Raman spectroscopy illumination and readout system

Abstract

Raman spectroscopy illumination and readout system comprising a measurement beam generation arrangement and a Raman scattering light extraction arrangement, wherein the measurement beam generation arrangement comprises a laser (1), a first aperture (3), a first lens (4), an illumination fiber (6) with end faces (5 and 8), a line filter (9), a microscope with a second lens (10), a second aperture (16), a beam splitter in the form of a dichroic mirror (18) and an objective (19), and wherein the beam path from the laser (1) via the first aperture (3), the first lens (4), the illumination fiber (6), the line filter (9), the microscope with the second lens (10), the dichroic mirror (18) and the objective (19) to a sample (20) can be generated, and wherein the Raman scattering light extraction arrangement comprises the objective (19) of the microscope, the beam splitter in the form of the dichroic mirror (18), an edge filter (22), a tube lens (24),a fiber bundle (28) with end faces (26 and 29) and a Raman spectrometer (30) and the beam path starting from the sample (20) via the objective (19), the dichroic mirror (18), the edge filter (22), the tube lens (24) and the fiber bundle (28) to the Raman spectrometer (30) can be generated, wherein the illumination fiber (6) is a highly multimode optical fiber with a square core several decameters long, which can be illuminated by the laser (1) via the first lens (4) and imaged in a reduced size on the sample (20) via the second lens (10) and the dichroic mirror (18), wherein the polarization and the coherence of the incoming light of the laser (1) can be disrupted by the large length of the illumination fiber (6), and the fiber bundle (28) is a dimensionally reducing bundle of optical fibers, the cross-section of which is on the sample (20) side facing square and on the,The side facing the Raman spectrometer (30) is designed in a linear configuration, characterized in that the coherence of the incoming light of the laser (1) can be disrupted by a vibration device (7) on the illumination fiber (6), in that the vibration of the interferences leads to temporal superposition in the illumination fiber (6), so that the coherence disappears and the sample (20) is irradiated with incoherent light, and wherein non-optical intermediate fibers with a round cross-section are located between light-conducting fibers with a square cross-section, which space the light-conducting fibers apart from each other on the side facing the Raman spectrometer (30).

Description

[0001] The present invention relates to a Raman spectroscopy illumination and readout system, for example for imaging Raman spectroscopy. Raman spectroscopy

[0002] Raman spectrometers with at least one external measuring probe have been known for some time, as illustrated, for example, by DE 94 14 467 U1 and the publication “Device for Raman Difference Spectroscopy”, Torsten Frosch, Tobias Meyer, Michael Schmidt and Jürgen Popp in Anal. Chem. 2007, 79, 6159-6166.

[0003] For example, DE 10 2004 015 946 B3 also discloses, in principle, a device and a method for generating and detecting a Raman spectrum of a medium to be examined. The device for generating and detecting a Raman spectrum of a substance to be examined comprises an optical fiber, wherein the first end of the optical fiber is connected via a means for coupling and decoupling light, i.e. via a suitable optical coupling element, to both an (excitation) light source and a detection unit for recording a Raman spectrum, and light is coupled out into the medium to be examined via an exposed part of the optical fiber and backscattered light from the medium to be examined is coupled into the fiber.

[0004] WO 2013 / 067 996 A1 discloses a suitable optical fiber for the filtered collection of Raman scattered radiation generated by monochromatically excited light sources, as well as a method for its production, which has a small outer diameter (< 1 mm) over path lengths > 1 m and a small bending radius of less than 5 mm.

[0005] The application of Raman spectroscopic investigations is very diverse, with not only a single fiber but also fiber bundles being used. For example, DE 10 2013 020 703 A1 discloses a device for generating and detecting radiation in a biological tissue sample and a Raman spectrum, comprising a laser light source, a detector, a detection and control unit, and a measuring probe containing a bundle of optical fibers with at least one fiber for placement on the surface of the biological tissue sample. Laser light can be coupled into one or more fibers of the bundle of optical fibers and guided to the surface of the biological tissue sample via a focusing element, and light emitted by the tissue sample can be guided to the detector and / or a Raman spectrometer via the focusing element.The focusing element is arranged at the head of the measuring probe in the form of a transparent optical lens and a transparent optical lens arranged directly next to it, by means of which the laser light of the fiber can be coupled in collimated as Raman excitation radiation and the Raman signal radiation can be coupled out into the external fiber.A transparent optical cube with a subsequent transparent optical lens is arranged downstream of the lens, so that the cube is positioned directly between the lens and the lens, wherein a filter layer, an absorber, a mirror and a filter are introduced into the cube in such a way that the background created in the fiber can be directed onto the absorber through the filter layer and excitation radiation can be guided through a lens onto the surface of the biological tissue sample, Raman signal radiation generated by the tissue sample can be collected and collimated by the lens, the Raman excitation and Raman signal radiation can be separated by the filter in the cube, so that the Raman signal radiation separated by the filter can be deflected in the cube by the mirror and coupled via the lens into the external fiber, which leads into the Raman spectrometer.

[0006] US Pat. No. 5,929,986 A discloses a method and an arrangement for the synchronous recording of Raman bands. The spectral dispersion of the light is achieved using an optical grating.

[0007] Likewise, the Raman imaging system described in US Pat. No. 6,002,476 A is based on the spectral separation of scattered light using a monochromator. The system is also distinguished by the fact that it can be coupled to an optical microscope, which allows for better lateral resolution in the chemical images.

[0008] From WO 2007 / 117 764 A3 a system and a method for image reconstruction in a fiber array spectral conversion system are known. Spectral converter (spectral converter)

[0009] A spectral converter in the sense of the invention is a bundle of optical fibers which has a planar arrangement on one side and a linear arrangement of the fiber end faces on the other side, so that the radiation arriving on the planar side is reduced to one dimension.

[0010] EP 1 984 714 A2 discloses a system and a method for image reconstruction in a fiber array spectral conversion system and US 6 002 476 A a Raman imaging system to which an optical microscope can be coupled, from EP 1 994 418 A2 a system and a method for a fiber array spectral converter based on polymorphic screening is known, EP 1 999 444 A1 discloses a system and a method for structured illumination and collection for improved optical confocality of the images of a Raman fiber array spectral converter and interactive Raman probing.

[0011] In imaging spectroscopy, four quantities must be recorded: the wavelength (or wavenumber), the intensity and two spatial coordinates at the sample level, with one additional quantity being added for time- and depth-resolved spectroscopy.

[0012] Raman spectroscopic imaging is predominantly performed by rasterizing the spatial information or by rasterizing the spectral information.

[0013] When scanning the spatial information, the sample or the beam path is moved, while when scanning the spectral information, tunable optical filters are used.

[0014] By using a fiber bundle as a dimension-reducing element, the screening process can be circumvented. The optical fibers are arranged in a plane (typically a square array) on one side of the bundle and as a line on the other side. The light emitted by the sample is imaged onto the fiber array via an optical imager. The line side is positioned in the slit of an imaging spectrometer so that the fiber end faces are imaged along one dimension onto a two-dimensional detector.

[0015] With two-dimensional detectors (e.g., CCDs), only one dimension is required for simultaneous spectral detection of polychromatic radiation, whereas the second dimension can be freely used to read out multiple spectra. This allows all spectra to be recorded simultaneously. In addition to the reduced measurement time, the simultaneity of recording itself is a decisive advantage over scanning methods. Furthermore, such systems offer very high spectral resolution through the use of a spectrometer and a diffraction-limited spatial resolution.

[0016] However, in order for these advantages to come into effect, the use of special lighting is required, which must meet the following conditions: • the illuminated area should correspond in size to the area imaged on the fiber array, • the excitation radiation should be distributed homogeneously over the illuminated area, • the intensity of the excitation radiation should be easy and continuously adjustable, • the aperture in the excitation beam path should be adjustable, • the excitation spectrum should be monochromatic and free from interference, • the sample should be irradiated with incoherent light, • the illuminated sample area and thus the one imaged onto the fiber array should be assigned to a microscope image, • Adjusting the polarization and illuminating with completely unpolarized light should be possible.

[0017] The first four conditions follow from the principle of Köhler illumination. Illuminating too large an area leads to contamination of the beam path with unwanted scattered radiation. Adjusting the aperture is necessary to adapt the beam path to the various objectives used for sample illumination and for collecting the scattered light.

[0018] Only homogeneous illumination enables sufficient software-supported normalization of the intensities of the individual pixels of the fiber array.

[0019] Crosstalk between the fibers leads to significant errors in the case of inhomogeneous illumination. A monochromatic radiation source is essential for recording spectra of spectral scattered radiation or luminescence. Additional radiation generated in the excitation beam path must be appropriately filtered.

[0020] For orientation, an additional microscopic image is required, which also shows the area imaged onto the fiber array, i.e., the illuminated area. For this purpose, the fiber array must be precisely positioned using a suitable measurement method. Incoherent light provides higher resolution and prevents disruptive interference patterns.

[0021] Since Raman scattering depends on the polarization of the exciting and analyzed radiation, it is often desirable to work with unpolarized light, as this minimizes the influence of polarization-dependent devices such as the spectrometer. The desired polarization can then be easily adjusted using an additional linear polarization filter.

[0022] If a liquid crystal tunable filter (LCTF) is used instead of a fiber array, it acts like a linear analyzer due to its nature, which is why illumination with unpolarized light represents a significant simplification.

[0023] A conventional simple expansion of a laser contradicts all these conditions and represents an inadequate solution in practice.

[0024] Although various fiber optic illumination systems are already known for other applications, these are not tailored to the above-mentioned conditions (the eight previous points) for imaging Raman spectroscopy.

[0025] The publication Schmälzlin, E.; et al.: “Raman imaging with a Fiber-Coupled Mulitchannel Spectrograph” in Sensors 2014, 14, 21968-21980 discloses a Raman spectroscopy illumination and readout system comprising • a measuring light beam generation arrangement and a Raman scattered light coupling arrangement, • wherein the measuring light beam generation arrangement comprises a laser, a lens, an illumination fiber with end faces, a line filter, a microscope with a lens, a pinhole, a beam splitter in the form of a dichroic mirror and an objective, and the beam path can be generated from the laser via the lens, the illumination fiber, the line filter, the microscope with a lens, the dichroic mirror and the objective to the sample, and • wherein the Raman scattered light coupling arrangement comprises the objective of the microscope, a beam splitter (filter) in the form of a dichroic mirror, an edge filter, a tube lens, a fiber bundle with end faces and a Raman spectrometer and the beam path can be generated from the sample via the objective, the dichroic mirror, the edge filter, the tube lens, the fiber bundle to the Raman spectrometer, wherein • the illumination fiber is a highly multimode optical fiber that can be illuminated with the laser via the lens and imaged in reduced size on a sample via the lens and the dichroic mirror, and • the fiber bundle is a dimension-reducing bundle of optical fibers, which has a square cross-section on the side facing the sample and a linear cross-section on the side facing the Raman spectrometer.

[0026] The disadvantage of this technical solution is that samples cannot be irradiated with incoherent light from the illumination fiber and that the adjustable power range is small, so that no Raman spectroscopy illumination and readout system is possible that also allows hyperspectral imaging in backscattering, in which the scattered light is collected by the objective through which it is also illuminated.

[0027] DE 10 2009 025 815 A1 discloses a method for measuring the shape of at least one section of an object, in particular a semi-transparent object such as at least one section of a tooth, using a light source for generating light with preferably a broadband spectrum, a device for generating a multifocal illumination pattern, an objective with large chromatic aberration for imaging foci of the illumination pattern onto the object, a detection device for determining the wavelength spectra of the foci confocally imaged onto the object via the objective, wherein the spectral peak position of each focus is determined from the respective wavelength spectrum, from which the extension of the object in the direction of the imaging beam (Z-coordinate) is calculated, wherein • the multifocal illumination pattern is formed by light guides arranged between the light source and the lens with large chromatic aberration, • the lens images the object-side ends of the light guides onto the object and light remitted from the object onto the object-side ends of the light guides and • light guided and remitted through the optical fiber is directed onto the detection device.

[0028] In order to separate the evaluation part and the measuring part from each other, the light guides forming the illumination pattern are arranged between the light source and the lens, the object-side ends of which are arranged in an imaging plane or an area of ​​the lens comprising imaging planes, wherein a deflection device for the light emerging from the light guide and remitted by the object is arranged between the illumination-side ends of the light guides and the detection device.

[0029] The multifocal illumination pattern is formed by light guides arranged between the light source and the lens with large chromatic aberration, whereby the lens images the object-side ends of the light guides onto the object and light remitted from the object onto the object-side ends of the light guides, and light guided and remitted through the light guides is directed onto the detection device.

[0030] Although this technical solution makes it possible to separate the evaluation part and the measuring part, it has the disadvantage that the object cannot be irradiated with incoherent light from the illumination fiber and that it cannot be used in the field of Raman spectroscopy as an illumination and readout system, which allows hyperspectral imaging in backscattering.

[0031] DD 2 00 440 A1 discloses an optical spectrometer for wavelength or intensity modulation, which enables registration of the first derivative of the optical signal with respect to the wavelength and does not require major modifications to existing spectrometers, essentially consisting of a radiation source, a monochromator or a spectrograph, a sample, a detector, an electronic detection and recording device and a modulation device, wherein the modulation device, which is arranged between the monochromator and the sample or in the imaging plane of the spectrograph in the case of wavelength modulation, and at any desired point in the beam path between the radiation source and the detector in the case of intensity modulation, consists of a fiber optic cable with a holding device, an electromechanical vibration device and an LF generator, wherein: • In the case of wavelength modulation, the holding device is attached to the optical fibre cable so that one end of the optical fibre cable, stimulated by the electro-mechanical vibration device, performs oscillations transverse to the optical axis of the spectrometer. • the fiber optic cable is preferably designed as a cross-section converter. • the electro-mechanical vibration device is a loudspeaker. • in the case of intensity modulation, the holding device is firmly attached to the optical fibre cable and a light beam in the spectrometer is periodically interrupted by oscillation of the oscillating surface attached to the electro-mechanical vibration device, and • the oscillating surface is a rectangle.

[0032] The registration of the first derivative of the optical signal with respect to the wavelength is made possible by directing the light beam onto the sample or the detection device using an optical fiber cable, which is preferably designed as a cross-section converter with a rectangular cross-section.

[0033] With wavelength modulation, the fiber optic cable is located between the dispersive element and the sample or detector. One end of the fiber optic cable is coupled to a vibrating mount firmly connected to the diaphragm of a loudspeaker. A frequency generator in the low-frequency range and possibly an audio amplifier are required to excite the loudspeaker. For lock-in detection, the reference voltage is obtained from the audio generator, enabling frequency- and phase-selective detection. Since most optical spectrometers today are equipped with light chopping technology, the electronic detection section of the spectrometer can be used.

[0034] The arrangement of the optical fiber cables in the optical axis of the spectrometer and the oscillations carried out transversely to the light beam result in a periodic change in the light wavelength, whereby the modulation amplitude is determined by the amplitude of the loudspeaker oscillations, the dispersion of the monochromator or spectrograph, the distance of the optical fiber from the dispersive device and the size of the optical fiber entrance opening.

[0035] To prevent the oscillations from one side of the fiber optic cable from propagating to the sample side, the fiber optic cable must be secured at a sufficient distance behind the loudspeaker mount.

[0036] With intensity modulation, both ends of the fiber optic cable are held firmly in place. The oscillating surface on the electromechanical vibration device is located with its front edge at rest in the center position in front of the opening of the fiber optic cable. Excitation of the vibration device then causes a periodic interruption of the beam path in the spectrometer via the oscillating surface. There are no restrictions on the possible arrangement of the modulation device for intensity modulation; this means it can be arranged anywhere in the beam path between the radiation source and the detector.

[0037] This technical solution has the disadvantage that the object cannot be irradiated with incoherent light from the fiber optic cable and that it cannot be used in the field of Raman spectroscopy as an illumination and readout system, which allows hyperspectral imaging in backscattering.

[0038] EP 2 196 839 A1 discloses a method using a multimode glass fiber for coupling a radiation source module to a confocal multifocal microscope, comprising guiding light emitted from a radiation source module through a multimode fiber to a confocal multifocal microscope, wherein a core diameter of the multimode fiber is not more than 800 µm, • wherein the radiation source module comprises one or more superluminescent diodes or one or more solid-state radiation sources or one or more lasers, • wherein the radiation source module comprises a source of coherent radiation, • and the method further comprises: randomizing the phase of the light before passing the light through the multimode fiber, and / or randomizing the phase of the light while passing the light through the multimode fiber, and / or randomizing the phase of the light emitted by the multimode fiber before the light is incident on the confocal multifocal microscope.

[0039] EP 2 196 839 A1 thus discloses a multimode optical fiber that guides light from a radiation source to a multifocal confocal microscope with appropriate efficiency. The core diameter of a multimode fiber is selected such that the etendue of the light emitted by the fiber is not significantly greater than the total electrical conductivity of the light guided through a plurality of pinholes in a pinhole array of the multifocal confocal microscope.

[0040] The use of a multimode fiber enables the use of a variety of radiation sources and wavelengths in a multifocal confocal microscope compared to a single-mode fiber.

[0041] This technical solution eliminates the need for a single-mode fiber to efficiently guide light from a radiation source module to a micro-endoscope in a multifocal confocal microscopy system, whereby the multimode fiber can be equipped with a phase randomizer comprising a vibrating mechanical driver to generate vibrations in a section of the multimode fiber (induced oscillations).These can lead to rapid variations in the optical path lengths of the individual beams in the multimode fiber, causing the coherence effects of the light to be random and thus the spatial modulation of the amplitudes of the image of the sample. This has the disadvantage that the sample cannot be irradiated with incoherent light from the illumination fiber and it cannot be used in the field of Raman spectroscopy as an illumination and readout system, which allows hyperspectral imaging in backscattering.

[0042] The publication Gift, AD, et al: “Near-infrared Raman Imaging Microscope Based on Fiber-bundle Image Compression.” In: Journal of Raman Spectroscopy, 30, 1999, 757-765 discloses a near-infrared Raman imaging microscope based on fiber-bundle image compression, which uses fiber-bundle image compression (FIC) to simultaneously collect a 3D Raman spectral image data cube, whereby through holographic filtering the spectral image, which is actually acquired with a CCD detection with a 2D array detector, is acquired as a Raman spectral image in the form of a 3D data cube in a single CCD detector frame.

[0043] In this technical solution, the 3D data cube is scanned by sampling the spectral dimension and stacking the resulting sequence of single-color images into a complete spectral image, using image reconstruction, which includes line scanning (LS) and Hadamard transform (HT), as the imaging technique.

[0044] In line scanning, a strip of the sample is illuminated, and a complete Raman spectrum is obtained at each point along that line. The illumination strip is then moved stepwise across the sample (or the sample is translated) to accumulate a 3D data cube.

[0045] In the HT Raman imaging method, the sample is globally illuminated with a mask. This mask serves to encode the spatial information, which is then compressed into a line at the spectrograph's entrance slit (using a cylindrical lens). A complete spectral image is reconstructed by transforming the data from a sequence of different HT masks.

[0046] The heart of the near-infrared Raman imaging microscope is the FTC fiber bundle, a bundle of 100 fibers arranged in a 10 x 10 square array on the acquisition side and a linear stack on the detection side, with the number of fibers that can be accommodated in a fiber bundle being determined by the size of the fibers and the height of the CCD.

[0047] Global illumination of the sample is performed using a single 300 nm excitation fiber. The laser is focused at one end of the excitation fiber, and the light emerging from the other end is projected onto the sample, uniformly illuminating a circular area containing the field of view of the FIC. The coupled laser light is directed onto a holographic super-notch filter at an angle of 10° to the normal, where it is reflected into the microscope by diffraction and projected onto the sample through the microscope objective. The Raman light scattered by the sample is collected and returned along the same path as the laser.

[0048] When it reaches HNF1, the Raman light is transmitted and directed onto the FIC fiber bundle, while the laser light is preferentially rejected by HNF1 (and a second notch filter, HNF2).

[0049] The microscope's optical tube contains two reflective components. One is a "hot mirror" that reflects 99% of the near-infrared Raman light back to the FIC fiber bundle and transmits most of the visible light emanating from the sample. This allows the sample to be viewed on an overhead CCD while the laser illuminates the sample. The other component is a partially silvered mirror. This is used to illuminate the sample with the microscope's overhead lamp.

[0050] The disadvantage of this technical solution of the excitation configuration is that a significant part of the laser power is lost in the transmission optics and due to the overfilling of the image area, so that only about 100 mW of the laser power reaches the square image area and thus the adjustable power range is small.

[0051] In addition, this technical solution is not suitable for irradiating the sample with incoherent light.

[0052] The invention is therefore based on the object of providing a Raman spectroscopy illumination and readout system which fulfills all eight conditions mentioned above, simultaneously irradiates the sample with incoherent light and still allows hyperspectral imaging in backscattering (backscattering means that the scattered light is collected by the objective lens through which the illumination is also carried out) and can be easily adjusted to fiber arrays of different sizes.

[0053] According to the invention, this object is achieved by the characterizing features of the 1st and 9th patent claims. Further advantageous embodiments of the invention are specified in the subordinate patent claims.

[0054] The essence of the invention is that the Raman spectroscopy illumination and readout system is designed such that the end of an optical fiber with a special cross-section (illumination fiber) is imaged via a microscope beam path onto the sample for imaging Raman spectroscopy, wherein the fiber bundle for coupling out the Raman scattered light is a dimension-reducing spectral converter in this optical system.

[0055] A spectral converter within the meaning of the invention is a bundle of optical fibers which, on the input side (i.e. facing the sample), has a flat arrangement of fibers in an array and, on the output side (i.e. facing the spectrometer), has a linear, ordered arrangement of the individual fiber end faces so that the radiation arriving on the flat side is reduced to one dimension (line). On the output side, spacers (e.g. dummy fibers) can be inserted between the individual optical fibers to reduce crosstalk. The linear arrangement of fibers is placed or imaged in the slit plane of the spectrometer so that, using a two-dimensional detector (CCD, charge-coupled device), the complete Raman spectra of all fibers can be recorded simultaneously.Overall, the spectral converter allows simultaneous recording of the Raman spectra of all positions in the sample area, which are imaged onto the input plane of the spectral converter.

[0056] However, this case described above for Raman spectroscopy can also be generalized to other types of optical spectroscopy.

[0057] The invention is explained in more detail below with reference to the exemplary embodiments and the figures. In the figures: Fig. 1: a schematic representation of an embodiment of the inventive structure of the Raman spectroscopy illumination and readout system (with a fiber bundle as spectral converter), Fig. 2: a cross-sectional view of the Fig. 1 illustrated lighting fiber, Fig. 3: a representation of an intensity distribution on the sample surface recorded with a beam profile camera using the Raman spectroscopy illumination and readout system according to Fig. 1 with the lighting fiber according to Fig. 2, Fig. 4: a schematic representation of another embodiment of a special fiber bundle, Fig. 5: a representation of the intensity distribution on the fiber array when using a homogeneously scattering sample in combination with the illumination fiber according to Fig. 2 and the structure according to Fig. 1, where the grid has the dimensions of the Fig. 4 illustrated fiber bundle, and Fig. 6: a representation of the polarization dependence of the transmission of the illumination fiber according to Fig. 2, where the laser is coupled into the illumination fiber at 532nm. (The triangles represent the polarization dependence of the laser. The squares refer to the measurement with the illumination fiber according to Fig. 2. For comparison, a comparison measurement was taken with a shorter fiber. The corresponding measured values ​​are shown as circles.

[0058] The Fig. The fiber optic Raman spectroscopy illumination and readout system for imaging Raman spectroscopy shown in Figure 1 comprises a measurement light beam generation arrangement and a Raman scattered light extraction arrangement.

[0059] The measuring light beam generation arrangement comprises a laser (1), a first pinhole (3), a first lens (4), an illumination fiber (6) with end faces (5) and (8), a vibration device (7), a line filter (9), a microscope with a second lens (10), a second pinhole (16), a beam splitter in the form of a dichroic mirror (18) and an objective (19), wherein the beam path can be generated from the laser (1) via the first pinhole (3), the first lens (4), the illumination fiber (6), the line filter (9), the microscope with the second lens (10), the dichroic mirror (18) and the objective (19) to the sample (20).

[0060] In this case, a telescope (2) can be arranged in the beam path between the laser (1) and the first pinhole (3), and a rotatable linear polarization filter (17) can be arranged in the beam path between the second pinhole (16) and the dichroic mirror (18).

[0061] Starting from a monochromatic light source in the form of a laser (1), the excitation light is coupled into the end face (5) of an illumination fiber (6) via a first lens (4) or an objective. The end face (5) of the input head of the illumination fiber (6) is located just behind the focal plane of the first lens (4) to prevent focus formation in the illumination fiber (6).

[0062] The first pinhole (3) is located in front of the first lens (4) [as seen in the beam path from the direction of the laser (1)]. This is used for quick, easy, and temporally stable adjustment of the power with which the sample is irradiated.

[0063] To increase the adjustable power range, a telescope (2) can be mounted in front of the first pinhole (3) [in the beam path as seen from the direction of the laser (1)]. The different focus diameter at the focal point of the first lens (4) resulting from this power adjustment, as well as the changing numerical aperture when coupled into the end face (5) of the input head of the illumination fiber (6), have no influence on the fulfillment of the other illumination conditions mentioned above.

[0064] The illumination fiber (6) is a highly multimode optical fiber with a length of several decameters. This length is crucial for generating depolarized light.

[0065] The front side (5) of the input head of the illumination fiber (6) and the front side (8) of the output head of the illumination fiber (6) have the same shape.

[0066] The shape of the cross-section of the fiber (6) is similar to the shape of the arrangement of the fibers of the fiber bundle (28) on the front side (26) of the entrance of the fiber array / fiber bundle (28) in the Raman scattered light decoupling arrangement.

[0067] The area of ​​the cross-section of the illumination fiber (6) is selected so that the guidance of a sufficient number of modes for homogenization is ensured.

[0068] The Raman scattered light coupling arrangement comprises the objective (19) of the microscope, a beam splitter (filter) in the form of a dichroic mirror (18), an edge filter (22), a tube lens (24), a fiber bundle (28) with the end faces (26 and 29) and a Raman spectrometer (30), wherein the beam path can be generated from the sample (20) via the objective (19), the dichroic mirror (18), the edge filter (22), the tube lens (24), the fiber bundle (28) to the Raman spectrometer (30).

[0069] A polarization filter (23) can be installed in the beam path between the edge filter (22) and the tube lens (24).

[0070] By coupling the illumination fiber (6) to a vibration device (7), it is set into a temporal oscillation. This oscillation leads to temporal superposition of the interference in the illumination fiber (6), so that the coherence disappears for exposure times greater than a multiple of the oscillation period.

[0071] The illumination fiber (6) itself generates an interference signal through excitation by the radiation emitted by the first laser (1). This signal is filtered out by a line filter (9), which blocks all wavelengths except those of the monochromatic light source in the form of the first laser (1). The monochromatic excitation light, emanating from the end face (8) of the output head of the illumination fiber (6), is imaged onto the sample (20) via a microscope with a second lens (10) and an objective (19).

[0072] The end face (8) of the illumination fiber (6) is located at the focal point of the second lens (10), and the sample (20) is located at the focal point of the objective lens (19). The focal length of the second lens (10) is selected such that, for a given objective lens (19) and tube lens (24), only that portion of the sample surface is irradiated which, after imaging by the objective lens (19) and tube lens (24) onto the end face (26) of the entrance of the fiber array / fiber bundle (28), corresponds to the area of ​​the latter.

[0073] Within the beam path of the infinity microscope formed by components (10) and (19), the monochromatic excitation light is reflected by a beam splitter, such as a dichroic mirror (18) at an angle of 45° or by an edge filter at a very small angle, which is usually less than 10°. The filter (18) transmits radiation with a wavelength greater than the excitation wavelength and reflects radiation with a wavelength less than or equal to the excitation wavelength.

[0074] A semi-transparent mirror (11), which can also be replaced by a folding mirror, makes it possible to couple in additional white light (incident light) for optimal microscopic illumination of the sample (20) via an illumination component (14). The illumination component (14) is an illumination system that meets the conditions for optimal microscopic illumination with white light (incident light onto the sample). This is generally achieved using Köhler illumination. The image plane of the illumination component (14) is transferred to the sample (20) by imaging with a third lens (13) and the objective (19).

[0075] To take white light images (incident or transmitted light images) of the sample (20), it is possible to direct the light emanating from the sample (20) to a camera (27) via a folding mirror (25) or a beam splitter.

[0076] The third lens (13) and the illumination component (14) are selected so that a sample area is irradiated (incident light on the sample) which, when imaged onto the camera (27), corresponds to the size of the camera chip. The second pinhole (16) serves to adjust the aperture in the illumination beam path when using the laser (1) so that exactly the full rear opening of the lens (19) is illuminated.

[0077] Furthermore, this second pinhole (16) can be used as an aperture stop when using the illumination component (14). The dichroic mirror (18) only reflects light with a wavelength less than or equal to the wavelength of the light source (1). Therefore, it can be replaced by a 50% beam splitter, either tiltably or using a rotating multiple-shot system.

[0078] A further illumination component (21) for white light (transmitted light through the sample) is provided for taking transmitted light images. Using a suitable illumination component (21), imaging transmission measurements can also be performed. By using a rotatable linear polarization filter (17), the sample (20) can be irradiated with linearly polarized light. By removing or folding out this polarization filter (17), the sample (20) is irradiated with unpolarized light.

[0079] In the beam path of the scattered light, formed by the objective lens (19) and the tube lens (24), a linear polarization filter (23) can be inserted for polarization-dependent measurements.

[0080] To suppress the Rayleigh radiation and the excitation light reflected from the sample, which emanates from the laser (1), an additional edge filter (22) is inserted.

[0081] The image plane of the camera (27) and the front side (26) of the entrance of the fiber array / fiber bundle (28) are both located in the focal plane of the tube lens (24).

[0082] By changing the objective lens (19), the excitation beam path and the beam path of the scattered light are changed in the same way, so that only the sample surface imaged by the end face (26) of the fiber array / fiber bundle (28) continues to be illuminated by the laser (1).

[0083] The end face (26) of the input of the fiber array / fiber bundle (28) consists of multimode fibers with, for example, a square core and a square cladding. The side length of the cladding is only exactly as much larger than the side length of the core as is necessary for light guidance.

[0084] The fibers of the fiber bundle (28) are arranged in a line on the end face (29) of the output of the fiber array / fiber bundle (28). The end face (29) is located exactly in the slit plane of the spectrometer (30).

[0085] Additional fibers that do not conduct light are woven into the fiber bundle (28). These intermediate fibers serve as spacers on the end face (29). In the fiber arrangement, there is always an intermediate fiber between every two fibers of the fiber array / fiber bundle (28). This prevents crosstalk caused by the imaging in the spectrometer.

[0086] The front side (26) of the entrance of the fiber array / fiber bundle (28) must be positioned exactly in the beam path so that it exactly overlaps with the dimensions of the illuminated area being imaged.

[0087] For adjustment, an additional laser (15) with a different wavelength than the laser (1) is introduced into the beam path via a beam splitter (12) or folding mirror. The additional laser (15) is adjusted so that the focus in the sample plane (20) is exactly in the center of the area illuminated by the laser (1). This can be verified with the camera (27).

[0088] Subsequently, by comparing the images obtained by the spectrometer (30), the front side (26) of the entrance of the fiber array / fiber bundle (28) is positioned so that only the fiber, or fibers, in the center are illuminated by the further laser (15).

[0089] In the following, the Fig. 1 to 6 the embodiment is explained in more detail.

[0090] The laser used (1), for example, is a frequency-doubled Nd:YAG laser, which emits a TEM00 mode at 532 nm.

[0091] The laser (1) is coupled into the illumination fiber (6) via a biconvex first lens (4) with a focal length of 2 cm.

[0092] The actual cross-section of the lighting fiber (6) is shown in Fig. 2. The side length of the square core (111) is 400 µm, and that of the square first cladding (222) is 500 µm. The second cladding (333) is circular and has a diameter of 650 µm. The illumination fiber (6) is 30 m long and terminated in FC connectors. This length is sufficient to generate almost completely depolarized light.

[0093] To illustrate this fact, the intensity at the end of the illumination fiber (6) was measured using an analyzer. Fig. Figure 6 shows the dependence on the analyzer position. The measured values ​​shown as solid circles refer to a reference measurement with a square fiber of only 2 m in length. The measured values ​​shown as solid triangles show the polarization-dependent intensity of the laser without fiber optic illumination. All measured values ​​are standardized for better comparability.

[0094] A vibration device similar to the vibration device of a commercially available toothbrush can be used as the vibration device (7).

[0095] In Fig. Figure 3 shows the distribution of excitation radiation on the sample achieved with this illumination. The measurement is performed using a beam profiler. The standard deviation of the intensity on the plateau is always less than 2%. The front end (8) is located at the center of the focal plane of the second lens (10), which has a focal length of 6 cm.

[0096] Together with the 10x objective (19) this results in a magnification of 3.3 times.

[0097] By imaging the scattered radiation onto the front side (26) of the entrance of the fiber array / fiber bundle (28), a magnification of a factor of 10 is added. This results from the magnification of the objective lens, since a suitable tube lens (24) is selected as the lens.

[0098] This results in a side length of the image of the illumination fiber (6) onto the fiber array / fiber bundle (28) of 1320 µm.

[0099] In Fig. 5 shows the actual dimensions of the image on the fiber array.

[0100] The fiber bundle (28) itself is in Fig.4. The 64 identical square fibers (2222) of the bundle each have a core with a side length of 100 µm and a cladding with a side length of 123 µm. This results in a side length of the fiber array (1111) of approximately 1 mm. On the side of the fiber bundle facing the spectrometer (30) (3333), there are two intermediate fibers (4444) between each two square fibers (2222). These fibers have a round cross-section and do not conduct light.

[0101] The diameter of the intermediate fibers is 60 µm. The intermediate fibers prevent crosstalk between the fibers during imaging onto the CCD.

[0102] The illumination component (14) for providing the white light (top light onto the sample) is directed to the sample via a third lens (13) with a focal length of 2 cm. The dichroic mirror only reflects light with a wavelength shorter than approximately 532 nm onto the sample. Therefore, it can be replaced with a 50% beam splitter by tilting it.

[0103] The additional laser (15) for alignment is a He-Ne laser with a wavelength of 633 nm, which is guided via two additional mirrors to a 50:50 beam splitter (12). The two additional illumination components (14) and (21) are commercial systems for Köhler illumination with white light.

[0104] The advantage of this Raman spectroscopy illumination system is, among other things, that accurate qualitative and quantitative statements are possible through appropriate normalization with a homogeneous scattering sample, precisely adjusted imaging, low crosstalk between the fibers and appropriate illumination.

[0105] Another advantage of this Raman spectroscopy illumination system for imaging Raman spectroscopy is that it can be adjusted to fiber arrays of different sizes without major financial and technical effort (replacement of a lens).

[0106] This inexpensive extension can be easily adapted to any Raman spectrometer and therefore has high market potential.

[0107] The advantage of the Raman spectroscopy illumination system is in particular that the illuminated area corresponds in size to the area imaged onto the fiber array, the excitation radiation is distributed homogeneously over the illuminated area, the intensity of the excitation radiation can be easily and continuously controlled, the aperture in the excitation beam path is adjustable, the excitation spectrum is monochromatic and free from interference, the sample can be irradiated with incoherent light, the illuminated sample area and thus the sample area imaged onto the fiber array can be assigned to a microscope image and that the polarization can be adjusted as well as illumination with completely unpolarized light.

[0108] It is also advantageous that the Raman spectroscopy illumination and readout system according to the invention can be used not only for imaging Raman spectroscopy, but also for imaging fluorescence, IR, absorption and transmission spectroscopy or combinations of these types of spectroscopy.

[0109] It is also within the scope of the invention that it can be used for the simultaneous recording of at least two spectra for the calculation of difference spectra based on the simultaneously measured reference spectrum, for the analysis of the smallest spectral shifts in optical spectroscopy.

[0110] All features presented in the description, the following embodiments, the claims and the drawings can be essential to the invention both individually and in any combination with one another. List of reference symbols 1 laser 2 telescope 3 first pinhole 4 first lens 5 Front side of the input head of the lighting fiber 6 lighting fiber 7 Vibration device 8 Front side of the output head of the lighting fiber 9 line filters 10 second lens 11 semi-transparent mirror or folding mirror 12 beam splitters 13 third lens 14 Lighting component (for white incident light) 15 second laser 16 second pinhole 17 rotatable linear polarizing filter 18 dichroic mirrors 19 lens 20 samples 21 Illumination component (for white transmitted light) 22 edge filters 23 second polarization filter 24 tube lens 25 folding mirrors 26 Front side of the entrance of the fiber bundle 27 Camera 28 fiber bundles 29 Front side of the fiber bundle outlet 30 Raman spectrometers 111 square core 222 square first coat 333 circular second mantle 1111 Fiber Array 2222 square, identical fibers of the fiber bundle 3333 side of the fiber bundle facing the spectrometer 4444 round intermediate fibers (non-optical)

Claims

[1] Raman spectroscopy illumination and readout system comprising a measuring light beam generation arrangement and a Raman scattered light decoupling arrangement, wherein the measuring light beam generation arrangement comprises a laser (1), a first pinhole (3), a first lens (4), an illumination fiber (6) with end faces (5 and 8), a line filter (9), a microscope with a second lens (10), a second pinhole (16), a beam splitter in the form of a dichroic mirror (18) and an objective (19), and the beam path starting from the laser (1) via the first pinhole (3), the first lens (4), the illumination fiber (6), the line filter (9), the microscope with the second lens (10), the dichroic mirror (18) and the objective (19) to a sample (20) can be generated, and wherein the Raman scattered light decoupling arrangement comprises the objective (19) of the microscope, the beam splitter in the form of the dichroic mirror (18), an edge filter (22), a tube lens (24),a fiber bundle (28) with end faces (26 and 29) and a Raman spectrometer (30), and the beam path can be generated from the sample (20) via the objective (19), the dichroic mirror (18), the edge filter (22), the tube lens (24) and the fiber bundle (28) to the Raman spectrometer (30), wherein the illumination fiber (6) is a several-decameter-long, highly multimode optical fiber with a square core, which can be illuminated with the laser (1) via the first lens (4) and imaged in a reduced size on the sample (20) via the second lens (10) and the dichroic mirror (18), wherein the polarization and the coherence of the incoming light of the laser (1) can be destroyed by the great length of the illumination fiber (6), and the fiber bundle (28) is a dimension-reducing bundle of optical fibers, which in its cross-section is the side facing the sample (20) square and on the,the side facing the Raman spectrometer (30) is linear, characterized by that the coherence of the incoming light of the laser (1) can be destroyed by a vibration device (7) on the illumination fiber (6), in which the oscillation of the interference leads to temporal superposition in the illumination fiber (6), so that the coherence disappears and the sample (20) is irradiated with incoherent light, and wherein non-light-conducting intermediate fibers with a round cross-section are located between light-conducting fibers with a square cross-section, which space the light-conducting fibers from one another on the side facing the Raman spectrometer (30). [2] Raman spectroscopy illumination and readout system according to claim 1, characterized bythat a telescope (2) is arranged in the beam path between the laser (1) and the first pinhole (3), a rotatable linear polarization filter (17) is arranged in the beam path between the second pinhole (16) and the dichroic mirror (18), and a second polarization filter (23) is arranged in the beam path between the edge filter (22) and the tube lens (24), wherein the sample (20) can be irradiated with linearly polarized light through the rotatable linear polarization filter (17) or with unpolarized light through the illumination fiber (6) by removing or folding out the polarization filter (17). [3] Raman spectroscopy illumination and readout system according to claim 1, characterized by that a light-conducting fiber and two adjacent non-light-conducting intermediate fibers with spacer function are arranged alternately one after the other to form the line shape of the side of the fiber bundle (28) facing the Raman spectrometer (30). [4] Raman spectroscopy illumination and readout system according to claim 1, characterized by that the illumination fiber (6) is provided with the vibration device (7) and the exposure times of a two-dimensional detector in the form of a CCD can be set to be greater than the period of the oscillations of the vibration device (7), so that interference patterns can be avoided and thus the coherence can be destroyed. [5] Raman spectroscopy illumination and readout system according to claim 1, characterized by that additional white light can be coupled into the beam path in the direction of the sample (20) by a semi-transparent mirror (11) from an illumination component (14) or that white light can be coupled through the sample (20) into the beam path leading out of the sample (20) by a further illumination component (21) arranged on the side of the sample (20) opposite the objective (19). [6] Raman spectroscopy illumination and readout system according to claim 1, characterized by that several light sources can be coupled simultaneously into the illumination fiber (6), which can be operated consecutively or simultaneously. [7] Raman spectroscopy illumination and readout system according to claim 1, characterized by that the light emanating from the sample (20) can be guided to a camera (27) via a folding mirror (25) or a beam splitter. [8] Method using a Raman spectroscopy illumination and readout system according to one or more of the preceding claims 1 to 7, in which the sample (20) is irradiated with monochromatic excitation light via the illumination fiber (6) and Raman scattered light is guided into the Raman spectrometer (30) by means of the fiber bundle (28), the fiber bundle (28) performing the function of a dimension-reducing spectral converter. [9] Method according to claim 8, characterized bythat the excitation light is imaged onto the sample (20) and from there onto the fiber bundle (28) through the optics of the microscope in such a way that exactly the square surface of the fiber bundle (28) is sharply illuminated. [10] Method according to claim 9, characterized by that the scattered radiation generated in the illumination fiber (6) is removed by a line filter (9) which is arranged after the illumination fiber (6) in the beam path from the laser (1). [11] Method using a Raman spectroscopy illumination and readout system according to one or more of the preceding claims 1 to 7, in which a second laser (15) for adjustment with a different wavelength than the laser (1) for excitation is introduced into the beam path via a beam splitter (12) or folding mirror and the second laser (15) is adjusted so that the focus in a plane of the sample (20) lies exactly in the center of the area illuminated by the laser (1) and by comparison with the images obtained by the spectrometer (30) the end face (26) of the entrance of the fiber bundle (28) is positioned so that only the fiber or fibers in the center are illuminated by the second laser (15),and subsequently the fiber bundle (28) is centered over a spectral image, and in which, by recording an image by a camera (27) with a monochromatic light source in the form of the laser (1) and a polychromatic white light source in the form of an illumination component (14), with a prior adjustment of the position of the fiber bundle (28), the area of the sample (20) imaged onto the fiber bundle (28), and thus each pixel in a hyperspectral image, can be precisely assigned in a microscopic image, wherein a third lens (13) and the illumination component (14) are selected such that an area of the sample (20) is irradiated with incident light from the laser (1), which area, after being imaged onto the camera (27), corresponds to the size of a chip of the camera (27).

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