US20060256426A1 - Device for controlling light radiation - Google Patents

Device for controlling light radiation Download PDF

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Publication number
US20060256426A1
US20060256426A1 US11/416,392 US41639206A US2006256426A1 US 20060256426 A1 US20060256426 A1 US 20060256426A1 US 41639206 A US41639206 A US 41639206A US 2006256426 A1 US2006256426 A1 US 2006256426A1
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light
specimen
detection
polarization
excitation
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Ralf Wolleschensky
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Carl Zeiss Microscopy GmbH
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Carl Zeiss MicroImaging GmbH
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Assigned to CARL ZEISS MICROIMAGING GMBH reassignment CARL ZEISS MICROIMAGING GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WOLLESCHENSKY, RALF
Publication of US20060256426A1 publication Critical patent/US20060256426A1/en
Priority to US11/998,418 priority Critical patent/US20080088907A1/en
Priority to US12/318,287 priority patent/US7872799B2/en
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    • 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
    • G02B27/28—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising
    • G02B27/283—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00 for polarising used for beam splitting or combining
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/12—Generating the spectrum; Monochromators
    • G01J3/1256—Generating the spectrum; Monochromators using acousto-optic tunable filter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G01J3/4406—Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/447—Polarisation spectrometry
    • 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
    • G01N21/6445—Measuring fluorescence polarisation
    • 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
    • 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
    • G02B21/0068—Optical details of the image generation arrangements using polarisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0224—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using polarising or depolarising elements

Definitions

  • the invention relates to a method and arrangements in microscopy, in particular fluorescence microscopy, laser scanning microscopy, fluorescence correlation spectroscopy and near-field scanning microscopy, for examining predominantly biological specimens, preparations and related components.
  • microscopy in particular fluorescence microscopy, laser scanning microscopy, fluorescence correlation spectroscopy and near-field scanning microscopy, for examining predominantly biological specimens, preparations and related components.
  • the beamed-in photons having a determined energy excite the dyed molecules, through the absorption of a photon, from the original state into an excited state.
  • This excitation is usually referred to as single photon absorption.
  • the dyed molecules that are excited in this way can return to the original state in various ways.
  • fluorescence microscopy the most important way is the transition with emission of a fluorescence photon. Owing to the Stokes shift, there is generally a red shift in the wavelength of the emitted photon, in comparison to the excitation radiation; thus, it has a longer wavelength. The Stokes shift makes it possible to separate the fluorescence radiation from the excitation radiation.
  • the fluorescent light is split off from the excitation radiation by suitable dichroic beam splitters in combination with blocking filters and is observed separately. This makes it possible to show individual cell parts that are dyed with different dyes. In principle, however, several parts of a preparation can also be dyed simultaneously with different dyes that bind in a specific manner (multiple fluorescence). Special dichroic beam splitters are used in turn to distinguish the fluorescence signals, emitted by the individual dyes.
  • excitation with a plurality of low-energy photons is also possible.
  • the sum of energies of the single photons is equal to approximately a multiple of the high-energy photon.
  • This type of excitation of dyes is known as multiphoton absorption (literature: Corle, Kino. Confocal Scanning Optical Microscopy and Related Imaging Systems . Academic Press, 1996).
  • the dye emission is not influenced by this type of excitation. That is, the emission spectrum undergoes a negative Stokes shift in multiphoton absorption; thus, it has a shorter wavelength compared to the excitation radiation.
  • the separation of the excitation radiation from the emission radiation is carried out in the same way as in single photon absorption.
  • LSM confocal laser scanning microscope
  • Lasers with different wavelengths are used in an LSM for specific excitation of different dyes in a preparation.
  • the choice of excitation wavelengths is governed by the absorption characteristics of the dyes to be examined.
  • the excitation radiation is generated in the light source module.
  • Various lasers for example, glass lasers, argon, argon/krypton, solid state lasers, TiSa lasers, diodes
  • the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength is carried out in the light source module, i.e., using an acousto-optic crystal.
  • the laser radiation subsequently reaches the scan module via a fiber or a suitable mirror arrangement.
  • the laser radiation generated in the light source, is focused in the preparation in a diffraction-limited manner by means of the objective via the scanners, scanning optics and tube lens.
  • the scanner is moved over the specimen point-by-point in x-y direction.
  • the pixel dwell times when scanning over the specimen are mostly in the range of less than one microsecond to several seconds.
  • the light In confocal detection (descanned detection) of the fluorescent light, the light, which is emitted from the focal plane (specimen) and from the planes located above and below the latter, reaches a dichroic beam splitter (MDB) via the scanners.
  • MDB dichroic beam splitter
  • This dichroic beam splitter separates the fluorescent light from the excitation light.
  • the fluorescent light is subsequently focused on a diaphragm (confocal diaphragm/pinhole), which is located precisely in a plane conjugate to the focal plane. In this way, fluorescent light components outside of the focus area are suppressed.
  • the optical resolution of the microscope can be adjusted by varying the size of the diaphragm.
  • Another dichroic blocking filter (EF) which again suppresses the excitation radiation, is located behind the diaphragm. After passing the blocking filter, the fluorescent light is measured by means of a point detector (PMT).
  • PMT point detector
  • the excitation of the dye fluorescence is carried out in a small volume, at which the excitation intensity is particularly high.
  • This area is only negligibly larger than the detected area when using a confocal arrangement.
  • descanned detection is carried out again, but this time the pupil of the objective is imaged in the detection unit (non-confocal descanned detection).
  • the LSM is suitable for examining thick preparations.
  • the excitation wavelengths are determined by the utilized dye with its specific absorption characteristics.
  • Dichroic filters adapted to the emission characteristics of the dye, ensure that only the fluorescent light, emitted by the respective dye, will be measured by the point detector.
  • an additional splitting of the fluorescent light of a plurality of dyes is carried out with the secondary beam splitters (DBS); and a separate detection of the individual dye emissions is carried out in various point detectors (PMT 1 - 4 ).
  • Flow cytometers are used for examining and classifying cells and other particles.
  • the cells are dissolved in a liquid and are pumped through a capillary.
  • a laser beam is focused in the capillary from the side.
  • the cells are dyed with different dyes or fluorescing biomolecules.
  • the excited fluorescent light and the backscattered excitation light are measured.
  • the fluorescence signal of the specimen is separated from the excitation light by means of dichroic beam splitters (MDB, see FIG. 1 ).
  • MDB dichroic beam splitters
  • the size of the cells can be determined from the backscattered signal. Different cells can be separated/sorted or counted separately by means of the spectral characteristics of the fluorescence of individual cells.
  • the sorting of the cells is carried out with an electrostatic field in different capillaries. The results, that is, for example, the quantity of cells with dye A in comparison to cells with dye B, are often displayed in histograms.
  • the flow rate is typically about 10 to 100 cm/s. Therefore, a highly sensitive detection is necessary. According to the prior art, a confocal detection is carried out in order to limit the detection volume.
  • line scanners are also used, instead of point scanners (literature: Corle, Kino. Confocal Scanning Optical Microscopy and Related Imaging Systems . Academic Press, 1996).
  • the basic construction corresponds in essence to that of an LSM, according to FIG. 1 .
  • a line is imaged in the specimen ( 3 ); and the specimen to be examined is scanned in only one direction (x or y).
  • the image acquisition rate can be significantly increased by scanning a line, instead of a point. Therefore, this scanning method can be used for observing high speed processes in real time (real time microscopy).
  • Arrangements for screening dyes are similar in their optical construction to a laser scanning microscope. However, they scan a significantly larger image field for examining macroscopic specimens, for example, screening of active ingredients on a biochip.
  • the edge length of the scan fields amounts to several 10 nm.
  • These scan fields can be achieved, e.g. by increasing the scan angles of the galvo-scanners, by arranging the specimen in an intermediate image of the microscope arrangement or by a special objective arrangement (macro-objective), which images the intermediate image on the specimen in a magnified manner.
  • the separation of the excitation light from the light emitted by the specimen is carried out by spectral separation using the Stokes shift by restricting the numerical aperture of the optics, used for specimen illumination and detection, or by splitting into different polarization directions.
  • Special dichroic beam splitters are used for the spectral separation of the excitation light from the light emitted by the specimen. As shown in FIG. 2 a , these dichroic beam splitters are usually constructed in such a way that they reflect the excitation light as efficiently as possible and transmit the light emitted by the specimen as efficiently as possible.
  • the reflection factor (reflectivity) is shown as a function of the wavelength.
  • the minimum spectral bandwidth (s) of the reflected wavelength range is about 10 nm; the edge steepness (f) is usually greater than 5 nm. Therefore, according to the prior art, the light emitted by the specimen can be efficiently separated with a dichroic beam splitter when using an excitation wavelength.
  • a special beam splitter must be created each time when using different dyes with different absorption characteristics.
  • dichroic beam splitters results in poor efficiency of the separation of the excitation light from the emitted light.
  • the separation of excitation light from emitted light by restricting the numerical aperture of the specimen illumination optics ( 4 in FIG. 2 b ) can be carried out, for example, by illuminating the specimen with a restricted aperture, so that only the near-axis beams ( 1 ) arrive in the direction of the specimen ( 2 ). Since the emission is carried out in all spatial directions, this light from the specimen ( 2 ) can be collected in the rest of the aperture area.
  • the separation of the excitation light from the emitted light is carried out subsequently by a partially fully reflecting (black area) plane plate ( 3 ).
  • the detection of the light emitted by the specimen is carried out in the beam direction ( 5 ).
  • EP 1353209 is that, on the one hand, the efficiency of detection and, on the other hand, the optical resolution of the arrangement is impaired due to the restriction of the aperture. These two parameters are connected in this regard. For example, in order to achieve a highly efficient separation, the optical resolution decreases.
  • U.S. Pat. No. 6,510,001, U.S. Pat. No. 6,654,165, U.S. Published Patent Application No. 2003/0133189 and German Patent DE 19936573 disclose optical devices, where a spectrally flexible separation of the detection light from the excitation light can be carried out in an adjustable manner without any movement of mechanical components ( FIG. 3 ).
  • the MDB is replaced by an acousto-optic modulator AOTF ( 17 , 4 ). It transmits the observation light ( 5 , 12 ), coming from the direction of the specimen, so that it arrives in the direction of the detector ( 15 ).
  • the excitation light ( 3 , 9 ) runs at an angle relative to ( 12 ) and is diffracted into the joint specimen beam path ( 5 ) by means of the AOTF. Therefore, the frequency of the AOTF must be adjusted in such a manner that the excitation beam path and the detection beam path run colinearly. If this is not guaranteed, then the result is a reduction in the detection efficiency, particularly in the case of a confocal detection, and/or aliasing errors, because when different wavelengths are used, the excitation spots are not stacked. Special compensation devices are described in U.S. Pat. No. 6,967,764 B2. The drawback with these arrangements lies in the need for a plurality of tunable optical components that have a negative impact on the overall transmission.
  • U.S. Pat. No. 6,947,127 B2 discloses a method and optical devices, with which an achromatic separation of the detection light from the excitation light can be carried out in a wide field or in a line-scanning microscope.
  • the light radiation which is excited in a specimen and/or which is backscattered and/or reflected by the specimen, is separated by focusing the specimen illumination in and/or in the vicinity of a pupil plane of the beam path between the specimen plane and the acquisition plane, and by providing means for a spatial separation of the illumination light from the detection light in this plane.
  • U.S. Published Patent Application No. 2004/0159797 A1 discloses a method and arrangement for changing the spectral composition and/or the intensity of the illumination light and/or the specimen light in an adjustable manner. Therefore, a spatial separation into radiation components of different polarization is carried out with the first polarization means (P 1 , P 3 ); a spectral, spatial splitting of at least one radiation component is carried out with the first dispersion means (D 1 ); the spectrally spatially split components are imaged (L 1 ) on an element S; the polarization state of at least one part of the spectrally spatially split radiation component is changed by the action of the element S; and a spatial separation and/or combination of radiation components of different polarization are/is carried out by the second imaging means (L 2 ) and the polarization means (P 2 , P 4 ).
  • a spatial combination of radiation components which are changed and not changed with respect to their polarization state, is advantageously carried out by the second dispersion means (D 2 ).
  • the drawback with this arrangement lies in the number of optical components for a spectral spatial splitting, by means of which the efficiency of the arrangement is reduced.
  • the manipulation of the polarization state of the spectral components at the element S is carried out with a linear array. Depending on the specified spectral resolution, this array is costly with regard to the electronic wiring.
  • the speed is restricted when using a spatial light modulator and amounts to a few 10 ms.
  • dispersive elements e.g. prisms or gratings
  • D 1 and D 2 which split the light radiation spatially and spectrally along the Y coordinate and combine it again, are disposed between 2 beam splitter cubes each (P 2 and P 1 or P 4 and P 3 ).
  • the optics L 1 and L 2 are positioned at a distance from their respective focal length f, which can also vary for the optics, between the dispersive elements D 1 or D 2 and an element for rotating the polarization, for example a spatial light modulator (SLM) S.
  • SLM spatial light modulator
  • the optics L 1 and L 2 together with the dispersive elements D 1 and D 2 are used to produce a spectral Fourier plane at the location of the SLM S. In this plane the spectral components of the light, coming from the direction 2 or the direction 1 , are separated spatially along the Y coordinate.
  • the SLM e.g. SLM640 of the company Jenoptik, Germany
  • the SLM comprises a number of strips (in the case of the SLM 640 there are 640 strips), which can be actively controlled individually.
  • the polarization direction of the light passing through can be varied.
  • the SLM's according to the prior art, are used in so-called pulse shapers (literature: Stobrawa et al. Applied Physics B72. pp. 627-630 (2001)). Therefore, the action of the SLM in combination with the dispersive elements results in a phase delay and/or a change in the amplitude of the spectral components of the light source.
  • the light source must be polarized linearly, because otherwise an energy loss occurs.
  • a plurality of adjustable lambda half-wave plates which are arranged in the Fourier plane, can also be used, for example.
  • the invention discloses a method and arrangements, by which the excitation light can be separated from the light radiation (e.g. fluorescence or luminescence), which is excited and/or backscattered in the specimen, in an especially advantageous manner with high efficiency.
  • the number of optical components in the beam path is reduced, as compared to U.S. Published Patent Application No. 2004/0159797 A1, so that the result is a higher efficiency of the optical arrangement.
  • the separation can be adjusted in a spectrally flexible manner without any movement of mechanical components and is, therefore, particularly suitable especially for use in multi-fluorescence microscopy, i.e. for simultaneous excitation of different dyes.
  • the optical resolution is not impaired by the arrangements, according to the invention.
  • the suppression of the stray light is improved by at least one order of magnitude. Accordingly, fast switching between several excitation wavelengths or spectral detection wavelength ranges—so-called multi-tracking, as described in U.S. Pat. No. 6,462,345 B1,—can be realized in an especially simple manner.
  • Another advantage is that laser power fluctuations, caused by an unstable coupling into a glass fiber, can be prevented by automatic control, so that the output can be held constant at the site of the specimen.
  • the illumination distribution can be manipulated at the site of specimen interaction. This makes it possible to scan so-called regions of interest (ROI) in real time.
  • ROI regions of interest
  • the illumination methods known from wide field microscopy, such as oblique illumination can be realized.
  • the solution can be used in image generating microscope systems as well as in analytic microscope systems.
  • the microscope systems are image generating systems, such as laser scanning microscopes for three dimensional examination of biological preparations with an optical resolution of up to 200 nm, near field scanning microscopes for high resolution examination of surfaces with a resolution of up to 10 nm, fluorescence correlation microscopes for quantitative determination of molecular concentrations and for measuring molecular diffusions. Also included are methods based on fluorescence detection for screening dyes and methods for flow cytometry.
  • the quantity of the dye signatures which may be used simultaneously, i.e., the quantity of the characteristics, for example, of cells that can be examined simultaneously, can be increased by means of the methods, according to the invention.
  • the spectral signatures of the individual dyes overlap extensively or are very close to one another, the detected wavelength range or numerical aperture must be limited, according to the prior art, for separate detection of the fluorescence signals of individual dyes. This reduces the sensitivity of detection, i.e., increases the noise of the detectors, because greater amplification must be used. This is avoided by the methods and arrangements of the invention.
  • FIG. 1 is a schematic diagram of a confocal laser scanning microscope (LSM).
  • LSM confocal laser scanning microscope
  • FIG. 2A is a graph in which the reflection factor (reflectivity) of a dichroic beam splitter of a prior art LSM is shown as a function of the wavelength.
  • FIGS. 2B and 2C show the separation of excitation light from emitted light in the specimen illumination optics of a prior art LSM.
  • FIG. 3 shows the separation of detected light from excitation light carried out in an adjustable manner.
  • FIG. 4 is a schematic diagram of a prior art arrangement for changing the spectral composition and/or the intensity of the illumination light and/or the specimen light in an adjustable manner.
  • FIG. 5 a is a schematic diagram of AOTF light components.
  • FIG. 5 b is a schematic diagram of a colinear AOTF forming part of the subject invention.
  • FIG. 6A is a schematic diagram which shows the effect of the subject invention in the excitation beam path.
  • FIG. 6B is a schematic diagram which shows the effect of the subject invention in the detection beam path.
  • FIG. 7 is a schematic diagram of the arrangement of FIGS. 6A and 6B in the Y-Z plane.
  • FIGS. 8A and 8B illustrate in schematic form another embodiment of a dichroic beam splitter (MDB) used in the present invention.
  • MDB dichroic beam splitter
  • FIGS. 9 a , 9 b and 9 c schematically illustrate yet another embodiment of a dichroic beam splitter (MDB) used in the present invention.
  • MDB dichroic beam splitter
  • FIG. 10 is a schematic diagram illustrating the inventive arrangement for a laser scanning microscope (LSM) in the X-Z plane.
  • LSM laser scanning microscope
  • FIG. 11 is a schematic diagram illustrating another inventive arrangement for a laser scanning microscope (LSM) in the X-Z plane.
  • LSM laser scanning microscope
  • FIG. 12 is a schematic diagram of a series of MDBs arranged in succession.
  • FIG. 13 is a graph showing the effect of an AOTF with an acoustic wave at frequency f 1 and amplitude A 1 .
  • the arrangements are especially suitable for fast multi-tracking with a spectrally adjusted, flexible separation of the excitation radiation from the detection light.
  • light radiation emitted by the specimen is light that is radiated from the specimen preferably in a large solid angle.
  • This light radiation is usually not polarized (unpolarized) and/or the magnitude of the polarization differs from the polarization of the excitation light. They are in particular fluorescent light, luminescent light and backscattered light that are excited in the specimen.
  • FIGS. 6A and 6B depict an arrangement for separating the excitation light from the detection light in a variable manner.
  • the partial image A) shows the effect of the arrangement in the excitation beam path; partial image B), in the detection beam path.
  • FIG. 6B shows in schematic form the construction of the arrangement for separating the excitation light from the detection light for the detection beam path; and FIG. 6A , for the excitation beam path.
  • the arrangement comprises in essence at least three polarizing beam splitter cubes P 1 to P 3 .
  • P 4 can be another polarizing beam splitter cube or a mirror.
  • beam splitter cubes examples include Glan laser polarizing beam splitters, birefringent materials or specially micro-structured beam splitters (e.g., MicroWires from the company Moxtek, Inc.; Orem, Utah, USA).
  • An acousto-optic element is located between the polarizing beam splitter cubes.
  • Specimen light LD which is coupled in at the coupling port KP 2 in the direction of the arrow, ( 2 ) is separated into two orthogonally reflected polarization components Pol 1 (circles in the drawing, pole direction in the observation direction) and continuous polarization components Pol 2 (arrows in the drawing, pole direction in the direction of the arrow) at the pole splitter P 2 .
  • the gray (I) and the black (II) symbols are supposed to represent lights of different wavelengths (e.g. black (II) fluorescence ( ⁇ 2 ) and gray (I) scattered excitation light ( ⁇ 1 )).
  • Pol 1 of different wavelengths arrives from P 2 via P 4 ; and Pol 2 arrives from P 2 directly at a number of regions of an acousto-optic tunable filter (AOTF) S; and in particular Pol 1 arrives at region b; and Pol 2 , at region a.
  • the AOTF rotates, for example, the polarization for the light radiation having wavelength ⁇ 2 (shaded black (II)) by, for example, exactly 90 deg. ( FIG. 4 ). Then the light reaches the pole splitters P 1 and P 3 , where the gray (I) and the black (II) components (i.e.
  • the fluorescence radiation and the excitation radiation are polarized orthogonally in both arms P 2 -P 1 or P 4 -P 2 ( FIG. 4 ). Therefore, the excitation light (gray (I) components) exits through the coupling ports KP 1 and KP 5 . Both polarization directions of the fluorescent light (black (II) components) exit jointly through the coupling port KP 4 .
  • Excitation light which passes (arrow) through the inlet KP 1 , is separated into orthogonal polarization components Pol 1 and Pol 2 , in KP 2 at P 1 .
  • the gray (I) and the black (II) symbols are supposed to represent in turn light of different wavelengths (e.g., black excitation light of wavelength ⁇ 2 and red excitation light of wavelength ⁇ 1 ).
  • Pol 2 arrives directly at the outlet KP 6 .
  • Pol 1 of different wavelengths ( ⁇ 1 , ⁇ 2 ) arrives from P 1 at the AOTF S.
  • the AOTF rotates, for example, the polarization for the light radiation ⁇ 2 falling (shaded black II) by, for example, exactly 90 deg.
  • the AOTF rotates the polarization by an angle that is, for example, not equal to 90 deg. (preferably in the range from 0 deg. to 180 deg.). Then the light reaches P 2 .
  • P 2 separates the components, as a function of the polarization, into the outlet KP 3 or into the outlet KP 2 .
  • the polarization for the wavelength ⁇ was rotated by exactly 90 deg. by the AOTF. Therefore, all light of this wavelength is passed through P 2 into the outlet KP 3 .
  • the polarization for the wavelength ⁇ 1 was rotated only by an angle not equal to 90 deg. Therefore, the light power is split into the two outlets KP 2 and KP 3 .
  • the division ratio is derived from the adjusted rotation angle of the polarization at the AOTF.
  • the light radiation, which enters through the inlet KP 1 can be spatially separated and adjusted in the various outlets KP 2 , KP 3 and KP 6 , independently of the magnitude of the polarization, and can, therefore, be further processed optically and separately.
  • the light radiation, which enters through the inlet KP 2 can be spatially separated into the various outlets KP 1 , KP 5 and KP 4 , independently of the magnitude of the polarization, and can, therefore, be further processed separately and optically. Therefore, the arrangement is suitable as the main color portions for separating the excitation beam path from the detection beam path.
  • Birefringent media with a specified or flexible polarization rotation can be used as the polarization-rotating elements.
  • Elements with flexible adjustment options are acousto-optic elements, like an AOTF, or electro-optical elements, like a Pockel cell.
  • Elements with specified polarization rotation are, for example, delay plates, like lambda/4 plates.
  • AOTFs with colinear acoustic and optic waves are especially suitable as the AOTF S. In contrast to non-colinear AOTFs, they can rotate the polarization without affecting the direction of the optic wave.
  • the acoustic wave (between couplers 3 and 4 ) is angled relative to the incident radiation ( 1 ). After the AOTF light components ( 2 a ), diffracted at the acoustic wave, and undiffracted light components ( 2 b ) occur.
  • colinear AOTF FIG.
  • an acoustic wave of a determined frequency is applied between the couplers (transducers 3 , 4 ) for rotating the polarization of a specific wavelength.
  • the amplitude of the acoustic wave determines the magnitude of the polarization rotation of the optic wave at the outlet 2 .
  • FIG. 13 shows the effect of the AOTF with an acoustic wave at frequency f 1 and amplitude A 1 , which causes, for example, a 90 deg. rotation of the polarization of the input light at wavelength lambdal.
  • the polarization direction of the light at other wavelengths is not changed.
  • FIG. 7 shows the arrangement from FIG. 6 in the Y-Z plane. Due to the optical elements preferably no deflection of the excitation light and/or the detection light is carried out.
  • FIG. 8 shows another advantageous design of the MDB, wherein birefringent media M 1 , M 2 are used as the polarization splitters. They can be birefringent crystals, like calcite. The function and the description of the ports is analogous to that of FIG. 6 . Only the polarization splitters are replaced with birefringent media.
  • the KPs are the above described coupling ports labeled with the respective reference numerals.
  • FIG. 9 shows another advantageous design of the MDB.
  • the arrangement uses a single polarization-splitting element P.
  • the light from port 2 e.g. specimen
  • S AOTF
  • the light passes twice through AOTF S; and, thus, the AOTF S is operated in reflection. Therefore, the reflecting surface is arranged at a small angle.
  • the reflecting surface can also be in an advantageous manner a surface of the AOTF crystal. By tilting S or the mirror at another angle, the light reaches the lens L; and beams, which run in the direction P, form parallel to the input beam.
  • both beams arrive at P from the port 4 (e.g. detector). If, however, the polarization at the AOTF is changed, then the polarization components arrive in the direction of port 1 (e.g. light source). Since the parallel shift of both polarization components at the ports 1 and 4 is extremely small, both components at, for example, 4 can be guided to a joint detector.
  • the parallel shift between ports 1 and 2 is chosen in such a manner that a spatial separation of the two beams is possible (e.g. by means of M 1 in the figure).
  • FIG. 10 shows in schematic form the inventive arrangement for a laser scanning microscope (LSM) in the X-Z plane.
  • LSM laser scanning microscope
  • the functional principle, described under 1 can be applied analogously to a microscope for separating fluorescence radiation from excitation radiation.
  • the specimen is illuminated with a point focus, which is moved by means of the scanners SX and SY in the XY plane.
  • the preferably linearly polarized light source LQ in the MDB is coupled in via the port 1 at P 1 .
  • the light of the light source LQ arrives preferably at an area a of the AOTF S. If the excitation light is supposed to reach the specimen, then the AOTF is switched in such a manner that the polarization direction of the light is rotated by 90 deg.; and the excitation light reaches the outlet 2 of the MDB.
  • the polarization direction of the excitation light is rotated by an angle that is not equal to 90 deg. Therefore, depending on the polarization direction, a part of the light reaches the outlet 2 ; and the rest of the component reaches the outlet 3 .
  • a monitor diode M 2 for determining the excitation light output, which, as the controlled variable, can be used to compensate for the intensity fluctuations, caused by coupling into a plurality of polarization direction of, for example, a glass fiber.
  • this operating mode can also be used for fast switching or attenuating individual wavelengths of the light source.
  • the linearly polarized excitation light coupled in the direction of the outlet 2 , reaches the scanners SX and SY, which are located in pupil planes of the microscope arrangement that are conjugate to one another and the back focal plane of the objective P 3 , so that the scanners can move the excitation point, which is focused in a diffraction-limited manner, in the XY plane of the specimen—that is, scan the specimen.
  • the imaging in the specimen is carried out by means of the scan optics SO, the tube lens TL and the objective O.
  • the relay optics RL generate the conjugate pupil planes SX and XY of the microscope arrangement.
  • the relay optics can also be dispensed with. For example, they can be omitted when the distance between SX and SY is decreased.
  • the light emitted by the specimen is collected by the optics O (e.g. a microscope objective) and imaged jointly with the tube lens TL in an intermediate plane ZB of the microscope unit. From there the light arrives in turn via the scanners SX/SY and the relay optics RL at the inlet 2 of the MDB. Since the light emitted by the specimen is usually unpolarized, it is separated into two orthogonal polarization directions Pol 1 and Pol 2 at the beam splitter P 2 . If, for example, fluorescent light is excited in the specimen, then because of the Stokes shift the spectrum of the light is spectrally shifted in comparison to the excitation light. Therefore, the AOTF S does not rotate the polarization in the areas a. and b.
  • the optics O e.g. a microscope objective
  • the element PO 3 is constructed as a mirror. Therefore, the fluorescent light reaches the outlet 4 . However, the backscattered unpolarized excitation light reaches outlet 5 , because the polarization is rotated, according to the adjustment of the excitation light, by the acoustic wave in the AOTF S.
  • the light of the specimen which reaches the outlet 4 of the MDB, is focused by means of imaging optics PO through a confocal diaphragm PH, so that the detection light, occurring outside of the focus, is suppressed.
  • the diaphragm can be dispensed with.
  • a detector DE 1 Behind the confocal diaphragm there is a detector DE 1 , which detects the light radiation excited in the specimen.
  • an emission filter (dichroic filter) F can be swiveled in for additionally suppressing the excitation light backscattered by the specimen, or for limiting the spectral detection area.
  • the polarization of the emitted light of the specimen is supposed to be detected (e.g. when determining fluorescence anisotropy), this can be carried out with two detectors.
  • PO 3 is constructed as a polarizer; and another detector DE 2 is disposed at the outlet 5 .
  • a lambda/2 plate L/2 is disposed between PO 3 and S; the polarization is rotated by 90 deg.
  • the respective polarization can be composed of two components, which are polarized orthogonally to one another. The two orthogonally polarized components are separated with DE 1 and DE 2 . Then the respective polarization can be derived by forming the ratio of the signals of the detectors DE 1 and DE 2 .
  • the backscattered or reflected excitation light of the specimen which travels through the outlet 5 of the MDB, can also be focused through a confocal diaphragm (PH) by means of imaging optics (PO), thus suppressing the detection light occurring outside of the focus.
  • a detector Behind the confocal diaphragm there is a detector (DE 2 ), which detects the excitation radiation, backscattered by the specimen.
  • the emission filter F ceases to be applicable.
  • FIG. 11 shows in schematic form another design of the inventive arrangement for a laser scanning microscope LSM in the X-Z plane, in which another light source LQ 2 , which does not run through the MDB 1 , is coupled.
  • the outlet 6 exhibits another monitor diode M 1 . If the excitation radiation is coupled not only in one polarization direction Pol 1 , but also in the polarization direction Pol 2 , then M 1 measures the coupled power. If the measurement signal M 1 diverges from a desired value, then the AOTF S can be actuated correspondingly in such a manner that another commensurate desired value is set at M 2 . Owing to this adjustment, fluctuations in the coupling efficiency, e.g., in a glass fiber, which is located between the light source LQ and the input 1 of the MDB, can be compensated for. In the case of polarization-achieving glass fibers the coupling efficiency and, thus, the light power, coupled in the direction of the specimen, can be influenced by disadjusting the coupling into the glass fiber or by coupling into different polarization directions.
  • the coupling of the light sources by the AOTF S may or may not be desired.
  • These light sources LQ 2 can be combined with a conventional beam splitter MDB 2 between the outlet 2 and the first scanner, e.g. SX, with the light sources LQ 1 , which travel through the AOTF S.
  • the detection is usually carried out in the wavelength range between 400 and 800 nm, i.e., for example, through the AOTF S or with detectors, according to the prior art.
  • the light which is emitted by the specimen and which impinges on the outlet 4 of the MDB 1 , can be split, according to the prior art, with dichroic beam splitters NFT into different confocal detectors (e.g. DE 1 and DE 2 ).
  • outlets can be also be exchanged accordingly.
  • the MDB is also suitable for scanning regions of special interest ROI (see EP 977069 A2). See FIG. 10 .
  • the laser light of specific wavelength and output is unblocked only for specific regions, which are selected beforehand by the user.
  • the wavelength or the adjustment of the excitation output is switched over by means of actuating in a suitable manner the AOTF S, with the result that the polarization state is changed accordingly.
  • the outlet ( 2 ) of the first MDB is coupled into the inlet ( 1 ) of the second MDB (see simplified schematic FIG. 12 ). Therefore, two light source modules (LQ 1 and LQ 2 ) can be coupled, for example, in a joint specimen beam path.
  • the function of the illustrated scanners can also be replaced with a corresponding scan table (object scanner).

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US20160246062A1 (en) * 2009-11-02 2016-08-25 Olympus Corporation Beam splitter apparatus, light source apparatus, and scanning observation apparatus
US20190346668A1 (en) * 2018-04-30 2019-11-14 University Of Central Florida Research Foundation, Inc. Multiple inclined beam line-scanning imaging apparatus, methods, and applications
US10795138B2 (en) 2015-10-07 2020-10-06 Deutches Krebsforschungszentrum Fluorescence microscope instrument comprising an actively switched beam path separator
US11036037B2 (en) * 2016-11-12 2021-06-15 The Trustees Of Columbia University In The City Of New York Microscopy devices, methods and systems
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EP3538941A4 (fr) 2016-11-10 2020-06-17 The Trustees of Columbia University in the City of New York Procédés d'imagerie rapide de grands échantillons à haute résolution
CN112711130B (zh) * 2020-10-31 2022-02-11 浙江大学 基于电光调制技术的相位调制荧光差分显微成像方法和装置
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EP2157472A1 (fr) * 2007-05-31 2010-02-24 Nikon Corporation Filtre accordable, dispositif de source de lumière et dispositif de mesure de distribution spectrale
US20100141950A1 (en) * 2007-05-31 2010-06-10 Nikon Corporation Tunable filter, light source apparatus, and spectral distribution measuring apparatus
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US20160246062A1 (en) * 2009-11-02 2016-08-25 Olympus Corporation Beam splitter apparatus, light source apparatus, and scanning observation apparatus
US10795138B2 (en) 2015-10-07 2020-10-06 Deutches Krebsforschungszentrum Fluorescence microscope instrument comprising an actively switched beam path separator
US11630292B2 (en) 2016-05-13 2023-04-18 Leica Microsystems Cms Gmbh Optical scanning microscope and examination method
US11036037B2 (en) * 2016-11-12 2021-06-15 The Trustees Of Columbia University In The City Of New York Microscopy devices, methods and systems
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DE102005020545A1 (de) 2006-11-09
US7872799B2 (en) 2011-01-18
US20090257107A1 (en) 2009-10-15
JP2006313357A (ja) 2006-11-16
US20080088907A1 (en) 2008-04-17

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