US20060097188A1 - Method for microscopy, and microscope - Google Patents
Method for microscopy, and microscope Download PDFInfo
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- US20060097188A1 US20060097188A1 US11/301,439 US30143905A US2006097188A1 US 20060097188 A1 US20060097188 A1 US 20060097188A1 US 30143905 A US30143905 A US 30143905A US 2006097188 A1 US2006097188 A1 US 2006097188A1
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- 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
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
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- 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
- G02B21/0064—Optical details of the image generation multi-spectral or wavelength-selective arrangements, e.g. wavelength fan-out, chromatic profiling
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- G—PHYSICS
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- G02B21/008—Details of detection or image processing, including general computer control
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- G—PHYSICS
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- 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
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- G01J2003/1282—Spectrum tailoring
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- 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
- G01J3/0229—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using masks, aperture plates, spatial light modulators or spatial filters, e.g. reflective filters
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- G—PHYSICS
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- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
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- 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
Definitions
- the invention concerns a method for microscopy.
- the invention furthermore concerns a microscope and a confocal scanning microscope.
- CARS coherent anti-Stokes Raman scattering
- STED stimulated emission depletion
- the lateral edge regions of the focus volume of the excitation light beam are illuminated with a light beam of another wavelength, called the stimulation light beam, that is emitted by a second laser, so that the specimen regions excited there by the light of the first laser are brought back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, resulting overall in improved resolution.
- the fluorescent photons attributable to a two-photon or multi-photon excitation process are detected.
- the probability of a two-photon transition depends on the square of the excitation light power level, and therefore occurs with high probability at the focus of the scanning illuminating light beam, since the power density is highest there.
- a further advantage of multi-photon excitation especially in confocal scanning microscopy lies in the improved bleaching behavior, since the specimen bleaches out only in the region of sufficient power density, i.e. at the focus of an illuminating light beam. Outside that region, in contrast to one-photon excitation, alnost no bleaching takes place.
- a specimen In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen.
- the focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction.
- Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements.
- the power level of the light coming from the specimen is measured as a function of the position of the scanning beam.
- the positioning elements are usually equipped with sensors to ascertain the present mirror position.
- a confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light.
- the illuminating light is coupled in via a beam splitter.
- the fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located.
- Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image.
- a three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.).
- the specimen stage or the objective is displaced after a layer has been scanned, thus bringing the next layer to be scanned into the focal plane of the objective.
- Spectral influencing of light pulses by amplitude modulation or phase modulation is known from the literature, e.g. from Rev. of Scientific Instruments 71 (5) pp. 1929-1960.
- Spectral modification of the laser pulses is usually used to shorten the pulses, to shape them optimally, or to control optically induced processes.
- the aforesaid methods are disadvantageous in that high light power levels are necessary, resulting on the one hand in great demands on the light source and on the other hand in undesirable damage to the specimen, for example due to bleaching.
- a further object of the invention is to provide a microscope which reliably and efficiently allows an investigation of a specimen exploiting nonlinear processes with reduced specimen impact.
- the invention also provides a microscope having a light source for generating pulsed illuminating light that comprises light from a spectral region, and having at least one detector for detecting the detection light proceeding from a specimen in a detection spectral region, wherein the detection spectral region lies within the spectral region; and the illuminating light contains no light from the detection spectral region having the same polarization properties.
- the invention has the advantage that the method according to the present invention exploits location-dependent optical nonlinearities but makes do with much lower light intensities, the use of the lowest possible light intensities having particular significance especially for biological specimens. Investigation of the specimen to a great depth is also possible.
- An aspect of the method according to the present invention is to influence, certain spectral components (specifically those from the detection spectral region) from the spectrum of ultrashort laser pulses (i.e. preferably picosecond and femtosecond laser pulses); to focus the illuminated light prepared in this fashion onto a specimen volume; and to detect in practically background-free fashion the detection light thereby produced, by nonlinear processes, in the region of the previously removed spectral components.
- the power level and (optionally) spectral distribution of this detection light is used for image production.
- the influencing is a removal of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
- the influencing contains a modification of the polarization state of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
- the modification of the polarization state can encompass, in particular, a rotation of a linear polarization. Rotation of the linear polarization direction makes the detection light in the detection spectral region distinguishable from the illuminating light.
- the influencing encompasses a spectral filtration.
- a spectral filter that removes from the illuminating light the light components of the illuminating light that comprise wavelengths within the detection spectral region.
- the illuminating light contains no light from the detection spectral region.
- a spectral filter is provided that modifies the polarization state of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
- the spectral filtration removes certain frequency regions from the spectrum of the illuminating light in order to create there a spectral window within which detection light produced as a result of nonlinear processes can be detected in background-free fashion.
- a further spectral filter is provided that allows only light of the wavelengths of the detection spectral region to arrive at the detector.
- the further spectral filter is preferably inverse with respect to the spectral filter.
- FIG. 1 shows a microscope according to the present invention
- FIG. 3 shows a further microscope according to the present invention
- FIG. 4 shows a further microscope according to the present invention
- FIG. 6 shows a further microscope according to the present invention.
- FIG. 1 schematically shows a microscope according to the present invention that is embodied as a scanning microscope.
- the optical components for guiding, directing, and focusing illuminating light beam 1 (generated by a pulsed laser 7 ) and detection light beam 3 , and the apparatuses for evaluating the detection light data and displaying an image of the specimen, are not shown in the interest of better clarity. These components are sufficiently familiar to one skilled in the art.
- the microscope contains a spectral filter 5 that removes from illuminating light beam 1 the light components of the illuminating light that comprise wavelengths within the detection spectral region.
- the light is spatially spectrally split using a first grating 9 , and then focused with first lens 11 onto a mask 13 which removes the spectral components that lie within the detection spectral region.
- Grating 9 and first mask 13 are located in the focal planes of lens 11 in a 4f arrangement.
- Mask 13 has transparent and opaque regions. It can be expressed as a static mask but also as a dynamically controllable mask (liquid crystal display, hinged mirror array).
- detection light beam 3 proceeding from specimen 21 is collimated by a condenser 23 and directed to a further spectral filter 25 .
- Further spectral filter 25 is embodied inversely with respect to spectral filter 5 through which illuminating light beam 1 passes; i.e. wherever light previously passed through, the light is now blocked. It contains a third lens 29 and a fourth lens 31 , as well as a third grating 33 and a fourth grating 35 ; also a second mask 37 that is the inverse of first mask 13 .
- FIG. 2 shows several spatial first masks 13 and second masks 37 that can be used in first spectral filter 5 and in second spectral filter 25 , second masks 37 being inverse with respect to first masks 13 .
- the transmitting regions can be limited even further.
- FIG. 3 shows a further microscope according to the present invention. It corresponds analogously, in terms of illumination, to the scanning microscope shown in FIG. 1 ; several detectors 39 , 41 , 43 , 45 arranged behind second mask 37 are provided for detection. A linear detector or an array of detectors (e.g. CCD) could also be used. After spectral splitting using grating 33 , the components of detection light beam 3 that comprise the same wavelength region as the components of illuminating light beam 1 that were removed by first mask 13 strike the several individual detectors 39 , 41 , 43 , 45 . In a particularly simple arrangement, the mask itself can even be omitted. Because the several detectors 39 , 41 , 43 , 45 are used, additional information is obtained as to the intensity with which the nonlinear processes are occurring in the various spectral regions; this can possibly be utilized for differentiated image production.
- a linear detector or an array of detectors e.g. CCD
- second spectral filter 25 at least some of the same optical elements as for first spectral filter 13 , by guiding the light beam through at least some of them a second time.
- etalon 63 is usually embodied as a resonator made up substantially of two semitransparent mirrors 65 , 67 at the spacing of the effective resonator length. It is useful if there is located, in the interior of the resonator, a controllable element 69 with which the effective resonator length can be regulated so that precise adaptation can be performed and with which any drift resulting e.g. from thermal longitudinal expansion can be controlled out.
- An element of this kind could be made of materials whose refractive index can be controlled externally, e.g. liquid crystals or ferroelectric crystals.
Abstract
A method for microscopy includes generating pulsed illuminating light including wavelengths in a spectral region. A detection spectral region within the spectral region is defined. Using a dynamically controllable mask, light components of the illuminating light that comprise wavelengths within the detection spectral region are influenced. A specimen is illuminated with the influenced illuminating light. Detection light proceeding from the specimen within the detection spectral region is detected.
Description
- This is a continuation of application Ser. No. 10/601,804, filed Jun. 23, 2003, which claims priority to German patent application 102 28 374.5. The subject matter of both of these applications is hereby incorporated by reference herein.
- The invention concerns a method for microscopy. The invention furthermore concerns a microscope and a confocal scanning microscope.
- For the investigation of biological specimens, it has been usual for some time to prepare the specimen using optical markers, in particular fluorescent dyes. Often, for example in the field of genetic research, several different fluorescent dyes are introduced into the specimen and become attached specifically to certain specimen constituents. From the fluorescence properties of the prepared specimen conclusions can be drawn, for example, as to the nature and composition of the specimen or the concentration of certain substances within the specimen.
- In scanning microscopy in particular, methods that exploit location-dependent nonlinearities of the specimen are used. This field includes, for example, coherent anti-Stokes Raman scattering (CARS), which is known inter alia from PCT Application WO 00/04352 A1. It must be noted, however, that illuminating light having at least two different illuminating light wavelengths, at high light power levels, is required for this method.
- Another method that makes use of nonlinear effects is so-called STED (stimulated emission depletion) microscopy, known for example from PCT/DE/95/00124. Here the lateral edge regions of the focus volume of the excitation light beam are illuminated with a light beam of another wavelength, called the stimulation light beam, that is emitted by a second laser, so that the specimen regions excited there by the light of the first laser are brought back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, resulting overall in improved resolution.
- In multi-photon scanning microscopy, the fluorescent photons attributable to a two-photon or multi-photon excitation process are detected. The probability of a two-photon transition depends on the square of the excitation light power level, and therefore occurs with high probability at the focus of the scanning illuminating light beam, since the power density is highest there. To achieve sufficiently high light power levels, it is useful to pulse the illuminating light and thereby achieve high peak pulsed light power levels. This technique is known, and is disclosed e.g. in U.S. Pat. No. 5,034,613 “Two-photon laser microscopy” and in German Unexamined Application DE 44 14 940. A further advantage of multi-photon excitation especially in confocal scanning microscopy lies in the improved bleaching behavior, since the specimen bleaches out only in the region of sufficient power density, i.e. at the focus of an illuminating light beam. Outside that region, in contrast to one-photon excitation, alnost no bleaching takes place.
- In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
- In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
- A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To allow acquisition of image data in layers, the specimen stage or the objective is displaced after a layer has been scanned, thus bringing the next layer to be scanned into the focal plane of the objective.
- Spectral influencing of light pulses by amplitude modulation or phase modulation is known from the literature, e.g. from Rev. of Scientific Instruments 71 (5) pp. 1929-1960. Spectral modification of the laser pulses is usually used to shorten the pulses, to shape them optimally, or to control optically induced processes.
- The aforesaid methods are disadvantageous in that high light power levels are necessary, resulting on the one hand in great demands on the light source and on the other hand in undesirable damage to the specimen, for example due to bleaching.
- It is therefore an object of the invention to provide a method for microscopy that reliably and efficiently allows exploitation of nonlinear processes with reduced specimen damage.
- The invention provides a method comprising the following steps:
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- generating pulsed illuminating light that comprises wavelengths which lie in a spectral region;
- defining a detection spectral region that lies within the spectral region;
- influencing the light components of the illuminating light that comprise wavelengths within the detection spectral region;
- illuminating a specimen with the illuminating light;
- detecting the detection light proceeding from the specimen within the detection spectral region.
- A further object of the invention is to provide a microscope which reliably and efficiently allows an investigation of a specimen exploiting nonlinear processes with reduced specimen impact.
- The invention also provides a microscope having a light source for generating pulsed illuminating light that comprises light from a spectral region, and having at least one detector for detecting the detection light proceeding from a specimen in a detection spectral region, wherein the detection spectral region lies within the spectral region; and the illuminating light contains no light from the detection spectral region having the same polarization properties.
- The invention has the advantage that the method according to the present invention exploits location-dependent optical nonlinearities but makes do with much lower light intensities, the use of the lowest possible light intensities having particular significance especially for biological specimens. Investigation of the specimen to a great depth is also possible.
- An aspect of the method according to the present invention is to influence, certain spectral components (specifically those from the detection spectral region) from the spectrum of ultrashort laser pulses (i.e. preferably picosecond and femtosecond laser pulses); to focus the illuminated light prepared in this fashion onto a specimen volume; and to detect in practically background-free fashion the detection light thereby produced, by nonlinear processes, in the region of the previously removed spectral components. The power level and (optionally) spectral distribution of this detection light is used for image production.
- In a preferred embodiment, the influencing is a removal of the light components of the illuminating light that comprise wavelengths within the detection spectral region. In another embodiment, the influencing contains a modification of the polarization state of the light components of the illuminating light that comprise wavelengths within the detection spectral region. The modification of the polarization state can encompass, in particular, a rotation of a linear polarization. Rotation of the linear polarization direction makes the detection light in the detection spectral region distinguishable from the illuminating light.
- In another preferred embodiment, the influencing encompasses a spectral filtration. Provided for this purpose, in an embodiment, is a spectral filter that removes from the illuminating light the light components of the illuminating light that comprise wavelengths within the detection spectral region. In this embodiment, the illuminating light contains no light from the detection spectral region. In another variant, a spectral filter is provided that modifies the polarization state of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
- The spectral filtration removes certain frequency regions from the spectrum of the illuminating light in order to create there a spectral window within which detection light produced as a result of nonlinear processes can be detected in background-free fashion.
- In a preferred embodiment, a further spectral filter is provided that allows only light of the wavelengths of the detection spectral region to arrive at the detector. The further spectral filter is preferably inverse with respect to the spectral filter.
- In an embodiment, the illuminating light is already generated in such a way that the detection spectral region lies within the spectral region, and so that the illuminating light contains no light from the detection spectral region. The detection spectral region or regions can, for example, be the spectral gaps between the equidistant modes of a mode-coupled pulsed laser.
- In another embodiment, the microscope is a scanning microscope, in particular a confocal scanning microscope.
- The subject matter of the invention is depicted schematically in the drawings and will be described below with reference to the Figures, identically functioning elements being labeled with the same reference characters. In the drawings:
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FIG. 1 shows a microscope according to the present invention; -
FIG. 2 shows masks for spectral filters; -
FIG. 3 shows a further microscope according to the present invention; -
FIG. 4 shows a further microscope according to the present invention; -
FIG. 5 shows a further microscope according to the present invention; -
FIG. 6 shows a further microscope according to the present invention. -
FIG. 1 schematically shows a microscope according to the present invention that is embodied as a scanning microscope. The optical components for guiding, directing, and focusing illuminating light beam 1 (generated by a pulsed laser 7) anddetection light beam 3, and the apparatuses for evaluating the detection light data and displaying an image of the specimen, are not shown in the interest of better clarity. These components are sufficiently familiar to one skilled in the art. - The microscope contains a
spectral filter 5 that removes from illuminatinglight beam 1 the light components of the illuminating light that comprise wavelengths within the detection spectral region. For that purpose, the light is spatially spectrally split using afirst grating 9, and then focused withfirst lens 11 onto amask 13 which removes the spectral components that lie within the detection spectral region.Grating 9 andfirst mask 13 are located in the focal planes oflens 11 in a 4f arrangement.Mask 13 has transparent and opaque regions. It can be expressed as a static mask but also as a dynamically controllable mask (liquid crystal display, hinged mirror array). Afterfirst mask 13, the various spectral components of the illuminating light are combined again, by means of a symmetrical arrangement of asecond lens 15 andsecond grating 17, into a common illuminatinglight beam 1. This illuminating light beam is then coupled into the microscope beam path and focused by an objective 19 ontospecimen 21 that is to be examined. The microscope scans, for example, by the fact that one or more mirrors in the beam path are embodied as scanning mirrors, and/or by moving the specimen stage. In the interior ofspecimen 21 at the location of the focus of illuminatinglight beam 1, nonlinear processes such as self-phase modulation, continuum generation, etc. take place, in which new light frequencies are generated that may also be present, inter alia, in the regions filtered out by the previous stop. After passage through the specimen,detection light beam 3 proceeding fromspecimen 21 is collimated by acondenser 23 and directed to a furtherspectral filter 25. Furtherspectral filter 25 is embodied inversely with respect tospectral filter 5 through which illuminatinglight beam 1 passes; i.e. wherever light previously passed through, the light is now blocked. It contains athird lens 29 and afourth lens 31, as well as athird grating 33 and afourth grating 35; also asecond mask 37 that is the inverse offirst mask 13. The components of illuminatinglight beam 1 still present indetection light beam 3 are thereby filtered out, so that ultimately only detection light produced at the specimen focus arrives atdetector 27. The power level of this light provides information, inter alia, about the nonlinear refractive indices at the specimen focus which depend on local conditions inspecimen 21, and is therefore suitable, as the focus is scanned overspecimen 21, as a signal for image-producing methods. -
FIG. 2 shows several spatialfirst masks 13 andsecond masks 37 that can be used in firstspectral filter 5 and in secondspectral filter 25,second masks 37 being inverse with respect tofirst masks 13. The transmitting regions can be limited even further. -
FIG. 3 shows a further microscope according to the present invention. It corresponds analogously, in terms of illumination, to the scanning microscope shown inFIG. 1 ;several detectors second mask 37 are provided for detection. A linear detector or an array of detectors (e.g. CCD) could also be used. After spectral splitting using grating 33, the components ofdetection light beam 3 that comprise the same wavelength region as the components of illuminatinglight beam 1 that were removed byfirst mask 13 strike the severalindividual detectors several detectors - It is also possible to use for second
spectral filter 25 at least some of the same optical elements as for firstspectral filter 13, by guiding the light beam through at least some of them a second time. -
FIG. 4 shows a further microscope according to the present invention. Instead ofgratings first prism 47,second prism 49,third prism 51, andfourth prism 53 are used for spectral splitting and combining. Illuminatinglight beam 1 generated bypulsed laser 7 is linearly polarized.Mask 13 rotates through 90 degrees the polarization direction of those components of illuminatinglight beam 1 that comprise wavelengths from the detection regions. The polarization influence is exerted by way of a suitably patterned and oriented birefringent fixed mask 13 (e.g. patterned λ/2 plate), or also by means of a dynamically controlledmask 13 that can be implemented, for example, using a liquid crystal display. After passage through the specimen, the detection light proceeding from the specimen is filtered through a secondspectral filter 25 in such a way that the illuminating light whose polarization was not rotated by firstspectral filter 5 is completely removed. This is done by the fact that in secondspectral filter 25, by way of a suitablesecond mask 37, the polarization state of the various spectral components is modified in such a way that all components deriving directly frompulsed laser 7 are once again given a common polarization, which is removed from the beam path by means of adownstream polarizer 55. In the concrete exemplary embodiment, those spectral components that had already experienced a polarization change in firstspectral filter 13 are once again rotated 90° in polarization in secondspectral filter 25.Polarizer 55 then removes from the beam all spectral components that have a polarization of 0°. The beam path then, as a rule, contains only light which was produced in the specimen by nonlinear processes, and whose intensity permits conclusions as to the local nonlinear refractive indices of the specimen at the focus and is therefore suitable for image production. In this exemplary embodiment it is also possible to dispense with certain parts of second spectral filter 25 (e.g.fourth lens 31 and fourth prism 53) if, for example, the detector(s) is/are equipped with polarizers and is/are arranged directly behindmask plane 25. -
FIG. 5 shows an embodiment in which the light polarized in the 0° or 90° direction (depending on wavelength) is split upstream from the specimen using apolarization splitter 57, after which the two light components of illuminatinglight beam 1 are focused from opposite directions ontospecimen 21 by afirst objective 59 and afurther objective 61. Here the objective for the one polarization direction is in each case simultaneously the condenser for the other polarization direction. After passage through the specimen and through a polarization rotator 62 (this number has already been assigned to the second objective, including in the Figure), which is embodied as a λ/2plate 63 that preferably rotates the polarization 90°, the two light components of the detection light are combined using the polarizing beam splitter; the light uninfluenced by the specimen is separated, by polarizingbeam splitter 57, from the light later to be detected in such a way that only the light just produced in the specimen is detected indetector 27. - In the exemplary embodiment in
FIG. 6 , the first spectral filter has been omitted. The light ofpulsed laser 7 is made up of lines lying very close together. This occurs in many usual picosecond and femtosecond lasers as an effect of mode coupling. The spectral line spacing usually corresponds here to the pulse frequency of the laser in question; for example, the spectrum of a titanium-sapphire femtosecond laser pulsing at a repetition rate of 80 MHz is made up of individual spectral lines at a spectral spacing of 80 MHz. Gaps in the spectrum are present between the individual spectral lines, so that the spectrum of thispulsed laser 7 is similar to the filtered spectra of the exemplary embodiments discussed previously. If components are present in these spectral regions after an excitation laser of this kind has passed throughspecimen 21, this is attributable to nonlinear processes; as in the case of the previous exemplary embodiments, this can be utilized for image production. Separation of the detection light produced by nonlinear processes from the excitation light can be accomplished, as in the previous exemplary embodiments, by spatial filtration; in this context, the use of monochromators, etc. of course also represents a spatial filtration. Alternatively and in particularly preferred fashion, what is used as secondspectral filter 25 is anetalon 63, which is constituted by afirst mirror 65 and asecond mirror 67 and which removes fromdetection light beam 3 all spectral components within a certain wavelength spacing (as is also possible, in the previous exemplary embodiments, with a suitable first spectral filter). In the case of the mode-coupled laser, the spectral distance within whichetalon 63 absorbs light must correspond exactly to the spectral spacing of the individual laser modes, which substantially means that the length ofetalon 63 must be matched to the effective resonator length of the mode-coupled laser. Since the etalon length is relatively long for the short-pulse lasers commonly in use at present,etalon 63 is usually embodied as a resonator made up substantially of twosemitransparent mirrors controllable element 69 with which the effective resonator length can be regulated so that precise adaptation can be performed and with which any drift resulting e.g. from thermal longitudinal expansion can be controlled out. An element of this kind could be made of materials whose refractive index can be controlled externally, e.g. liquid crystals or ferroelectric crystals. Appropriate regulation of the etalon's resonator length could also be accomplished by way of a movable end mirror. Instead of the resonator length of the etalon, the length of the short-pulse laser resonator could, of course, also be regulated. - The invention has been described with reference to exemplary embodiments. It is self-evident, however, that changes and modifications can be made without thereby leaving the range of protection of the claims below.
Claims (20)
1. A method for microscopy comprising:
generating pulsed illuminating light including wavelengths in a spectral region;
defining a detection spectral region within the spectral region;
influencing, using a dynamically controllable mask, light components of the illuminating light that comprise wavelengths within the detection spectral region;
illuminating a specimen with the influenced illuminating light; and
detecting detection light proceeding from the specimen within the detection spectral region.
2. The method as defined in claim 1 , wherein the dynamically controllable mask includes at least one of a liquid crystal display and a hinged mirror array.
3. The method as defined in claim 1 , wherein the influencing includes a removal of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
4. The method as defined in claim 1 , wherein the influencing includes a modification of the polarization state of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
5. The method as defined in claim 4 , wherein the modification of the polarization state encompasses a rotation of a linear polarization.
6. The method as defined in claim 4 , further comprising modifying the polarization state of light components of the detection light.
7. The method as defined in claim 1 , wherein the influencing encompasses a spectral filtration.
8. The method as defined in claim 1 , wherein a pulsed laser is provided for generating the pulsed illuminating light.
9. The method as defined in claim 1 further comprising allowing, using a further spectral filter, only light of wavelengths of the detection spectral region to arrive at the detector, wherein further spectral filter is at least partially inverse with respect to the spectral filter.
10. A microscope comprising:
a light source configured to generate pulsed illuminating light that includes light from a spectral region;
at least one detector configured to detect detection light proceeding from a specimen in a detection spectral region, the detection spectral region being within the spectral region; and
a spectral filter including a dynamically controllable mask configured to influence light components of the illuminating light that comprise wavelengths within the detection spectral region.
11. The microscope as defined in claim 10 , wherein the dynamically controllable mask includes at least one of a liquid crystal display and a hinged mirror array.
12. The method as defined in claim 10 , wherein the dynamically controllable mask is configured to remove the light components of the illuminating light that comprise wavelengths within the detection spectral region.
13. The method as defined in claim 10 , wherein the dynamically controllable mask is configured to modify the polarization state of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
14. The method as defined in claim 13 , wherein the modification of the polarization state encompasses a rotation of a linear polarization.
15. The method as defined in claim 13 , further comprising a further spectral filter configured to modify the polarization state of light components of the detection light.
16. The microscope as defined in claim 10 , further comprising a further spectral filter configured to allow only light of wavelengths of the detection spectral region to arrive at the detector, wherein the further spectral filter is at least partially inverse with respect to the spectral filter.
17. The microscope as defined in claim 10 , wherein the light source includes a pulsed laser.
18. A microscope comprising:
a light source configured to generate pulsed illuminating light that includes light from a spectral region;
at least one detector configured to detect detection light proceeding from a specimen in a detection spectral region, the detection spectral region being within the spectral region;
a spectral filter configured to remove, from the illuminating light, light components of the illuminating light that comprise wavelengths within the detection spectral region; and
a further spectral filter configured to allow only light of wavelengths of the detection spectral region to arrive at the detector, wherein the further spectral filter is at least partially inverse with respect to the spectral filter.
19. The microscope as defined in claim 18 , further comprising a third spectral filter configured to modify the polarization state of the light components of the illuminating light that comprise wavelengths within the detection spectral region.
20. The method as defined in claim 18 wherein the light source includes a pulsed laser.
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US11/301,439 US20060097188A1 (en) | 2002-06-25 | 2005-12-13 | Method for microscopy, and microscope |
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US7005654B2 (en) | 2006-02-28 |
DE10228374A1 (en) | 2004-01-15 |
US20040065845A1 (en) | 2004-04-08 |
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