EP2195612A2 - Fluoreszenzlichtmikroskopisches messen einer probe mit rotverschobenen stokes-linien - Google Patents
Fluoreszenzlichtmikroskopisches messen einer probe mit rotverschobenen stokes-linienInfo
- Publication number
- EP2195612A2 EP2195612A2 EP08837241A EP08837241A EP2195612A2 EP 2195612 A2 EP2195612 A2 EP 2195612A2 EP 08837241 A EP08837241 A EP 08837241A EP 08837241 A EP08837241 A EP 08837241A EP 2195612 A2 EP2195612 A2 EP 2195612A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- light
- wavelength
- optical fiber
- pulses
- fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- 239000000835 fiber Substances 0.000 claims abstract description 51
- 238000001228 spectrum Methods 0.000 claims abstract description 40
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 29
- 238000001069 Raman spectroscopy Methods 0.000 claims abstract description 14
- 239000013307 optical fiber Substances 0.000 claims description 51
- 238000000034 method Methods 0.000 claims description 40
- 230000005284 excitation Effects 0.000 claims description 15
- 230000003287 optical effect Effects 0.000 claims description 12
- 230000001960 triggered effect Effects 0.000 claims description 6
- 238000003384 imaging method Methods 0.000 claims description 3
- 230000001360 synchronised effect Effects 0.000 claims description 3
- 230000009022 nonlinear effect Effects 0.000 claims 2
- 238000000926 separation method Methods 0.000 abstract 1
- 238000000386 microscopy Methods 0.000 description 13
- 230000000694 effects Effects 0.000 description 7
- 230000003595 spectral effect Effects 0.000 description 6
- 239000004038 photonic crystal Substances 0.000 description 4
- 238000010870 STED microscopy Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000000799 fluorescence microscopy Methods 0.000 description 3
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 239000010936 titanium Substances 0.000 description 2
- 229910052719 titanium Inorganic materials 0.000 description 2
- 238000001429 visible spectrum Methods 0.000 description 2
- 208000025174 PANDAS Diseases 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
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- 230000002349 favourable effect Effects 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
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Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
-
- 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
- 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
- G01N21/65—Raman scattering
-
- 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
- G01N21/65—Raman scattering
- G01N2021/653—Coherent methods [CARS]
- G01N2021/655—Stimulated Raman
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
- H01S3/00—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range
- H01S3/30—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range using scattering effects, e.g. stimulated Brillouin or Raman effects
- H01S3/302—Lasers, i.e. devices using stimulated emission of electromagnetic radiation in the infrared, visible or ultraviolet wave range using scattering effects, e.g. stimulated Brillouin or Raman effects in an optical fibre
Definitions
- the invention relates to a method for fluorescence light microscopic measurement of a sample having the features of the preamble of independent claim 1 and to a fluorescence light microscope having the features of the preamble of independent claim 16.
- fluorescence light microscopy In fluorescence light microscopy, light of different wavelengths is required in order to excite different fluorescent dyes for fluorescence in order to deliberately deplete different fluorescent dyes (for example in STED fluorescence microscopy) in order to convert different fluorescent dyes into a dark state in a targeted manner (for example in US Pat GSD fluorescence light microscopy) or on the one hand to excite the same dye in overlapping, but different spatial areas on the one hand to fluorescence and on the other hand by stimulated emission again to de-excite or in a dark state.
- STED fluorescence microscopy in order to deliberately deplete different fluorescent dyes
- a targeted manner for example in US Pat GSD fluorescence light microscopy
- several different monochromatic light sources can be used to provide the light of different wavelengths.
- the monochromatic light sources suitable for this purpose are generally high-quality lasers since the light is required in high-intensity pulses, correspondingly constructed fluorescent light microscopes are very costly. For some wavelengths, it is fundamentally difficult to find suitable lasers of sufficient quality. It is known from DE 10 2005 020 003 A1, inter alia, to use a gas laser as light source in fluorescence light microscopy, which has a plurality of usable emission lines or at least can be tuned to a plurality of emission lines. However, gas lasers are expensive to buy and expensive to operate. In addition, the emission lines that can be used with a gas laser at best cover a small spectral range so far that all absorption lines of possible fluorescent dyes having a good cross-section that fall within this spectral range can be addressed.
- a fluorescent light microscope is known in which light of a certain wavelength is provided for transferring a fluorescent dye in a sample from one state to another state by light of a different wavelength from a laser, in particular a mode-locked titanium sapphire Laser, which emits at 800 nm wavelength, is coupled into an optical element that distributes the intensity of the coupled light of a wavelength to a continuous spectrum having smaller and larger wavelengths than the coupled light and which is referred to as supercontinuum.
- the optical element may, for example, be an optical fiber fiber tapering over a length of 30 mm to 90 mm in cross-section (a so-called “tapered fiber”), which has a total length of 1 m, or a microstructured optical fiber with photonic band gap (US Pat. "photonic band gap”), which is also called photonic crystal fiber ("photonic crystal fiber”).
- a photonic crystal fiber the photonic band gap is created by a honeycomb-like microstructure around a very small fiber core of only about 2 microns in diameter.
- the typical length of a photonic crystal fiber (PCF) is 38 cm.
- a spectral distribution in the form of a supercontinuum makes it possible to select light of any wavelength for transferring a fluorescent dye from one state to another from the supercontinuum.
- the available light power at each wavelength of the supercontinuum is extremely reduced compared to the output power of the exciting laser.
- a considerable effort is required to provide a supercontinuum with an at least approximately homogeneous intensity distribution and in particular a temporally stable intensity distribution.
- the development of corresponding fluorescent light microscopes from the patent application of the idea to commercial products took more than five years.
- the corresponding fluorescent light microscopes are expensive, which is, inter alia, the titanium sapphire laser with the necessary output power and the PCF used in the commercial product.
- DE 103 47 712 A1 discloses a light source for fluorescence microscopy, including STED microscopy, in which the wavelength can be influenced by means of a photonic fiber or an optical parametric oscillator (OPO).
- OPO optical parametric oscillator
- the desired wavelength is generated by a non-linear-optical 3-wave frequency conversion process in which the irradiated frequency of the pump wave is split into two frequencies (signal and idler).
- a Raman amplifier arrangement based on a standard single-mode fiber is known in which Stokes waves are generated by means of stimulated Raman scattering of a high-power optical pump signal in order to amplify an incoming optical signal.
- EP 1 662 296 A1 describes a device with which incident light is spectrally split based on dispersion and polarization effects. This device is also proposed as a light source for use in STED microscopy, wherein light is separated from an incident laser beam of such a wavelength that corresponds exactly to the wavelength of the fluorescent light of interest from a sample, so that this light for stimulation by stimulated emission particularly suitable while the remaining light of the laser beam is used as the excitation light.
- WO 03/016781 A2 a device for generating light in a desired wavelength range is known, with which the excitation of a sample in an even smaller range than in STED microscopy should be possible.
- the light emission of the device is based on photoinduced excitation of surface plasmons of a metal layer.
- the invention is based on the object, a method for fluorescence light microscopic measurement of a sample having the features of the preamble of the independent claim
- Claim 16 show, in which without great and costly effort and without - A -
- Stability problems light of different wavelengths is available to convert different fluorescent dyes from one state to another state.
- the object is achieved by a method for fluorescence light microscopic measurement of a sample having the features of independent claim 1 and by a fluorescent light microscope having the features of independent claim 16.
- the dependent claims 2 to 15 relate to preferred embodiments of the new method, while the dependent claims 17 to 30 are directed to preferred embodiments of the new fluorescent light microscope.
- the optical fiber is selected and the light is coupled into the optical fiber with such intensity that stimulates Raman scattering in the optical fiber to such an extent that the light spectrum adjacent to a line of the coupled wavelength is at least has a redshifted Stokes line whose intensity half-width is less than half its distance to the line of the light spectrum adjacent to it in the blue; and the particular wavelength for transferring the fluorescent dye is selected from one of the red-shifted Stokes lines.
- the new method utilizes the effect of stimulated Raman scattering in an optical fiber which, with intensity of the coupled light tuned to the length of the optical fiber, results in the formation of red-shifted Stokes lines adjacent to the line of injected light. At least in the new method, such a Stokes line is generated, wherein the number of Stokes lines increases with increasing intensity of the coupled-in light and increasing length of the optical fiber.
- the Stokes lines with the largest wavelengths, that is, the most red-shifted Stokes lines are less sharply formed. However, even the most red-shifted Stokes line has such a low half-width that over a small wavelength range of a few nanometers it comprises a high percentage of the intensity of the coupled-in light.
- the most red-shifted Stokes line between this and the line of the Coupled light lying further Stokes lines are sharper. Each of them meets the criterion given above in terms of their full width at half maximum and, over a band of a few nanometers, provides a few percent of the intensity of the injected light.
- the distance of the red-shifted Stokes lines from each other or to the line of the coupled-in light is 10-20 nm, typically 12-15 nm, when the wavelength of the coupled-in light is about 500 nm. The exact value depends on the material and structure of the optical fiber and the wavelength of the injected light.
- the Stokes lines are so close together that any fluorescent dye whose relevant absorption line falls between the wavelength of the injected light and the wavelength of the most red-shifted Stokes line can be served with one or even more of the Stokes lines.
- a large portion of the intensity of the coupled-in light is stably available over a small wavelength range within the half-width of the Stokes lines, that is, well discriminable, the position of the Stokes lines and their relative intensity component on the light spectrum being stable without special measures would have to be taken.
- Advantages of the new method are not present until there are several Stokes lines in the light spectrum emerging from the optical fiber. Already a single Stokes line next to the line of the coupled light doubles the possibilities of using light from the emerging light spectrum.
- red-shifted Stokes lines can be generated at regular intervals by means of stimulated Raman scattering in a single mode fiber.
- Agrawal, Ch. 8: Stimulated Raman Scattering (Agrawal, Govind P., Nonlinear Fiber Optics, 2nd ed., San Diego, CaNf., 1995).
- Stimulated Raman Scattering (Agrawal, Govind P., Nonlinear Fiber Optics, 2nd ed., San Diego, CaNf., 1995).
- fluorescent light microscopes have been developed at great expense, in which a light spectrum is generated with a supercontinuum.
- the new method has significant advantages in providing a much higher light intensity at the wavelengths of each Stokes line.
- the supposed absence of light with wavelengths between the individual Stokes lines or between the first Stokes line and the line of the coupled-in light is of no greater importance because the lines are sufficiently close to one another. Added to this are the stability of the position of the Stokes lines and their relative intensity. Retrospective, it is therefore It is surprising why, in practical development, the well-known effect of stimulated Raman scattering as the basis for a light source with several wavelengths in fluorescence light microscopy has been completely ignored.
- the optical fiber does not have to be a complex microstructured or locally strongly tapered fiber in the new method. Rather, it is preferably a conventional single mode fiber with at least approximately constant diameter or at least no pronounced local taper and no microstructure around a fiber core of minimum diameter.
- the single mode fiber used in the new method is comparatively long. This is especially true when several or even many red-shifted Stokes lines are to be generated in order to provide light over a wider spectral range for the transfer of fluorescent dyes from one state to another state.
- the single-mode fiber may have a cut-off wavelength that is not far on the blue side, e.g. B. less than 70 nm, away from the wavelength of the injected light.
- the minimum length of a conventional single-mode fiber in the new process is 9 m. In order to obtain several redshifted Stokes lines, however, a minimum length of 19 m is preferred. In order for the number of usable redshifted Stokes lines to be on the order of 10, a single mode fiber length of about 30 meters or more is useful. With 10 red-shifted Stokes lines, a wavelength range of about 150 nm is covered by the entire light spectrum at the output of the single-mode fiber, which has the wavelength of the coupled-in light as a further line.
- the optical fiber in the new process is a microstructured fiber, its length should be kept as short as possible, since such optical fibers have a very high length-dependent attenuation unlike conventional single-mode fiber.
- the optical fiber is still a microstructured fiber in the new process, it is preferred to use a gas-filled hollow core in which the choice of gas or its composition in the hollow core allows the redshift, ie the Stokes line, to be influenced.
- a gas-filled hollow core in which the choice of gas or its composition in the hollow core allows the redshift, ie the Stokes line, to be influenced.
- it can be used to produce only one Stokes line, for which a comparatively short length is sufficient.
- the two-line light spectrum emerging from the microstructured fiber can then be coupled into a conventional single-mode fiber to form additional red-shifted Stokes lines to each of these lines.
- the intensity of the light of the other wavelength coupled into the optical fiber should be at least 50 watts peak power, which may be sufficient for the formation of a Stokes line. To generate several or even many Stokes lines, the intensity should reach 500 watts and preferably at least 1000 watts peak power.
- the peak power value required to produce a given number of Stokes lines for a given optical fiber also depends on the diameter of the optical fiber. This determines, together with the peak power, the intensity of the coupled-in light in the optical fiber.
- pulses of 0.5-5 ns duration are coupled into the optical fiber.
- the duration of the pulses of the coupled-in light in the new method corresponds approximately to the duration of the pulses of the light emerging from the single-mode fiber.
- Pulses of 0.5-5 ns duration, especially pulses of 1-3 ns duration are of particular interest for the transfer of fluorescence dyes between two states in fluorescence light microscopy, if such transfers are to be driven to saturation, which is the case on several occasions.
- the light spectrum in the novel process may be at least 5, preferably at least 7, and most preferably at least 10 redshifted Stokes lines. Even with even more red-shifted Stokes lines, each of these Stokes lines has an intensity component of several percent of the intensity of the coupled-in light.
- the Stokes lines are red-shifted. That is, they have a longer wavelength than the light coupled into the optical fiber. In other words, the other wavelength of the injected light should be at the blue end of the visible spectrum.
- the other wavelength of the injected light should be at the blue end of the visible spectrum.
- Such frequency-doubled lasers can be constructed as low-cost solid-state lasers due to the laser fundamental frequency in the range of 1 ⁇ m.
- the light of the other wavelength can be provided in particular with a so-called microchip laser.
- This is a low-cost available solid-state laser type, which may be, for example, a frequency-doubled Nd: YAG laser.
- a frequency doubled Nd: YAG laser typically has an emission wavelength of 532 nm
- microchip lasers that emit at other wavelengths in a range of 450-600 nm, especially in a range between 500 and 550 nm. This can be used to influence the position of the individual redshifted Stokes lines.
- Microchip lasers are particularly well suited for emission of pulses of about 1-3 ns duration. This represents an optimum duration of the pulses for use in high-resolution fluorescence light microscopy.
- the pulse repetition rate in a conventional microchip laser can be slightly lower at 1-100 KHz than is desirable for fluorescence light microscopy, where a pulse repetition rate in the megahertz range is preferred. However, a pulse repetition rate of 1 MHz can also be achieved with microchip lasers of sufficient peak power.
- Another possibility is to divide the emerging from the optical fiber light pulses of one, for the transfer of the respective fluorescent dye from its one in its other state required wavelength and merge staggered again. The person skilled in the art, the necessary techniques for this are known. This includes spectrally fanning out a single Stokes line and part of the spectral ones Delaying components prior to recombining the spectral components by half the time interval of the pulses.
- Another way to increase the pulse repetition rate of the light of interest at one wavelength, which has one wavelength of tolerance, is to couple different non-simultaneous light pulses of different wavelengths into the same or other optical fiber and Stokes lines from the different light pulses as light pulses of one wavelength for transferring the fluorescent dye from one state to the other state. If the two different wavelengths, with which the non-simultaneous light pulses are coupled into the optical fiber, the distance of the Stokes lines, even light pulses of exactly one wavelength can be generated in this way with increased repetition rate of the pulses. However, if the Stokes lines are produced in different single-mode fibers, they must at least slightly differ in wavelength in order to be aligned with the same polarization on a matching optical axis can.
- an excitation light source in particular a laser diode, which provides excitation light for the respective fluorescent dye in synchronism with the non-pulses of the one wavelength with which the fluorescent dye is locally de-excitation-triggered, this can occur when coupling not coincident light pulses of different wavelengths
- the same or other optical fiber (s) take place without these non-simultaneous light pulses must be synchronized with each other, because a laser diode can be clocked easily in the high megahertz range.
- the two light sources for the non-simultaneous light pulses of different wavelengths can therefore travel freely relative to each other.
- the invention can also be used in conventional fluorescence light microscopy, but also in other techniques of fluorescence light microscopy with an increase in spatial resolution by switching of fluorescent dyes between different states, such as, for example, GSD fluorescence light microscopy and RESOLFT fluorescent light microscopy.
- a particularly interesting field of application of the new method is the so-called Fluorescence Lifetime Imaging (FLIM), in which fluorescence lifetime images of the sample in the time or frequency domain are generated from the fluorescent light from the sample.
- FLIM Fluorescence Lifetime Imaging
- the new method easily provides excitation light of various, sufficiently finely graded wavelengths to operate Fluorescence Lifetime Imaging on a variety of colors.
- the optical fiber is selected and the laser couples light into the optical fiber at such an intensity that Raman scattering is stimulated in the optical fiber to such an extent that the light spectrum has at least one red-shifted Stokes line in addition to the coupled wavelength whose intensity half-width is less than half its distance from the line of the light spectrum adjacent to it in the blue; and the wavelength-selective means, but the light of the particular wavelength for transferring the fluorescent dye from one of the red-shifted Stokes lines.
- a frequency increaser combining different light pulses to increase the pulse repetition rate of light of the one wavelength of interest may have a dispersive optical element for this combination.
- suitable dispersive optical elements include prisms and prism arrays.
- acousto-optic filters which are used in the reverse direction in order to bring together light pulses of only slightly different wavelengths on one optical axis are particularly preferred.
- Fig. 1 schematically shows the structure of a first embodiment of a fluorescent light microscope according to the present invention.
- Fig. 2 shows a spectrum of light available in the fluorescent light microscope of Fig. 1.
- FIG. 3 shows a schematic representation of another embodiment of a fluorescence light microscope according to the invention.
- FIG. 4 shows the parts of a fluorescence light microscope according to the invention which are essential for a variant of the fluorescence light microscope according to FIG. 3.
- the fluorescence light microscope 1 outlined in FIG. 1 is used for fluorescence light microscopic measurement of a sample 2 in which a structure of interest is labeled with a fluorescent dye.
- the fluorescent dye is excited with excitation light 3 by a laser diode 4 in the focal region of an objective 5 to fluorescence.
- Fluorescence light 6 emitted therefrom by the fluorescent dye from the focus region is detected in a spatially resolving manner by a detector 7, that is to say in association with a specific location of the sample 2.
- the fluorescent dye in the sample 2 is deoxidized.
- the wavelength of the depletion light 8 is greater than the wavelength of light 10 of a laser 15 formed as a microchip laser 11, which is coupled into an optical fiber 13 in the form of spaced-apart pulses.
- the microchip laser 11 is a frequency doubled laser 15, which emits in the blue region of the visible spectrum.
- One of the Stokes lines is selected with a wavelength-selective element 14 and forms the de-excitation light 8.
- FIG. 2 outlines the light spectrum 17 emerging from the single-mode fiber 12 in the fluorescence light microscope 1.
- this light spectrum 17 was produced by irradiating pulses of a wavelength of 532 nm (frequency-doubled Nd: YAG) and a pulse duration of 1.5 ns and a pulse repetition rate of 7 kHz at 2OmW average power and 1.9 kW peak power in a standard single-mode optical fiber ( Schaefter and Kirchhoff, cut-off wavelength approx. 470 nm, mode field diameter (core diameter) approx. 5 ⁇ m, numerical aperture 0.1 1, polarization-preserving panda fiber) of 30 m length.
- the light spectrum 17 has a first line 18 of the light 10 coupled into the single-mode fiber 12 at the wavelength 532 nm, at which the microchip laser 1 1 emitted. This is followed by a plurality of red-shifted Stokes lines 19 to 28, each comprising a few percent of the total intensity of the light spectrum 17. Although the most red-shifted Stokes lines 26 to 28 have an increasing half-width. However, they also cover several percent of the total intensity of the light spectrum 17 over a range of a few nanometers.
- the distance 29 between the red-shifted Stokes lines 19 to 28 and the first red-shifted Stokes lines 19 in relation to the line 18 is about 12-15 nm in each case. That is, in the fluorescent light microscope of FIG. 1, the de-excitation light 8 can be detected in steps of 12-15 nm over the entire region of the spectrum 17 be adjusted in terms of its wavelength. In this case, because each of the Stokes lines has a half-width 30, which is smaller than half the distance 29, with the selected Stokes line large portions of the intensity of the light 10 from the laser 1 1 is used for the excitation light 8. In a conventional Mikrochiplaser 1 1, these proportions are so large that they can be split to double the rate of the pulses of the laser 11 and directed at a time offset to the sample 2.
- FIG. 3 outlines another way of increasing the rate of the pulses of depletion light 8 versus the rate of pulses of a single microchip laser 11.
- two microchip lasers 1 1 'and 1 1 are provided here, which emit light 10' or 10" with different wavelengths.
- the light 10 'or 10 "of both microchip lasers 1 1' and 1 1" is connected to a beam splitter 31, the z. B. may be formed as an acousto-optical filter 32, merged on an optical axis and coupled into the single-mode fiber 12.
- the distance of the wavelength of the light 10 'from the wavelength of the light 10 is about the same as the distance between the Stokes lines 19 to 28 shown in FIG.
- the de-excitation light 8 has a doubled pulse rate behind the wavelength-selective element 14, which here is a narrow-band color filter 34.
- the laser diode 4, which controls the excitation light 8 is thereby triggered as a function of both microchip lasers 1 1 'and 11 ", which can travel freely relative to one another, that is, they need not be synchronized with one another.
- the fluorescent light microscope 1 according to FIG. 3 uses only one single-mode fiber 12 for increasing the pulse rate of the pulses of the depletion light 8
- the subsequent to the phase filter 9 part of the fluorescent light microscope of FIG. 4 is not shown and corresponds to FIG. LIST OF REFERENCE NUMBERS
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Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102007048135A DE102007048135B4 (de) | 2007-10-05 | 2007-10-05 | Fluoreszenzlichtmikroskopisches Messen einer Probe mit rotverschobenen Stokes-Linien |
PCT/EP2008/063171 WO2009047189A2 (de) | 2007-10-05 | 2008-10-01 | Fluoreszenzlichtmikroskopisches messen einer probe mit rotverschobenen stokes-linien |
Publications (1)
Publication Number | Publication Date |
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EP2195612A2 true EP2195612A2 (de) | 2010-06-16 |
Family
ID=40262141
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP08837241A Withdrawn EP2195612A2 (de) | 2007-10-05 | 2008-10-01 | Fluoreszenzlichtmikroskopisches messen einer probe mit rotverschobenen stokes-linien |
Country Status (5)
Country | Link |
---|---|
US (1) | US8039815B2 (de) |
EP (1) | EP2195612A2 (de) |
JP (1) | JP2011503525A (de) |
DE (1) | DE102007048135B4 (de) |
WO (1) | WO2009047189A2 (de) |
Families Citing this family (12)
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DE202009007250U1 (de) | 2009-05-20 | 2009-11-26 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Feldveränderungsmittel zur Erzeugung komplementärer Lichtintensitätsmuster |
DE102009056058B4 (de) * | 2009-11-25 | 2021-08-12 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Vorrichtung zur Erzeugung von Anregungsstrahlung für selbstkalibrierende faseroptische Raman-Sensoren, Messeinrichtung zum Erfassen der Temperatur eines Messobjekts und Raman-Sensoranordnung |
DE102010037190B4 (de) | 2010-08-27 | 2015-11-26 | Leica Microsystems Cms Gmbh | Vorrichtung zum zeitlichen Verschieben von Weißlichtlaserpulsen |
FR2966292B1 (fr) * | 2010-10-18 | 2013-01-18 | Centre Nat Rech Scient | Methode et dispositif d'emission laser pour l'analyse spectroscopique d'un echantillon |
JP2012237714A (ja) * | 2011-05-13 | 2012-12-06 | Sony Corp | 非線形ラマン分光装置、顕微分光装置及び顕微分光イメージング装置 |
CN102564642B (zh) * | 2012-02-21 | 2013-08-07 | 中国计量学院 | 融合拉曼放大效应的光纤拉曼频移器的全分布光纤传感器 |
AU2013352075B2 (en) * | 2012-11-28 | 2018-03-01 | Trustees Of Princeton University | Detection systems and methods using coherent anti-Stokes Raman spectroscopy |
DE202013006817U1 (de) * | 2013-07-30 | 2014-10-31 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Gepulste Laserlichtquelle für die Fluoreszenzanregung |
JP6103008B2 (ja) * | 2015-09-09 | 2017-03-29 | ソニー株式会社 | 非線形ラマン分光装置、顕微分光装置及び顕微分光イメージング装置 |
JP6729878B2 (ja) | 2016-04-05 | 2020-07-29 | ウシオ電機株式会社 | 多光子励起用スーパーコンティニウム光生成光源、多光子励起用スーパーコンティニウム光生成方法、多光子励起蛍光顕微鏡及び多光子励起方法 |
DE102021128556A1 (de) | 2021-11-03 | 2023-05-04 | Amphos GmbH | STED-Mikroskop |
DE102021005684A1 (de) | 2021-11-16 | 2023-05-17 | Jörn Volkher Wochnowski | STED-Verfahren mit Hohllichtwellenleitern |
Family Cites Families (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5491344A (en) * | 1993-12-01 | 1996-02-13 | Tufts University | Method and system for examining the composition of a fluid or solid sample using fluorescence and/or absorption spectroscopy |
JP3390755B2 (ja) * | 1998-09-29 | 2003-03-31 | 科学技術振興事業団 | 波長可変短パルス光発生装置及び方法 |
DE10012881B4 (de) * | 2000-03-16 | 2008-09-04 | Nokia Siemens Networks Gmbh & Co.Kg | Ramanverstärkeranordnung |
US7190705B2 (en) * | 2000-05-23 | 2007-03-13 | Imra America. Inc. | Pulsed laser sources |
US6885683B1 (en) * | 2000-05-23 | 2005-04-26 | Imra America, Inc. | Modular, high energy, widely-tunable ultrafast fiber source |
DE20122791U1 (de) * | 2000-06-17 | 2007-11-29 | Leica Microsystems Cms Gmbh | Scanmikroskop |
US6818907B2 (en) * | 2000-10-17 | 2004-11-16 | The President And Fellows Of Harvard College | Surface plasmon enhanced illumination system |
DE60204864D1 (de) * | 2001-04-11 | 2005-08-04 | Univ Southampton Highfield | Quellen und verfahren zur erzeugung von optischen pulsen |
JP4212471B2 (ja) * | 2001-07-03 | 2009-01-21 | プレジデント アンド フェローズ オブ ハーバード カレッジ | 偏光コヒーレントアンチストークスラマン散乱顕微鏡使用のシステム及び方法 |
US6870663B2 (en) * | 2001-08-28 | 2005-03-22 | Nippon Telegraph And Telephone Corporation | Wavelength tunable light source and pulse light source |
DE102004009066B4 (de) * | 2003-02-25 | 2007-01-25 | Toptica Photonics Ag | Vorrichtung zur Erzeugung abstimmbarer Lichtimpulse |
US7224518B2 (en) * | 2003-02-25 | 2007-05-29 | Toptica Photonics Ag | Fiber-optic amplification of light pulses |
US7064824B2 (en) * | 2003-04-13 | 2006-06-20 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | High spatial resoulution imaging and modification of structures |
JP2005084266A (ja) * | 2003-09-05 | 2005-03-31 | Kawasaki Heavy Ind Ltd | 光制御装置および光制御方法 |
DE10347712A1 (de) * | 2003-10-14 | 2005-05-12 | Leica Microsystems | Rastermikroskop |
WO2005094275A2 (en) * | 2004-03-25 | 2005-10-13 | Imra America, Inc. | Optical parametric amplification, optical parametric generation, and optical pumping in optical fibers systems |
DE102005020003B4 (de) * | 2005-04-27 | 2007-10-11 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Fluoreszenzmikroskop |
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2008
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- 2008-10-01 JP JP2010527448A patent/JP2011503525A/ja not_active Withdrawn
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2010
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JP2011503525A (ja) | 2011-01-27 |
WO2009047189A2 (de) | 2009-04-16 |
US8039815B2 (en) | 2011-10-18 |
DE102007048135A1 (de) | 2009-04-16 |
US20100187438A1 (en) | 2010-07-29 |
DE102007048135B4 (de) | 2012-02-16 |
WO2009047189A3 (de) | 2009-09-03 |
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