths relative to one another is to be understood in each case as relative variation of the beam paths relative to one another.
  • the microscope may further comprise tiltable scanning elements, in particular scanning mirrors, which may serve to deflect the beam paths of the at least two wavelengths together, wherein in such a deflection, the relative offset or the relative tilting of the beam paths of the at least two wavelengths remains unchanged.
  • tiltable scanning elements in particular scanning mirrors, which may serve to deflect the beam paths of the at least two wavelengths together, wherein in such a deflection, the relative offset or the relative tilting of the beam paths of the at least two wavelengths remains unchanged.
  • the scanning mirror can be used to generate a so-called virtual light sheet.
  • One-dimensional focus areas formed in the sample to be observed can be moved through the scanning mirror, so that the virtual light sheet builds up sequentially in a plane perpendicular to the observation direction of the sample.
  • a system of scanning mirrors may be provided which allows the virtual light sheet to be displaced along the viewing direction and thus to generate a computer-aided three-dimensional representation of the sample.
  • the illumination optics can have a transverse color defect, so that the foci of the at least two wavelengths are formed at a different distance from the optical axis of the illumination optics on the sample side of the illumination optics.
  • the color cross-correction device counteracts this relative offset of the foci of the at least two wavelengths.
  • the color cross-correction device counteracts the offset of the beam paths through the illumination optics such that said offset is compensated by the illumination optics.
  • the compensation means that the foci of the at least two wavelengths are offset relative to each other by means of the color cross correction device in such a way that they are located on the sample side of the illumination optical system at a substantially equal distance from the optical axis of the illumination optical system.
  • the color cross-correction device can in particular be arranged in the region of an expanded beam waist, that is to say in a section of the beam path or the beam paths with little divergence or convergence.
  • the color cross-correction device comprises a highly dispersive thin-film filter.
  • a filter may, for example, comprise a periodic structure of dielectric materials of different refractive index, which may for example be in the form of a so-called chirped structure (see chirped mirrors) or a resonator structure.
  • the color cross-correction device can comprise at least one optical element, on whose sample side the beam paths of the at least two wavelengths can be tilted relative to the illumination side.
  • Such optical elements are, for example, prisms and / or gratings, the angular dispersion produced by these optical elements, i. the dependent of the respective wavelength angle of at least two wavelengths to each other, is transformed by an imaging optics with an odd number of optical elements in an offset of the beam paths of the at least two wavelengths.
  • a deflection element such as, for example, a deflection prism and / or a deflection mirror can also be provided, by means of which the wavelengths acted upon by the angular dispersion can be redirected on average back to the original propagation direction.
  • Such a structure of an optical deflection device by means of deflecting mirrors or by means of at least one deflecting mirror and a deflecting prism can also be used for the color cross-correction device with a highly dispersive thin-film filter.
  • the filter In the case of the filter, the beam path of both laterally offset wavelengths can thus be redirected on average to the original propagation direction.
  • At least two optical color cross correction elements are provided, of which at least one optical color cross correction element is designed to be relatively tiltable relative to the other color cross correction element.
  • a tiltable arrangement of at least one of the optical color cross-correction elements has the advantage that the offset of the beam paths of the at least two wavelengths can be set.
  • the at least two optical color cross-correction elements can preferably be operated in transmission.
  • two highly dispersive thin-film filters can be provided.
  • the color cross-correction elements are preferably mounted rotatably about an axis, wherein the axis may preferably be oriented parallel to the light sheet and / or perpendicular to that plane which is defined by the optical axis of the illumination optics and the direction of the transverse chromatic aberration.
  • At least one of the at least two optical color cross-correction elements is designed as a transmitting, substantially plane-parallel plate.
  • both color cross-correction elements can be designed as a plane-parallel transmitting plate.
  • An essentially plane-parallel plate has the advantage that, given non-normal incidence of light of different wavelengths, the different spectral components are offset from each other, with all wavelength components experiencing a common offset through the plane-parallel plate.
  • substantially plane-parallel 'is to be understood that the transmitting plate may have a slight wedge (so-called wedged optical elements). This has the advantage that Fabry Perot effects can be avoided.
  • An optical element with a slight wedge is to be regarded as plane-parallel in the context of this description.
  • two of the at least two optical color cross correction elements are configured as transmitting, substantially plane-parallel plates, the at least two plane-parallel transmitting plates having a different refractive index and / or thickness. If the two plates are designed with different refractive index and / or thickness, it is possible to compensate the common beam offset of the beam paths of the at least two wavelengths, but without compensating for the relative offset of the beam paths of the at least two wavelengths as well.
  • the two plane-parallel transmitting plates of different refractive index and / or thickness can each be oriented at different angles to the beam paths, in particular one plate in the mathematically positive direction sense (counterclockwise), the other plate in the mathematically negative direction sense (clockwise) tilted be. This makes it possible to compensate for the absolute displacement of the beam paths of the at least two wavelengths to the original propagation direction.
  • the different angles to the original propagation direction produce a different angular separation of the at least two wavelengths and also a different offset, which is compensated by making the plane-parallel plate with the smaller angular separation and the smaller offset thicker.
  • the at least two optical color cross-correction elements have a different material dispersion. Such a configuration is particularly advantageous when a relatively large offset of two different wavelengths is desired to each other.
  • the material dispersion is a material parameter by means of which the splitting of two different wavelengths or the salt of two different wavelengths can be set to one another.
  • Low Abbe number (e.g., flint glass) highly dispersive materials can be used for the relative offset of the beam paths of the at least two wavelengths, whereas low Abbe number low dispersion materials such as crown glass can be used to increase the common beam displacement of both wavelengths compensate, but without completely compensate for the relative offset of the beam paths of the at least two wavelengths to each other.
  • low Abbe number low dispersion materials such as crown glass
  • crown glasses and flint glasses have a similar refractive index
  • two plane-parallel plates of these two materials will generate a comparable absolute offset of both different wavelengths
  • the higher dispersive flint glass will generate a larger relative offset of the optical paths of the at least two wavelengths to one another.
  • the relative offset of the beam paths of the at least two wavelengths to one another can be further increased if the plane-parallel plate with less dispersion is arranged at a smaller angle to the original propagation direction in the beam path and has a greater thickness than the plane-parallel plate with greater material dispersion.
  • the at least two optical color cross-correction elements are coupled to each other in a motion-transmitting manner. This has the advantage that the relative offset of the beam paths of the at least two wavelengths to each other is variably adjustable and at the same time can be compensated by the motion-transmitting coupling of the at least two optical color cross-correction elements of the changed absolute offset of the beam paths to the original propagation direction.
  • the relative offset of the beam paths of the at least two wavelengths after transmission through the first optical color cross-correction element is dependent on the angle of the illumination-side color cross-correction element to the original propagation direction of the light. A larger angle leads to a larger relative offset of the beam paths of the at least two wavelengths, but also to an increased absolute offset of the beam paths. len réelle to the original direction of propagation.
  • the motion-transmitting coupling of the sample-side color optical cross-correction element can be designed such that it substantially compensates for the varied absolute offset of the beam paths relative to the original propagation direction.
  • the at least two optical color cross-correction elements can be mechanically, z. B. be coupled by a transmission, motion-transmitting.
  • the motion transmitting coupling may also be electrically, e.g. B. by means of two electric motors, in particular stepper motors.
  • Such a motion-transmitting coupling can also be used in the abovementioned embodiments of the microscope with a highly dispersive thin-film filter or an optical element, such as prism or grating, which angles the beam paths of the at least two wavelengths.
  • the elements which tilt the mutually at least two wavelengths can be displaceable individually and / or against each other along the propagation direction.
  • prisms can be displaceable along or against an insertion direction, the insertion direction pointing from a prism base to an apex.
  • the color cross-correction device can be designed as a device which can be introduced into the beam paths of the at least two wavelengths, wherein at the sample-side output of the color cross correction only a relative offset of the beam paths of at least two wavelengths to each other, but no absolute offset Beam paths to the original propagation direction occurs.
  • a color longitudinal correcting device wherein the beam paths of the at least two different wavelengths on the illumination side of the color longitudinal correcting device have a divergence or convergence which differs from the divergence or convergence of the beam paths of the at least two wavelengths differ on the sample side of the color longitudinal correction device.
  • the displacement of the foci of the at least two wavelengths along or against the optical axis of the illumination optical system can be used, for example, for both foci in To overlap with each other, so that a defined by the overlapping foci area can be illuminated simultaneously with both wavelengths.
  • the color longitudinal correction device can in particular be provided alone, that is to say without the color cross-correction device in a microscope.
  • the illumination optics of the microscope can have a longitudinal color aberration, wherein the color longitudinal correction device allows the displacements of the focal points of different wavelengths relative to one another along the optical axis to be additionally and specifically displaced by the longitudinal color aberration on the specimen side of the illumination optics and these z. B. overlap. A color cross defect can still occur on the sample side of the illumination optics.
  • the color longitudinal correction device has at least one refractive and at least one diffractive optical element.
  • Refractive optical elements may, for example, be designed as spherical lenses.
  • Diffractive optical elements also called DOEs
  • the microstructure has different optical path lengths for transmitted light depending on the position on the DOE, so that partial beams passing through the DOE are phase modulated and constructive or destructive interference of the light by the DOE is caused. While a normal dispersion spherical lens has a shorter focal length for short wavelength light than for long wavelength light, the ratio of focal lengths for a diffractive lens is opposite.
  • the color longitudinal correction device on the illumination and the sample side can have a substantially identical absolute convergence or divergence of the beam paths of the at least two wavelengths, whereas the beam paths of the at least two different wavelengths on the sample side of the color longitudinal correction device have a relative change which may have divergence or convergence with each other.
  • the at least one refractive optical element and / or the at least one diffractive optical element has a variably adjustable focal length. This has the advantage that both the absolute position of the Foki the beam paths of the at least two wavelengths, as well as the relative position of the Foki the beam paths of the at least two wavelengths is adjustable to each other.
  • both the refractive optical element and the diffractive optical element can be variably adjustable.
  • the correction of a longitudinal chromatic aberration may be possible for different combinations of two wavelengths without the effective focal length, i. H. For example, the position of the light sheet is changed.
  • a plurality of color longitudinal correction devices can also be provided in cascade in order to be able to compensate for the longitudinal chromatic aberration for more than two wavelengths.
  • the cascading of color longitudinal correction devices can be done independently of or in addition to a cascading of the color cross correction devices.
  • the refractive optical element may be a liquid-filled lens which is variable in its focal length via pressure or a piezoelectric element.
  • the diffractive, preferably tunable, optical element may comprise a diffractive tunable lens, such.
  • a diffractive Moire lens a diffractive liquid crystal lens or an Alvarez Lohmann lens.
  • the at least one diffractive optical element is designed as a spatial light modulator.
  • a spatial light modulator also called SLM (spatial light modulator)
  • SLM spatial light modulator
  • the spatial light modulator can be designed to be transmissive or reflective. Since a reflective spatial light modulator requires folded beam paths and a larger distance between the light modulator and the refractive optics, a transmissive spatial light modulator is preferably provided.
  • a spatial light modulator can generally be applied to computer-generated diffraction patterns, which, for example, can be easily subjected to optimization in a computer-aided manner in order to set the desired relative displacement of the focuses on the sample side of the illumination optics.
  • the comparative diffractive lens may be set to a fixed value, wherein deviations of the variable refractive lens to the desired preset value may be compensated by the variable diffractive lens provided by the spatial light modulator.
  • the beam paths of the at least two wavelengths extend in non-paraxial peripheral regions of the illumination optics, at least one deflecting mirror being provided on the specimen side of the illumination optics and the beam paths being deflected by substantially 90 °.
  • the microscope may have an observation optics, for example in the form of an eyepiece, which is preferably arranged coaxially to the illumination optics.
  • the beam paths of the illumination and observation optics can be oriented parallel to one another.
  • the illumination optics can be arranged substantially collinear to a detection optics, so that the illumination optics can also be used paraxial or confocal illumination.
  • the same illumination optics can thus be used by using the non-paraxial edge regions of the illumination optics and the deflection mirrors for the lateral illumination of the sample as well as for the coaxial illumination of the sample (transmitted light microscopy / confocal microscopy).
  • two deflecting mirrors can be provided, which are arranged diametrically opposite the inspection area. This can enable illumination of at least one of the two sides or illumination of the sample on both sides.
  • a color cross-correction can be effected by the optical color cross-correction element, wherein optionally an additional color longitudinal correction by the optical color longitudinal correction element is possible.
  • the color cross error and the color length error are independent of each other and overlap linearly.
  • the color longitudinal correcting device can be located both in the beam path of the paraxial and in the beam path of the non-paraxial regions of the illumination optics, so that a color longitudinal correction can take place both in the case of lateral sample illumination and in the case of coaxial sample illumination.
  • the optical color cross-correction device can be configured in such a way that it is located only in the beam path of the at least two wavelengths extending in the non-paraxial edge regions of the illumination optics, so that color lateral correction by the optical color cross-correction device can take place only with lateral sample illumination.
  • the aforementioned scanning mirrors can deflect both the paraxial beam paths and the non-paraxial beam paths.
  • the above-mentioned optical retrofit kit for a microscope can be designed as a separate unit which can be retrofitted in microscopes to illuminate a sample in the microscope with at least two different wavelengths, the focal position of the beam paths of at least two different wavelengths to move each other transversely to the optical axis of the illumination optics.
  • the optical retrofit kit can be configured in a further embodiment thereof such that the offset of the foci of the at least two wavelengths transversely to the optical axis of the illumination optics caused by the color cross correction element on the sample side of the illumination optics causes the offset of the foci essentially caused by the illumination optics is directed opposite to each other transversely to the optical axis of the illumination optical system.
  • the optical retrofit kit can also be configured such that when it is combined with an existing microscope, a microscope is obtained which is identical to an embodiment of a microscope according to the invention described above.
  • the optical retrofit kit may thus comprise a highly dispersive thin-film filter, comprising at least one optical element, on whose sample side the beam paths of the at least at least two wavelengths with respect to the illumination side are tilted relative to one another, at least two optical color cross-correction elements are provided, of which at least one optical color cross-correction element is designed to be relatively tiltable relative to the other color cross-correction element and at least one or two of the at least two optical color cross-correction elements are transmissive, provide substantially plane-parallel plate, which may have a different thickness or different material dispersion and / or may be coupled to each other to transmit movement.
  • a color longitudinal correcting device is provided, the beam paths of the at least two different wavelengths on the illumination side of the color longitudinal correcting device having a divergence or convergence which is dependent on the divergence or convergence of the beam paths of the at least two wavelengths relative to one another the sample side of the color-longitudinal correction device differ. If an illumination optical unit of an existing microscope has a longitudinal chromatic aberration, then this longitudinal chromatic aberration can be compensated with this embodiment of the optical retrofit kit.
  • the optical retrofit kit can only comprise a color longitudinal correction device, so that only a longitudinal chromatic aberration of an existing illumination optical unit can be corrected.
  • the color-length correction device of the optical retrofit kit as well as a previously mentioned embodiment of the microscope according to the invention, have at least one refractive and at least one diffractive optical element, wherein the at least one refractive optical element and / or the at least one diffractive optical element can have a variably adjustable focal length and wherein the at least one diffractive optical element can be designed as a spatial light modulator.
  • the at least one refractive optical element and / or the at least one diffractive optical element can have a variably adjustable focal length and wherein the at least one diffractive optical element can be designed as a spatial light modulator.
  • Figure 1 is a schematic representation of the color longitudinal error and color cross error in a confocal microscope.
  • Figure 2 is a schematic representation of the color longitudinal error and the color cross error in a light disc microscope.
  • 3A shows a first embodiment of the color cross-correction element.
  • 3B shows a second embodiment of the color cross correction element
  • 3C shows a third embodiment of the color cross correction element
  • Fig. 5A is a schematic representation of the color longitudinal error in a diffractive optical element
  • 5B is a schematic representation of the color longitudinal error in a refractive optical element comprising a tunable convex lens
  • 5C shows a color longitudinal correction device in a first correction state
  • FIG. 5D shows the color longitudinal correction device of FIG. 5C in a second correction state
  • Fig. 6A shows a part of the color cross-correction device of a third embodiment
  • Fig. 6B is a part of the color cross-correction device of the third embodiment of Fig. 6A with lower angular dispersion.
  • the microscope 1 shows a schematic representation of a microscope 1, in particular a confocal microscope 3, which has an illumination optical unit 5, lenses 7 and further optical elements 9.
  • the further optical elements 9 are symbolized by a rectangle.
  • the microscope 1 further comprises a light source 11, which in other embodiments of the microscope 1 may also be part of the illumination optics 5, a sample holder 15, on which a sample 15a can be arranged, and an observation optics 17, via which the light illuminated by the illumination optics 5 Sample 15a can be observed.
  • the confocal microscope 3 shown in FIG. 1 has an eyepiece 19 for observation, wherein in other embodiments of the confocal microscope 3 preferably a recording, processing and display of the sample 15a via digital image sensors and monitors can take place.
  • Fig. 1 does not show the sequential image structure of confocal microscopy or a correspondingly necessary raster and scanning device.
  • a magnification 13 shows a refractive optical element 21 of the illumination optical system 5, which is designed as a lens 7.
  • two beam paths 23 extend, the different beam paths 23 representing a short-wave beam path 23a and a boring beam path 23b.
  • the beam paths 23 are shown schematically by edge beams of beams 25 and the short-wave beam 23a, which is shown by a solid line corresponds to the beam 23 of short-wave radiation of a first wavelength 27a, which is shorter than a wavelength 27 of long-wave radiation of a second Wavelength 27b.
  • the absolute wavelength difference of both wavelengths 27a, 27b is less relevant for the following description, only the relation of both wavelengths 27a, 27b is necessary for the following considerations.
  • shortwave and longwave light as well as blue and red light are used synonymously for the first wavelength 27a and the second wavelength 27b.
  • the blue 27 a and the red light 27 b essentially have an identical beam path 23.
  • the lens 7 is configured in the embodiment of the microscope 1 shown in FIG. 1 as a biconvex lens 7a and focuses the light incident on the lens 7 in a focus region 31 which is a focal point only in the approximate case of the geometric optics of radiation beams 25.
  • the blue light 27a is focused in a first focus area 31a and the red light 27b in a second focus area 31b.
  • blue and red focus area are used synonymously with the first 31a and second focus area 31b.
  • the lens 7, which is part of the illumination optics 5, has two mutually independent, linearly overlapping chromatic aberrations 37.
  • a longitudinal color aberration 37a also called lateral chromatic aberration, manifests itself in a lateral offset 39 between the blue focal region 31a and the red focal region 31b along an optical axis 41 or along a direction parallel to the optical axis 41.
  • a transverse chromatic aberration 37b also called a transverse chromatic aberration, manifests itself in a transverse offset 43 of the blue focus area 31a and the red focus area 31b relative to one another in a direction perpendicular to the optical axis 41.
  • an optical axis of the observation optics 41a also extends in the embodiment shown.
  • the observation optics 17 is set to an observation plane 47, so that only objects which are located in the observation plane 47 are sharply imaged by the observation optics 17.
  • FIG. 2 also shows a microscope 1, wherein the microscope 1 shown is a lens microscope 2.
  • the optical disk microscope 2 also comprises an illumination optical system 5 and an observation optical system 17.
  • the principal differences in the illumination compared to the confocal microscope 3 shown in FIG. 1 are shown in the magnification 13.
  • the magnification 13 shows a lens 7 and a deflecting element 51 in the form of a deflecting mirror 53, which are part of the illumination optics 5.
  • the illumination optics 5 of the light-sheet microscope 2 also comprises a plurality of lenses 7, further optical elements 9 and a light source 11.
  • the light-sheet microscope 2 comprises a shell 55, which is filled with immersion liquid 57, so that an observation optics 17, in particular a microscope objective 17a with a high numerical aperture can be used.
  • FIG. 2 shows an observation lens 7b which is located in the immersion liquid 57.
  • the observation lens 7b forms an observation plane 47, wherein in the representation shown in FIG. 2 the observation plane 47 is correctly illuminated only by the red light 27b, while the further plane 47a illuminated by the blue light 27a is located behind the observation plane 47 in the viewing direction 59 is and thus can no longer be sharply imaged by the observation lens 7b.
  • the longitudinal chromatic aberration 37a leads in the light-sheet microscope 2 to a distribution of the intensity of the wavelengths 27, which varies along an illumination direction 61.
  • FIG. 2 only the stationary illumination in a light-sheet microscope 2 is shown.
  • the structure of the light sheet, i. the two-dimensional illuminated area is effected by a tilting of the beam paths 23 about a scanning axis 63 by means of a deflecting element not shown in FIG. 2 (see FIG. 4).
  • FIG. 3A shows a first embodiment of a color cross-correction device 65, which comprises two optical color cross-correction elements 67 in the form of transmitting, substantially plane-parallel plates 68.
  • a first color cross-correction element 67a has a thickness 69 that is greater than a thickness 71 of a second color cross-correction element 67b.
  • first color cross correction element 67a is tilted clockwise 73 against an original propagation direction 75 at a first tilt angle 77a.
  • the second color cross-correction element 67b is tilted in the direction of rotation counterclockwise 79 against the original propagation direction 75 at a second tilt angle 77b.
  • the ray paths 23 of the blue 27a and the red light 27b on the illumination side 29 of the color cross-correction device 65 are identical and define the original propagation direction 75.
  • the first color cross-correction element 67a generates, due to its material dispersion 33a, an angular dispersion 81 which generates a relative offset 85 between the blue 27a and the red light 27b on a sample side 81 of the first color cross-correction element 67a.
  • an absolute offset 85a of both wavelengths 27a, 27b with respect to the original propagation direction 75 occurs.
  • a reference point for determining the absolute offset 85a is defined centrally in FIG. 3A between the blue 27a and the red light 27b.
  • the relative offset 85 is larger and not drawn to scale with respect to the absolute offset 85a.
  • the absolute offset 85a is greater than the relative offset 85.
  • the second color cross-correction element 67b Since the second color cross-correction element 67b is tilted counter to the original propagation direction 75 in the counterclockwise direction 79, this causes the absolute offset 85a to be reduced or completely compensated due to the material dispersion 33b (which may differ from the material dispersion 33a).
  • the absolute offset 85a On the illumination side 29 of the second color cross-correction element 67b, i. at a sample-side output 84 of the second color cross-correction element 67b to the sample side 83 of the second color cross-correction element 67b, only a relative offset 85b occurs.
  • the second color lateral correction element 67b completely compensates for the absolute offset 85a of the first color cross correction element 67a.
  • the two color cross correction elements 67 each have a motion transmission element 133, which is connected to a synchronization unit 135.
  • the synchronization unit 135 controls a variable rotation of the first color cross correction element 67a and synchronizes an opposite rotation of the second color cross correction element 67b.
  • the motion transmitting members 133 are configured as stepping motors 133a.
  • the synchronization can be done for example by means of a transmission.
  • 3B shows a second embodiment of the color cross-correction device 65.
  • This comprises a deflecting element 51 designed as a deflecting prism 87, a highly dispersive thin-film filter 89 and a deflecting element 51 configured as a deflecting mirror 53.
  • the blue 27a and the red light 27b On the illumination side 29 of the color cross-correction device 65, the blue 27a and the red light 27b have an identical beam path 23.
  • the beam paths 23 of the blue 27a and red light 27b are only schematically offset from one another.
  • the deflecting prism 87 directs the coaxial beam paths 23 onto the highly dispersive thin-film filter 89, which generates an angular dispersion 81 which, when leaving the highly dispersive thin-film filter 89, results in a relative offset 85 between the blue 27a and the red light 27b.
  • the highly dispersive thin film filter 89 has a dispersive structure 91 (eg, a periodic structure, a chirped structure, or a resonator structure), the blue light 27a in the highly dispersive thin film filter 89 is deflected less than the red light 27b.
  • a dispersive structure 91 eg, a periodic structure, a chirped structure, or a resonator structure
  • the beam paths 23a and 23b are deflected in the direction of the deflection prism 87, which outputs the two beam paths 23a, 23b substantially centered to the original propagation direction 75 on the sample side 83 of the color cross-correction device 65.
  • the absolute offset 85a is thus approximately zero and the two beam paths 23a and 23b have only the relative offset 85b to one another.
  • a third embodiment of the color cross correction device 65 is shown.
  • the two color cross-correction elements 67 are designed as prisms 131.
  • Both prisms 131 are rotatable about a respective axis of rotation 151a, 151b and / or displaceable along or against an insertion direction 157.
  • the insertion direction 157 extends from a prism base 159 to an apex 161, independently of the rotation of the prism 131.
  • Both prisms 131 can have different insertion directions 157.
  • the first 67a and second color cross-correction element 67b in this case have a different material dispersion 33a, 33b from each other.
  • the material dispersion 33a is larger than the material dispersion 33b.
  • the high material dispersion prism 131a deflects the first 27a and second wavelengths 27b by a first central deflection angle 153a.
  • a relative splitting 155 is formed due to the first material dispersion 33a.
  • Prism 131 of lesser material dispersion 33b deflects both first 27a and second wavelengths 27b by a second central deflection angle 153b, which deflection preferably compensates for the deflection about the first center deflection angle 153a, so that a resulting second propagation direction 75b is only laterally parallel to a first propagation direction 75a is shifted, but is oriented substantially parallel to this.
  • Optical axes 41b, 41c are also offset in parallel.
  • FIG. 4 shows an illumination optical unit 5 with installed color cross-correction device 65.
  • the two beam paths 23a, 23b are collinear and centered on the original propagation direction 75 and are focused by a focusing element 7e (shown here in the form of a lens 7d).
  • the color cross-correction device 65 is arranged between the focusing element 7e and intermediate focus areas 31c. As described above, the color cross correction device 65 introduces a relative offset 85 of the blue 27a to the red light 27b.
  • An absolute offset 85a is substantially compensated by the two oppositely tilted color cross correction elements 67. After the color transverse correction elements 67, the two beam paths 23a, 23b pass through a lens 7d, which leads to a slight tilting of the beam paths relative to each other.
  • FIG. 4 further shows a deflecting mirror 53 configured as a scanning mirror 93, which can be tilted about a scanning axis 63 emerging from the plane of the drawing and about a scanning axis 63a lying in the scanning mirror 93.
  • a deflecting mirror 53 configured as a scanning mirror 93, which can be tilted about a scanning axis 63 emerging from the plane of the drawing and about a scanning axis 63a lying in the scanning mirror 93.
  • the beam paths 23a, 23b are transmitted through said lenses 7 and focused in a wavelength-dependent focus area 31. Since the illumination optics 5 has only one color cross-correction device 65, the blue light 27a is focused at the same distance to the optical axis 41 as the red light 27b. An existing longitudinal color error 37a is independent of the correction of the transverse color error (not shown) and therefore continues to occur.
  • FIG. 4 shows that the lenses 7 of the illumination optical system 5 transmit the beam paths 23a, 23b in non-paraxial edge regions 8 through the corresponding lenses 7.
  • FIG. 5A shows a diffractive optical element 95, which is designed as a diffractive optical lens 97, in particular as a tunable diffractive lens 99, which can be generated, for example, by means of a spatial light modulator 101.
  • the beam paths 23a, 23b which run coaxially with one another, strike the tunable diffractive lens 99, the beam paths 23a, 23b are focused in accordance with the setting of the tunable diffractive lens 99.
  • FIG. 5B shows a refractive optical element 109, which is designed as a tunable lens system 111.
  • the tunable lens system 111 includes a concave lens 7c and a tunable convex lens 113.
  • the tunable lens system 111 in the setting shown in Fig. 5B, has the effect of a concave lens 7c, i. the coaxial beam paths 23a and 23b are not focused to give a blue virtual focus area 115 (the virtual focus of the blue light 27a) and a red virtual focus area 117 (the virtual focus of the red light 27b).
  • the blue light 27a has the negative focal length 105
  • the red light 27b has the negative focal length 103
  • the amount of the focal length of the blue light 105 is smaller than the amount of the focal length of the red light 103.
  • FIG. 5C shows a color longitudinal correction device 123 comprising the tunable diffractive lens 99 of FIG. 5A and the tunable lens system 111 of FIG. 5B.
  • the color longitudinal correction device 123 is shown in a first setting 125.
  • the divergence 121 generated by the tunable lens system 111 is substantially compensated by the convergence generated by the tunable diffractive lens 99.
  • the color-length correction device 123 has a very long focal length (which can be in the range of several meters).
  • the color longitudinal correction device 123 essentially maintains the collimation of the transmitted beam paths 23a, 23b.
  • the pairing of a positive focal length of the tunable diffractive lens 99 and an equal but negative focal length of the tunable lens system 111 thus does not alter the absolute convergence 119 or the absolute divergence 121 of the beam paths 23a, 23b.
  • a relative convergence / divergence difference 127 between the blue 23a and the red optical path 23b can be set.
  • the color longitudinal correction device 123 is shown in a second setting 129. Even in the second setting 129, the absolute convergence 119 or the absolute divergence 121 of the beam paths 23a, 23b entering the color longitudinal correcting device 123 is not changed and remains substantially zero.
  • the color-length corrector 123 has a relative divergence difference 127 that differs from the relative convergence difference 127 of the first setting of FIG. 5C.
  • the blue light 27a is substantially collimated, while the red light 27b has a convergence 121.
  • the red light 27b is substantially collimated, while the blue light 27a has a convergence 121.
  • FIGS. 6A and 6B show a second and third embodiment of the color cross correction device 65, wherein in both embodiments, a prism 131 for generating the angular dispersion 81 is used.
  • the angular dispersion 81 is generated on the basis of the material dispersion 33 of the prism 131, as described above using the plane-parallel plate as an example.
  • a different size angular dispersion 81 can be generated.
  • the beam paths 23a, 23b are deflected in such a way that both beam paths 23a, 23b have substantially no further tilting relative to the original propagation direction 75, but that a relative tilting of the blue beam path 23a to the red beam path 23b continues to be present.
  • An optical retrofit kit 150 may include one of the color cross-corrector 65 shown in FIGS. 3A, 3B, 6A, and 6B.
  • color longitudinal correction device 123 shown in FIGS. 5C and 5D may be part of the optical retrofit kit 150.

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Abstract

L'invention concerne un microscope (1), en particulier un microscope à feuillet de lumière (2) ou un microscope confocal (3), et un équipement optique complémentaire (150) destiné à un microscope (1), en particulier à un microscope à feuillet de lumière (2) ou à un microscope confocal (3), comportant une optique d'éclairage (5) servant à transmettre de la lumière présentant au moins deux longueurs d'onde (27a, 27b) provenant d'au moins une source lumineuse (11) respectivement le long d'un chemin optique (23a, 23b) fonction de la longueur d'onde d'un côté éclairage (29) de l'optique d'éclairage (5) vers un côté échantillon (83) de l'optique d'éclairage (5). Les microscopes (1) de l'état de la technique présentent une aberration chromatique longitudinale (37a) et/ou une aberration chromatique transversale (37b) qui réduit la qualité de reproduction du microscope (1). Le microscope (1) selon l'invention ou l'équipement optique complémentaire (150) résout ce problème en ce que le microscope (1) ou l'équipement optique complémentaire (150) présente un dispositif de correction chromatique transversale (65) muni d'au moins un élément de correction chromatique transversale (67), les chemins optiques (23a, 23b) des deux ou plus de deux longueurs d'onde différentes (27a, 27b) présentant au niveau de la sortie (84) côté échantillon de l'élément de correction chromatique transversale (65) un décalage (43) parallèlement l'un à l'autre et/ou un basculement l'un vers l'autre par rapport au côté éclairage (29), qui se traduit sur le côté échantillon (83) de l'optique d'éclairage (5) par un décalage (43) des foyers (31a, 31b) des deux ou plus de deux longueurs d'onde (27a, 27b) transversalement à un axe optique (41) de l'optique d'éclairage (5).
EP17749427.5A 2016-08-02 2017-08-02 Microscope, en particulier microscope à feuillet de lumière ou microscope confocal, et équipement complémentaire pour microscope Pending EP3494429A1 (fr)

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DE102019208232A1 (de) * 2019-06-05 2020-12-10 Carl Zeiss Microscopy Gmbh Optische Anordnung und Verfahren zur Korrektur von Zentrierfehlern und/oder Winkelfehlern
WO2021041804A1 (fr) * 2019-08-29 2021-03-04 Arizona Board Of Regents On Behalf Of The University Of Arizona Microscope à feuille de lumière chromatique
DE102022103459A1 (de) 2022-02-15 2023-08-17 Carl Zeiss Microscopy Gmbh Strahlteilerbaugruppe, verfahren zu deren dimensionierung und mikroskop

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JP2000010012A (ja) 1998-06-19 2000-01-14 Olympus Optical Co Ltd 顕微鏡照明光学系
DE19951482C2 (de) 1999-10-26 2003-01-09 Zeiss Carl Jena Gmbh Fluoreszenzmikroskop
DE102004058833A1 (de) * 2004-12-06 2006-06-08 Leica Microsystems Cms Gmbh Optische Anordnung für ein Mikroskop und ein Mikroskop
JP4242862B2 (ja) 2005-09-12 2009-03-25 オリンパス株式会社 光学特性可変光学素子及び光学特性可変光学素子を備えた表示装置
US20080246919A1 (en) 2007-04-09 2008-10-09 Ron Smith Ophthalmic Endoilluminator with Hybrid Lens
EP2169442B1 (fr) * 2007-07-19 2015-10-28 Nikon Corporation Microscope confocal de type à balayage
JP5054595B2 (ja) 2008-04-04 2012-10-24 日本放送協会 レーザプロジェクタ
DE102008022493A1 (de) * 2008-05-07 2009-11-12 Carl Zeiss Microlmaging Gmbh Vorrichtung und Verfahren zum evaneszenten Beleuchten einer Probe
EP2720075B1 (fr) 2012-10-12 2017-11-29 Spectral Applied Research Inc. Microscopie de fluorescence à réflexion interne totale (TIRF) simultanément à travers de multiples longueurs d'onde
DE102013213781A1 (de) 2013-03-20 2014-09-25 Leica Microsystems Cms Gmbh Verfahren und optische Anordnung zum Manipulieren und Abbilden einer mikroskopischen Probe
JP6253400B2 (ja) 2013-12-26 2017-12-27 オリンパス株式会社 画像形成方法、及び、画像形成装置
LU92505B1 (de) 2014-07-22 2016-01-25 Leica Microsystems Verfahren und vorrichtung zum mikroskopischen untersuchen einer probe
DE102014116782A1 (de) * 2014-11-17 2016-05-19 Carl Zeiss Microscopy Gmbh Detektorvorrichtung für ein Mikroskop

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US20190258052A1 (en) 2019-08-22
JP7184752B2 (ja) 2022-12-06

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