WO1999040413A1 - Method for time-resolved measurement of energy spectra of molecular states and device for carrying out said method - Google Patents

Method for time-resolved measurement of energy spectra of molecular states and device for carrying out said method Download PDF

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Publication number
WO1999040413A1
WO1999040413A1 PCT/EP1999/000688 EP9900688W WO9940413A1 WO 1999040413 A1 WO1999040413 A1 WO 1999040413A1 EP 9900688 W EP9900688 W EP 9900688W WO 9940413 A1 WO9940413 A1 WO 9940413A1
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Prior art keywords
sample
measuring
measurement
measurements
infrared
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PCT/EP1999/000688
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German (de)
French (fr)
Inventor
Klaus Gerwert
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RUBITEC Gesellschaft für Innovation und Technologie der Ruhr-Universität Bochum mbH
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Publication of WO1999040413A1 publication Critical patent/WO1999040413A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/45—Interferometric spectrometry
    • G01J3/453—Interferometric spectrometry by correlation of the amplitudes
    • G01J3/4535—Devices with moving mirror
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/2889—Rapid scan spectrometers; Time resolved spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/42—Absorption spectrometry; Double beam spectrometry; Flicker spectrometry; Reflection spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N2021/3595—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using FTIR
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated

Definitions

  • the invention relates to a method for the time-resolved measurement of energy spectra of molecular states in externally excited reactions and changes within molecules by measuring the interaction of infrared (IR) radiation with the molecules of the system, in particular in the case of non-cyclically operating systems.
  • IR infrared
  • the invention also relates to an apparatus for performing this method.
  • spectroscopic methods For the investigation of dynamic processes at the molecular level with high time resolution, the use of spectroscopic methods is generally known, in which the interaction of electromagnetic radiation, for example infrared radiation of low energy, with molecules of the system in question is evaluated.
  • electromagnetic radiation for example infrared radiation of low energy
  • the time-resolved acquisition of the spectrum takes place after initiation of the reaction to be examined by external energy supply, for example by irradiating the sample with an intense laser light flash.
  • time-resolved Raman spectroscopy and time-resolved FTIR (Fourier Transform Infrared) difference spectroscopy are currently used, for example. While in Raman spectroscopy the frequency, i.e. If the energy difference between incident and scattered IR light is measured, the FTIR method measures characteristic IR absorptions. By using differential methods, the energy states of interest of the functionally important groups can be determined with high structural resolution against the inevitable background noise.
  • IR absorption measurements are to be carried out for a predetermined number of energy measurement values each for preset measurement times.
  • an energy spectrum is run through at a predetermined time after the excitation, for example by a laser flash, in which the entire IR measurement range is scanned with an adjustable interferometer.
  • the time resolution is limited to about 40 ms by the speed of the moving mirror in the interferometer. Better time resolutions can be achieved with the so-called step-scan technique.
  • An IR wavelength, ie a measurement energy is set and held on the interferometer while the sample is excited by a laser flash. The decay of the IR absorption is now detected by means of appropriate detectors.
  • the complete spectrum can be obtained by adjusting the energy on the interferometer step by step (“Step"), after which each time there is another excitation by a flash of light and the temporal development of the IR absorption is measured (“Scan”) .
  • the time resolution of this measurement method is only limited by the rise time of the detector used and is in the ns range, which is almost three orders of magnitude better than with the aforementioned fast scan technique.
  • the step-scan technique particularly in connection with the implementation of FTIR when excited by intense laser pulses, places a considerable strain on the sample substance due to the energy input. This fact is of fundamental importance for measurements on systems that do not operate cyclically, which are shifted from an initial state to an end state by the excitation light flash, without then automatically returning to the initial state.
  • the invention proposes, based on the above-mentioned methods known from the prior art, that
  • a multiplicity of spatially separated localized sample segments is defined within an area-wide sample
  • a plurality of the sample segments are sequentially locally excited one after the other and the intensity of the infrared radiation after the interaction with the sample is measured as a measurement signal for a respectively preset spectral scan parameter
  • the scan parameter is varied step by step between the measurement of individual sample segments in such a way that all measurement values within the scan measurement range are carried out with a predetermined number of measurements.
  • the particular advantage of the method according to the invention is that to record a dynamic spectrum, which requires a large number of external excitations both in the fast scan and in the step scan method, a number of excitations and scan numbers corresponding to the previously defined sample segments Measurements can be carried out on a single sample without it having to be exchanged.
  • the excitation radiation ie the laser flash
  • Both the laser flash and the IR measurement radiation are limited to a diameter in the order of 100-500 ⁇ m within a sample segment.
  • the individual sample segments as a whole form a measurement grid and are expediently dimensioned such that the sample in the other sample segments is not influenced by irreversible excitation in one sample segment. As a result, the heat load is also relatively low.
  • the scan parameter ie. H. as the measurement parameter, the mirror position X; of the moving mirror on the interferometer, i.e. the IR energies in a Fourier-transformed representation.
  • the reaction is then started within a predetermined sample segment by irradiation of a laser flash and the resulting IR absorption changes are recorded as a measured value over time. Then another sample segment, which has not yet been excited within the measurement sequence, is selected and the aforementioned measurement is repeated.
  • the IR wavelength on the interferometer between successive excitations i. H. Measurements on different sample segments vary in each case until all the desired measuring points within the scan measuring range have been passed.
  • a particular advantage of the method according to the invention is that complete spectra on systems which do not operate cyclically, for example caged ATP, can be recorded on a single, areally extended sample without having to change them between two measurements.
  • a sample diameter of the order of magnitude from a few millimeters to a few centimeters a sufficient number of sample segments can be defined, each of which is individually subjected to an irreversible measurement process. Due to the small local measuring range in relation to the total sample area, only a relatively small amount of energy is coupled in by the laser flash, so that the change in state of the sample is only locally limited in each case.
  • the sample segments in the method according to the invention are small surface areas of a sample which are formed by a homogeneous, continuous thin layer of the sample substance. This can be prepared relatively simply by pressing the sample substance between IR-transparent windows to a thickness of a few // m. As a result, absorption measurements in transmission can be carried out particularly well. It is also conceivable to design the sample segments separately from one another.
  • the measuring method according to the invention can be used for FTIR measurements as well as for FT Raman measurements as well as for other local measuring methods. It is equally possible to carry out fast scan methods in which the measurement time is passed step by step as the scan measurement parameter.
  • a device for performing the aforementioned method essentially starts from a previously described measurement setup, for example an FTIR microscope as specified in Applied Spectroscopy 51, No. 4, 1 997, pp. 558-562.
  • the associated control measuring and evaluation units which are required to carry out scan measurements, are also known from the prior art.
  • the sample is expanded in terms of area, the sample area is a multiple of the measurement field, and that the sample is arranged in a scanning device in which the measurement field can be moved onto different sample segments within the sample area .
  • the scanning device is, for example, a cross table in which the sample is clamped and on which the X-Y coordinates of the sample can be set such that all predefined sample segments can be reached sequentially with the excitation and measuring beam.
  • the measuring head for coupling the excitation radiation and coupling out the measuring signal is spatially held in order to achieve the relative movement and whether the sample is moved or vice versa.
  • the sample itself it is advantageous for the sample itself to be arranged on a sample holder which can be moved by motor in the plane of the sample surface, ie a cross table. This results in a simpler optical and mechanical structure than if the measuring head itself had to be moved.
  • An FTIR spectrometer is preferably equipped with a sample holder according to the invention. Attachment to a Raman spectrometer or other spectrometric measuring device is, however, also conceivable.
  • the measurements can take place both in transmission and in reflection. Samples in which the sample substance is inserted with a layer thickness of a few ⁇ m between two extensive, IR-transparent windows are particularly well suited for transmission measurements.
  • an angle mirror is expediently arranged in front of the IR measurement optics, with which the laser flash irradiated from the side is directed perpendicularly onto the sample surface in the sample segment to be measured in each case.
  • a quartz prism is preferably used for this, since it easily withstands the high load from the laser flashes.
  • the IR measuring optics have a Cassegrain objective.
  • the quartz prism used as the deflection element is then preferably attached in front of the secondary mirror of the Cassegrain objective.
  • By means of an inclined reflection surface it is then possible to radiate a laterally irradiated laser beam perpendicularly into the sample surface.
  • the profile of the laser beam is expediently limited by a spatial filter, for example in the form of a pinhole.
  • 1 schematically a top view of a sample
  • 2 an FTIR spectrum
  • Fig. 4 a schematic structure of a complete FTIR measuring device.
  • FIG. 1 shows a schematic plan view of a sample 1, the sample substance being in the form of a round disk lying in the XY plane. This is for example squeezed between IR-transparent CaF 2 windows, not shown in detail, with a layer thickness of a few ⁇ m.
  • a measurement grid is defined in the XY direction.
  • the measuring fields x (n) drawn in as a filled circle lie on the intersection points of the measuring grid, which are in succession according to the method according to the invention, for example in a measuring sequence x (n), x (n + 1), x (n + 2), ... excited locally by a laser flash and measured using FTIR, for example.
  • FIG. 2 schematically shows an energy spectrum recorded in the FTIR step scan method.
  • the energy of the IR radiation is passed through step by step as a scan parameter.
  • the excitation and measurement of the IR intensity for the IR measurement energies x (n), x (n + 1),... Take place locally on the corresponding sample segments in accordance with the method shown in FIG. provided measuring grids.
  • sample 1 is moved relative to the measuring device when recording the spectrum so that the corresponding sample segment is in the beam path of the measuring device before a measurement.
  • FIG. 3 shows a measuring head 2 with which the IR measuring radiation radiated by the sample 1 from below can be detected via a Cassegrain mirror optic.
  • an optical deflection element for example a quartz prism 3
  • the measuring head 2 is designed as a Cassegrain lens, the quartz prism 3 being attached in front of the central secondary mirror.
  • each sample segment x (n) can thus be positioned in front of the measuring head 2 in such a way that it can be locally excited by a laser flash and then measured by measuring the intensity of the IR measuring radiation.
  • Fig. 4 shows schematically an FTIR measurement setup. This shows the arrangement of an IR measuring radiation source 5 that can be adjusted with respect to the energy via an interferometer, a laser 6, an IR detector 7 and the associated control and measuring electronics 8.
  • the device shown essentially represents a known system for carrying out FTIR measurements, which is expanded by a controllable cross table for carrying out the method according to the invention.
  • the advantages of the method according to the invention which have already been explained can thereby be realized.

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Abstract

The invention relates to a method for the time-resolved measurement of the energy spectra of molecular states in externally initiated reactions and changes within molecules by measuring the interaction of IR radiation with the molecules of the system, especially for systems which do not function cyclically. The invention also relates to a device for carrying out this method. The aim of the invention is to record a complete spectrum over a short measuring period using step-scan measuring techniques and by simple means. To this end, a number of spatially separate localised sample segments are defined within a sample which is stretched out so that it is flat; a plurality of the sample segments are locally activated in sequence, one after the other and the intensity of the infrared radiation after interaction with the sample for a pre-set spectral scan parameter is measured as a measuring signal; and the scan parameter is varied in steps between measuring the individual sample segments so as to run through all of the measuring values within the scan measuring range with a predetermined number of a measurements.

Description

Verfahren zur zeitaufgelösten Messung von Energiespektren molekularer Zustände und Vorrichtung zur Durchführung des Verfahrens Method for the time-resolved measurement of energy spectra of molecular states and device for carrying out the method
Die Erfindung betrifft ein Verfahren zur zeitaufgelösten Messung von Energiespektren molekularer Zustände bei extern angeregten Reaktionen und Änderungen innerhalb von Molekülen durch Messung der Wechselwirkung von lnfrarot-(IR)-Strahlung mit den Molekülen des Systems, und zwar insbesondere bei nicht zyklisch arbeitenden Systemen. Außerdem bezieht sich die Erfindung auf eine Vorrichtung zur Durchführung dieses Verfahrens.The invention relates to a method for the time-resolved measurement of energy spectra of molecular states in externally excited reactions and changes within molecules by measuring the interaction of infrared (IR) radiation with the molecules of the system, in particular in the case of non-cyclically operating systems. The invention also relates to an apparatus for performing this method.
Die Erfassung zeitaufgelöster Energiespektren molekularer Zustände ist für das Verständnis der Wirkungsweise von Proteinen auf molekularer Ebene von zentraler Bedeutung. Neben statischen Strukturuntersuchungen mit atomarer Auflösung tritt dabei die dynamisch, d. h. bezüglich der zeitlichen Abfolge aufgelöste Messung der Reaktionskinetik in den Vordergrund, wodurch organische Stoffwechselvorgänge nachvollziehbar werden. Als Beispiel seien dazu sogenannte Retinalproteine, wie Rhodopsin oder Bakterio- rhodopsin, genannt, welche lichtangeregte Protonenpumpen darstellen. Nahezu alle anderen Stoffwechselprozesse gehen ebenfalls mit strukturellen Änderungen der daran beteiligten Proteine, d. h. Reaktionen auf molekularer Ebene einher.The acquisition of time-resolved energy spectra of molecular states is of central importance for understanding the mode of action of proteins at the molecular level. In addition to static structural investigations with atomic resolution, the dynamic, d. H. with regard to the chronological sequence of the measurement of the reaction kinetics in the foreground, which makes organic metabolic processes understandable. As an example, so-called retinal proteins, such as rhodopsin or bacteriorhodopsin, may be mentioned, which represent light-excited proton pumps. Almost all other metabolic processes also involve structural changes in the proteins involved, i.e. H. Reactions at the molecular level.
Zur Untersuchung dynamischer Vorgänge auf molekularer Ebene mit hoher Zeitauflösung ist die Anwendung spektroskopischer Methoden allgemein geläufig, bei denen die Wechselwirkung elektromagnetischer Strahlung, bei- spielsweise Infrarotstrahlung niedriger Energie, mit Molekülen des betreffenden Systems ausgewertet wird. Die zeitaufgelöste Erfassung des Spektrums erfolgt nach Initiierung der zu untersuchenden Reaktion durch externe Energiezufuhr, beispielsweise durch Bestrahlung der Probe mit einem intensiven Laser-Lichtblitz.For the investigation of dynamic processes at the molecular level with high time resolution, the use of spectroscopic methods is generally known, in which the interaction of electromagnetic radiation, for example infrared radiation of low energy, with molecules of the system in question is evaluated. The time-resolved acquisition of the spectrum takes place after initiation of the reaction to be examined by external energy supply, for example by irradiating the sample with an intense laser light flash.
Zur Untersuchung struktureller Veränderungen auf molekularer Ebene wird zur Zeit beispielsweise zeitaufgelöste Raman-Spektroskopie sowie zeitaufgelöste FTIR(Fourier Transform Infrarot)-Differenz-Spektroskopie angewandt. Während bei der Raman-Spektroskopie der Frequenz-, d.h. der Energieunterschied zwischen einfallendem und an der Probe gestreutem IR-Licht gemessen wird, werden bei der FTIR-Methode charakteristische IR-Absorptionen gemessen. Durch die Anwendung von Differentialmethoden lassen sich die interessierenden Energiezustände der funktionell wichtigen Gruppen vor dem unvermeidlichen Hintergrundrauschen mit hoher struktureller Auflösung bestimmen.To investigate structural changes at the molecular level, time-resolved Raman spectroscopy and time-resolved FTIR (Fourier Transform Infrared) difference spectroscopy are currently used, for example. While in Raman spectroscopy the frequency, i.e. If the energy difference between incident and scattered IR light is measured, the FTIR method measures characteristic IR absorptions. By using differential methods, the energy states of interest of the functionally important groups can be determined with high structural resolution against the inevitable background noise.
Für die Aufnahme eines kompletten FTIR-Spektrums sind IR-Absorptions- messungen bei einer vorgegebenen Anzahl von Energie-Messwerten jeweils für voreingestellte Meßzeiten durchzuführen. Bei der sogenannten Fast-Scan- Meßmethode wird jeweils zu einer vorgegebenen Zeit nach der Anregung, beispielsweise durch einen Laserblitz, ein Energiespektrum durchgefahren, in dem der gesamte IR-Meßbereich mit einem einstellbaren Interferometer über- strichen wird. Die Zeitauflösung ist dabei durch die Geschwindigkeit des beweglichen Spiegels im Interferometer auf etwa 40ms begrenzt. Bessere Zeitauflösungen erreicht man mit der sogenannten Step-Scan-Technik. Dabei wird am Interferometer eine IR-Wellenlänge, d. h. eine Meßenergie, eingestellt und gehalten, während die Probe durch einen Laserblitz angeregt wird. Über entsprechende Detektoren wird nun das Abklingen der IR-Absorption erfaßt. Das vollständige Spektrum erhält man dabei, indem als Scan-Parameter die Energie am Interferometer jeweils schrittweise ("Step") verstellt werden, wonach jeweils erneut eine Anregung durch einen Lichtblitz erfolgt und die zeitliche Entwicklung der IR-Absorption gemessen wird ("Scan"). Die Zeitauflösung diese Meßmethode wird lediglich durch die Anstiegszeit des verwendeten Detektors begrenzt und liegt im ns-Bereich, ist also nahezu um drei Größenordnungen besser als bei der vorgenannten Fast-Scan-Technik. Durch die Step-Scan-Technik, insbesondere im Zusammenhang mit der Durchführung von FTIR bei Anregung durch intensive Laserpulse, wird die Probensubstanz durch die eingekoppelte Energie erheblich belastet. Grundsätzliche Bedeutung erhält dieser Umstand bei Messungen an nicht zyklisch arbeitenden Systemen, die durch den Anregungs-Lichtblitz von einem Ausgangszustand in einen Endzustand versetzt werden, ohne danach selbsttätig in den Ausgangszustand zurückzukehren. Bei den bisher bekannten Vorrichtungen zur Durchführung von FTIR-Step-Scan-Meßverfahren, wie sie beispielsweise in der Zeitschrift Applied Spectroscopy 51 , Nr. 4, 1 997, Seiten 558 - 562, beschrieben sind, ist es dabei erforderlich, vor jedem Reaktionsstart die durch die vorherige Messung irreversibel veränderte Probe jeweils auszutauschen. Dies führt natürlich dazu, daß die Aufnahme vollständiger Spektren einen enormen Arbeits- und Zeitaufwand mit sich bringt.In order to record a complete FTIR spectrum, IR absorption measurements are to be carried out for a predetermined number of energy measurement values each for preset measurement times. In the so-called fast-scan measurement method, an energy spectrum is run through at a predetermined time after the excitation, for example by a laser flash, in which the entire IR measurement range is scanned with an adjustable interferometer. The time resolution is limited to about 40 ms by the speed of the moving mirror in the interferometer. Better time resolutions can be achieved with the so-called step-scan technique. An IR wavelength, ie a measurement energy, is set and held on the interferometer while the sample is excited by a laser flash. The decay of the IR absorption is now detected by means of appropriate detectors. The complete spectrum can be obtained by adjusting the energy on the interferometer step by step ("Step"), after which each time there is another excitation by a flash of light and the temporal development of the IR absorption is measured ("Scan") . The time resolution of this measurement method is only limited by the rise time of the detector used and is in the ns range, which is almost three orders of magnitude better than with the aforementioned fast scan technique. The step-scan technique, particularly in connection with the implementation of FTIR when excited by intense laser pulses, places a considerable strain on the sample substance due to the energy input. This fact is of fundamental importance for measurements on systems that do not operate cyclically, which are shifted from an initial state to an end state by the excitation light flash, without then automatically returning to the initial state. In the previously known devices for carrying out FTIR step scan measurement methods, such as are described, for example, in the magazine Applied Spectroscopy 51, No. 4, 1 997, pages 558-562, it is necessary to do this before each reaction start to replace the irreversibly modified sample by the previous measurement. This naturally leads to the fact that the recording of complete spectra involves an enormous amount of work and time.
Daraus ergibt sich die Aufgabe der Erfindung, ein Verfahren zur Durchfüh- rung von Step-Scan-Meßverfahren, hier insbesondere auf der Grundlage von FTIR, anzugeben, bei dem ein vollständiges Spektrum in kürzerer Meßzeit mit geringem Arbeitsaufwand aufgenommen werden kann.This results in the object of the invention to provide a method for performing step-scan measurement methods, here in particular based on FTIR, in which a complete spectrum can be recorded in a shorter measurement time with little effort.
Zur Lösung der vorgenannten Problematik schlägt die Erfindung ausgehend von dem vorgenannten, nach dem Stand der Technik bekannten Verfahren vor, daßTo solve the above-mentioned problems, the invention proposes, based on the above-mentioned methods known from the prior art, that
• innerhalb einer flächenhaft ausgedehnten Probe eine Vielzahl von räumlich getrennten lokalisierten Probensegmenten definiert wird,A multiplicity of spatially separated localized sample segments is defined within an area-wide sample,
• eine Mehrzahl der Probesegmente sequentiell nacheinander lokal angeregt wird und dabei als Meßsignal jeweils die Intensität der Infrarot- Strahlung nach der Wechselwirkung mit der Probe für einen jeweils voreingestellten spektralen Scan-Parameter gemessen wird,A plurality of the sample segments are sequentially locally excited one after the other and the intensity of the infrared radiation after the interaction with the sample is measured as a measurement signal for a respectively preset spectral scan parameter,
• der Scan-Parameter schrittweise zwischen der Messung einzelner Probensegmente so variiert wird, daß mit einer vorgegebenen Anzahl von Messungen alle Meßwerte innerhalb des Scan-Meßbereichs durchgefahren werden. Der besondere Vorteil des erfindungsgemäßen Verfahrens liegt darin, daß zur Aufnahme eines dynamischen Spektrums, welches sowohl im Fast-Scan- als auch im Step-Scan-Verfahren eine Vielzahl von externen Anregungen erfordert, eine den vorab definierten Probensegmenten entsprechende Anzahl von Anregungen und Scan-Messungen an einer einzigen Probe durchführbar sind, ohne daß diese ausgetauscht werden müßte. Erfindungsgemäß wird dabei die Anregungsstrahlung, d. h. der Laserblitz, innerhalb eines Probensegments lokal auf der Probe fokussiert. Sowohl der Laserblitz als auch die IR- Meßstrahlung werden innerhalb eines Probensegments auf einen Durchmesser in der Größenordnung von 100 - 500μm begrenzt. Die einzelnen Probensegmente bilden in ihrer Gesamtheit ein Meß-Raster und sind zweckmäßigerweise so bemessen, daß durch eine irreversible Anregung in einem Probesegment die Probe in den anderen Probensegmenten nicht beeinflußt wird. Dadurch ist auch die Wärmebelastung relativ gering.• The scan parameter is varied step by step between the measurement of individual sample segments in such a way that all measurement values within the scan measurement range are carried out with a predetermined number of measurements. The particular advantage of the method according to the invention is that to record a dynamic spectrum, which requires a large number of external excitations both in the fast scan and in the step scan method, a number of excitations and scan numbers corresponding to the previously defined sample segments Measurements can be carried out on a single sample without it having to be exchanged. According to the invention, the excitation radiation, ie the laser flash, is focused locally on the sample within a sample segment. Both the laser flash and the IR measurement radiation are limited to a diameter in the order of 100-500 μm within a sample segment. The individual sample segments as a whole form a measurement grid and are expediently dimensioned such that the sample in the other sample segments is not influenced by irreversible excitation in one sample segment. As a result, the heat load is also relatively low.
Zur Aufnahme eines Spektrums im FTIR-Step-Scan-Verfahren wird als Scan- Parameter, d. h. als Meßparameter, die Spiegelposition X; des beweglichen Spiegels am Interferometer, also die IR-Energien in fourier-transformierter Darstellung, eingestellt. Anschließend wird die Reaktion innerhalb eines vorbestimmten Probensegments durch Einstrahlung eines Laserblitzes gestartet und die sich dabei ergebenden IR-Absorptionsänderungen als Meßwert über die Zeit erfaßt. Anschließend wird ein anderes Probensegment, welches bisher innerhalb der Meßsequenz noch nicht angeregt worden ist, ausgewählt und die vorgenannte Messung wiederholt. In Abhängigkeit von der gewünschten Anzahl der Mittelungen für einen Energiewert wird die IR-Wellenlänge am Interferometer zwischen aufeinanderfolgenden Anregungen, d. h. Messungen an unterschiedlichen Probensegmenten jeweils variiert, und zwar bis alle gewünschten Meßpunkte innerhalb des Scan-Meßbereichs durchgefahren sind.To record a spectrum using the FTIR step scan method, the scan parameter, ie. H. as the measurement parameter, the mirror position X; of the moving mirror on the interferometer, i.e. the IR energies in a Fourier-transformed representation. The reaction is then started within a predetermined sample segment by irradiation of a laser flash and the resulting IR absorption changes are recorded as a measured value over time. Then another sample segment, which has not yet been excited within the measurement sequence, is selected and the aforementioned measurement is repeated. Depending on the desired number of averages for an energy value, the IR wavelength on the interferometer between successive excitations, i. H. Measurements on different sample segments vary in each case until all the desired measuring points within the scan measuring range have been passed.
Ein besonderer Vorteil des erfindungsgemäßen Verfahrens liegt darin, daß vollständige Spektren an nicht zyklisch arbeitenden Systemen, beispielsweise caged ATP, an einer einzigen, flächig ausgedehnten Probe aufgenommen werden können, ohne diese jeweils zwischen zwei Messungen auswechseln zu müssen. Bei einem Probendurchmesser in der Größenordnung von einigen Millimetern bis hin zu einigen Zentimetern läßt sich nämlich eine hinreichende Anzahl von Probensegmenten definieren, die jeweils einzeln einem irreversiblen Meßvorgang unterzogen werden. Durch den im Verhältnis zur Gesamtprobefläche kleinen lokalen Meßbereich wird durch den Laserblitz jeweils nur eine relativ geringe Energie eingekoppelt, so daß die Zustandsänderung der Probe jeweils nur lokal begrenzt ist. Indem die Probe und der Einkopplungsbereich der Anregungs- und Meßstrahlung zwischen zwei Messungen jeweils relativ zueinander verfahren werden, läßt sich an einer einzigen Probe eine der Anzahl der Probensegmente entsprechende Vielzahl von Messungen durchführen.A particular advantage of the method according to the invention is that complete spectra on systems which do not operate cyclically, for example caged ATP, can be recorded on a single, areally extended sample without having to change them between two measurements. With a sample diameter of the order of magnitude from a few millimeters to a few centimeters, a sufficient number of sample segments can be defined, each of which is individually subjected to an irreversible measurement process. Due to the small local measuring range in relation to the total sample area, only a relatively small amount of energy is coupled in by the laser flash, so that the change in state of the sample is only locally limited in each case. By moving the sample and the coupling area of the excitation and measuring radiation between two measurements relative to each other, a large number of measurements corresponding to the number of sample segments can be carried out on a single sample.
Die Probensegmente beim erfindungsgemäßen Verfahren sind kleine Flächenbereiche einer Probe, die durch eine homogene, durchgehende dünne Schicht der Probensubstanz gebildet werden. Diese läßt sich relativ einfach dadurch präparieren, daß die Probensubstanz zwischen IR-durchlässige Fenster auf eine Dicke von wenigen //m eingepreßt wird. Dadurch lassen sich Absorptionsmessungen in Transmission besonders gut durchführen. Es ist auch denkbar, die Probensegmente voneinander separat zu gestalten.The sample segments in the method according to the invention are small surface areas of a sample which are formed by a homogeneous, continuous thin layer of the sample substance. This can be prepared relatively simply by pressing the sample substance between IR-transparent windows to a thickness of a few // m. As a result, absorption measurements in transmission can be carried out particularly well. It is also conceivable to design the sample segments separately from one another.
Innerhalb einer Meßsequenz, in deren Verlauf alle oder zumindest ein Teil der zu erfassenden Meßwerte durchgefahren werden, kann vorzugsweise vorge- sehen werden, daß jeweils zwei aufeinanderfolgende Messungen jeweils nicht unmittelbar in der Probenfläche benachbarte Probensegmente betreffen. Dadurch erreicht man zum einen eine gleichmäßig verteilte Energieeinkopplung durch die Laserblitze in die gesamte Probe und vermeidet dadurch lokale Beanspruchungen durch Erwärmung. Zum anderen werden durch eine stochastische Verteilung der betrachteten Probensegmente systematische Fehler durch Inhomogenitäten der Probe reduziert.Within a measurement sequence, in the course of which all or at least some of the measurement values to be recorded are run through, it can preferably be provided that two successive measurements each do not relate directly to adjacent sample segments in the sample area. On the one hand, this achieves an evenly distributed coupling of energy through the laser flashes into the entire sample, thereby avoiding local stresses caused by heating. On the other hand, systematic errors due to inhomogeneity of the sample are reduced by a stochastic distribution of the sample segments under consideration.
Das erfindungsgemäße Meßverfahren kann sowohl für FTIR-Messungen, als auch für FT-Raman-Messungen, als auch für sonstige lokale Meßverfahren verwendet werden. Die Durchführung von Fast-Scan-Methoden, bei denen als Scan-Meßparameter die Meßzeit schrittweise durchgefahren wird, ist gleichermaßen möglich. Eine Vorrichtung zur Durchführung des vorgenannten Verfahrens geht im wesentlichen aus von einem vorbeschriebenen Meßaufbau aus, beispielsweise einem FTIR-Mikroskop wie in Applied Spectroscopy 51 , Nr. 4, 1 997, S. 558 - 562, angegeben. Dieser weist eine Anregungsstrahlquelle, beispielsweise einen Laser, optische Elemente zur Fokussierung der Anregungsstrahlung auf ein Meßfeld einer Probe, eine Infrarotstrahlungsquelle, entsprechende optische Elemente zur Erfassung eines aus dem Meßfeld der Probe abgegebenen Infrarot-Meßsignals, einen Infrarot-Detektor sowie einen verstellbaren Energiefilter, beispielsweise ein Interferometer, auf, der im Strahlengang vor dem Infrarot-Detektor angeordnet ist und an dem die jeweilige Infrarot-Meßenergie einstellbar ist. Die dazugehörigen Steuer-Meß- und Auswerteeinheiten, die zur Durchführung von Scan-Messungen erforderlich sind, sind nach dem Stand der Technik ebenfalls bekannt.The measuring method according to the invention can be used for FTIR measurements as well as for FT Raman measurements as well as for other local measuring methods. It is equally possible to carry out fast scan methods in which the measurement time is passed step by step as the scan measurement parameter. A device for performing the aforementioned method essentially starts from a previously described measurement setup, for example an FTIR microscope as specified in Applied Spectroscopy 51, No. 4, 1 997, pp. 558-562. This has an excitation beam source, for example a laser, optical elements for focusing the excitation radiation onto a measuring field of a sample, an infrared radiation source, corresponding optical elements for detecting an infrared measuring signal emitted from the measuring field of the sample, an infrared detector and an adjustable energy filter, for example an interferometer, which is arranged in the beam path in front of the infrared detector and on which the respective infrared measurement energy can be set. The associated control measuring and evaluation units, which are required to carry out scan measurements, are also known from the prior art.
Zur Umsetzung des vorgenannten, erfindungsgemäßen Verfahrens wird erfindungsgemäß vorgesehen, daß die Probe flächenhaft ausgedehnt ist, die Probenfläche ein Vielfaches des Meßfelds beträgt, und daß die Probe in einer Scan-Einrichtung angeordnet ist, in der das Meßfeld auf verschiedene Probensegmente innerhalb der Probenfläche verfahrbar ist.To implement the aforementioned method according to the invention, it is provided according to the invention that the sample is expanded in terms of area, the sample area is a multiple of the measurement field, and that the sample is arranged in a scanning device in which the measurement field can be moved onto different sample segments within the sample area .
Bei der erfindungsgemäßen Scan-Einrichtung handelt es sich beispielsweise um einen Kreuztisch, in den die Probe eingespannt wird, und an dem die X- Y-Koordinaten der Probe derart einstellbar sind, daß mit dem Anregungs- und Meßstrahl alle vordefinierten Probensegmente sequentiell erreichbar sind.The scanning device according to the invention is, for example, a cross table in which the sample is clamped and on which the X-Y coordinates of the sample can be set such that all predefined sample segments can be reached sequentially with the excitation and measuring beam.
Grundsätzlich ist es gleichgültig, ob zur Erzielung der Relativbewegung der Meßkopf zur Ankopplung der Anregungsstrahlung und Auskopplung des Meßsignals räumlich festgehalten wird und die Probe verfahren wird oder umgekehrt. Es ist jedoch vorteilhaft, daß die Probe selbst auf einem motorisch in der Ebene der Probenfläche verfahrbaren Probenhalter angeordnet ist, d. h. einem Kreuztisch. Daraus ergibt sich ein einfacherer optischer und mechanischer Aufbau, als wenn der Meßkopf selbst verfahren werden müßte. Vorzugsweise wird ein FTIR-Spektrometer mit einer erfindungsgemäßen Pro- benhalterung ausgerüstet. Die Anbringung an einem Raman-Spektrometer oder einer sonstigen spektrometrischen Meßeinrichtung ist jedoch ebenfalls denkbar.In principle, it does not matter whether the measuring head for coupling the excitation radiation and coupling out the measuring signal is spatially held in order to achieve the relative movement and whether the sample is moved or vice versa. However, it is advantageous for the sample itself to be arranged on a sample holder which can be moved by motor in the plane of the sample surface, ie a cross table. This results in a simpler optical and mechanical structure than if the measuring head itself had to be moved. An FTIR spectrometer is preferably equipped with a sample holder according to the invention. Attachment to a Raman spectrometer or other spectrometric measuring device is, however, also conceivable.
Die Messungen können sowohl in Transmission als auch in Reflexion erfolgen. Besonders gut geeignet für Transmissionsmessungen sind Proben, bei denen die Probensubstanz mit einer Schichtdicke von einigen μm zwischen zwei flächenhaft ausgedehnten, IR-durchlässigen Fenstern eingebracht ist. Zur Einkopplung der Anregungsstrahlung, d. h. des Laserblitzes, koaxial zum IR-Strahlengang ist zweckmäßigerweise vor der IR-Meßoptik ein Win- kelspiegei angeordnet, mit dem der von seitlich eingestrahlte Laserblitz senkrecht auf die Probenoberfläche im jeweils zu messenden Probensegment gelenkt wird. Bevorzugt wird hierzu ein Quarzprisma verwendet, da dieses der hohen Belastung durch die Laserblitze ohne weiteres dauerhaft standhält.The measurements can take place both in transmission and in reflection. Samples in which the sample substance is inserted with a layer thickness of a few μm between two extensive, IR-transparent windows are particularly well suited for transmission measurements. To couple the excitation radiation, d. H. of the laser flash, coaxial to the IR beam path, an angle mirror is expediently arranged in front of the IR measurement optics, with which the laser flash irradiated from the side is directed perpendicularly onto the sample surface in the sample segment to be measured in each case. A quartz prism is preferably used for this, since it easily withstands the high load from the laser flashes.
Eine vorteilhafte Weiterbildung der erfindungsgemäßen Vorrichtung sieht vor, daß die IR-Meßoptik ein Cassegrain-Objektiv aufweist. Das als Umlenkelement verwendete Quarzprisma wird dann bevorzugt vor dem Fangspiegel des Cassegrain-Objektivs angebracht. Mittels einer schrägstehenden Reflexionsfläche ist es dann möglich, einen seitlich eingestrahlten Laserstrahl senkrecht in die Probenfläche einzustrahlen.An advantageous development of the device according to the invention provides that the IR measuring optics have a Cassegrain objective. The quartz prism used as the deflection element is then preferably attached in front of the secondary mirror of the Cassegrain objective. By means of an inclined reflection surface, it is then possible to radiate a laterally irradiated laser beam perpendicularly into the sample surface.
Zur Verringerung der in die Probe bei einer Anregung mit einem Laserblitz jeweils eingebrachten Energiemenge wird das Profil des Laserstrahls zweckmäßigerweise durch einen Raumfilter, beispielsweise in Form eines Pinholes, eingegrenzt.In order to reduce the amount of energy introduced into the sample during excitation with a laser flash, the profile of the laser beam is expediently limited by a spatial filter, for example in the form of a pinhole.
Das erfindungsgemäße Verfahren sowie eine Vorrichtung zur Durchführung des Verfahrens werden im folgenden anhand der Zeichnungen näher erläutert. Hierzu zeigen im einzelnenThe method according to the invention and an apparatus for carrying out the method are explained in more detail below with reference to the drawings. Show this in detail
Fig. 1 : schematisch eine Draufsicht auf eine Probe; Fig. 2: ein FTIR-Spektrum;1: schematically a top view of a sample; 2: an FTIR spectrum;
Fig. 3: eine schematische Darstellung eines kombinierten FTIR-Meßkopfes mit einem erfindungsgemäßen Probenhalter;3: a schematic representation of a combined FTIR measuring head with a sample holder according to the invention;
Fig. 4: einen schematischen Aufbau einer vollständigen FTIR-Meßvorrichtung.Fig. 4: a schematic structure of a complete FTIR measuring device.
Fig. 1 zeigt eine schematische Aufsicht auf eine Probe 1 , wobei die Probensubstanz die Form einer in der X-Y-Ebene liegenden, runden Scheibe hat. Diese ist beispielsweise zwischen im einzelnen nicht dargestellten, IR- durchlässigen CaF2-Fenstern mit einer Schichtdicke von einigen μm eingequetscht.1 shows a schematic plan view of a sample 1, the sample substance being in the form of a round disk lying in the XY plane. This is for example squeezed between IR-transparent CaF 2 windows, not shown in detail, with a layer thickness of a few μm.
Wie dargestellt wird in XY-Richtung ein Meßraster definiert. Jeweils auf den Kreuzungspunkten des Meßrasters liegen die als gefüllter Kreis eingezeichneten Meßfelder x(n), die gemäß dem erfindungsgemäßen Verfahren nachein- ander, beispielsweise in einer Meßsequenz x(n), x(n + 1 ), x(n + 2), ... lokal durch einen Laserblitz angeregt und beispielsweise mittels FTIR gemessen werden.As shown, a measurement grid is defined in the XY direction. The measuring fields x (n) drawn in as a filled circle lie on the intersection points of the measuring grid, which are in succession according to the method according to the invention, for example in a measuring sequence x (n), x (n + 1), x (n + 2), ... excited locally by a laser flash and measured using FTIR, for example.
Fig. 2 zeigt schematisch ein im FTIR-Step-Scan-Verfahren aufgenommenes Energiespektrum. Dabei wird als Scan-Parameter die Energie der IR-Strahlung schrittweise durchgefahren. Für jeden Energiewert erfolgt mindestens eine Anregung durch einen Laserblitz, was schematisch durch die Blitze bei den Energiewerten x(n) und x(n + 1 ) dargestellt ist. Nach jeder Anregung wird die IR-Intensität l(t), d. h. die Absorption zum Zeitnullpunkt t = 0 sowie danach in zeitlichen Intervallen t(i) gemessen. Auf diese Weise erhält man ein voll- ständiges Energiespektrum.2 schematically shows an energy spectrum recorded in the FTIR step scan method. The energy of the IR radiation is passed through step by step as a scan parameter. For each energy value there is at least one excitation by a laser flash, which is shown schematically by the flashes for the energy values x (n) and x (n + 1). After each excitation, the IR intensity l (t), i.e. H. the absorption at the time zero t = 0 and then measured at time intervals t (i). In this way you get a complete energy spectrum.
Gemäß dem erfindungsgemäßen Verfahren erfolgt die Anregung und die Messung der IR-Intensität für die IR-Meßenergien x(n), x(n + 1 ), ... jeweils lokal an den entsprechenden Probensegmenten gemäß dem in Fig. 1 darge- stellten Meßraster. Hierzu wird die Probe 1 bei der Aufnahme des Spektrums relativ zur Meßvorrichtung jeweils so verfahren, daß vor einer Messung jeweils das entsprechende Probensegment im Strahlengang der Meßvorrichtung steht.According to the method according to the invention, the excitation and measurement of the IR intensity for the IR measurement energies x (n), x (n + 1),... Take place locally on the corresponding sample segments in accordance with the method shown in FIG. provided measuring grids. For this purpose, sample 1 is moved relative to the measuring device when recording the spectrum so that the corresponding sample segment is in the beam path of the measuring device before a measurement.
Fig. 3 zeigt einen Meßkopf 2, mit dem die von unten durch die Probe 1 transmissiv gestrahlte IR-Meßstrahlung über eine Cassegrain-Spiegeloptik erfaßbar ist. Zur koaxialen Einkopplung des Laserblitzes ist zentral am Meßkopf 2 ein optisches Umlenkelement, beispielsweise ein Quarzprisma 3 angeordnet, mit dem ein radial eingestrahlter Laserblitz senkrecht von oben auf die Probe 1 eingestrahlt werden kann. Der Meßkopf 2 ist als Cassegrain- Objektiv ausgebildet, wobei das Quarzprisma 3 vor dem zentralen Fangspiegel angebracht ist.FIG. 3 shows a measuring head 2 with which the IR measuring radiation radiated by the sample 1 from below can be detected via a Cassegrain mirror optic. For the coaxial coupling of the laser flash, an optical deflection element, for example a quartz prism 3, is arranged centrally on the measuring head 2, with which a radially irradiated laser flash can be irradiated onto the sample 1 vertically from above. The measuring head 2 is designed as a Cassegrain lens, the quartz prism 3 being attached in front of the central secondary mirror.
Während der Meßkopf 2 während der Messung seine räumliche Position beibehält, ist die Probe 1 auf einem erfindungsgemäßen Kreuztisch 4 in XY- Richtung verfahrbar eingespannt. Mittels eines nicht dargestellten, rechnergesteuerten motorischen Antriebs kann damit jedes Probensegment x(n) vor dem Meßkopf 2 so positioniert werden, daß es lokal durch einen Laserblitz angeregt und anschließend durch Intensitätsmessung der IR- Meßstrahlung gemessen werden kann.While the measuring head 2 maintains its spatial position during the measurement, the sample 1 is clamped on a cross table 4 according to the invention so as to be movable in the XY direction. By means of a computer-controlled motor drive (not shown), each sample segment x (n) can thus be positioned in front of the measuring head 2 in such a way that it can be locally excited by a laser flash and then measured by measuring the intensity of the IR measuring radiation.
Fig. 4 zeigt schematisch einen FTIR-Meßaufbau. Darin ist die Anordnung einer über ein Interferometer bezüglich der Energie einstellbaren IR-Meßstrah- lungsquelle 5, eines Lasers 6, eines IR-Detektors 7 sowie dazugehöriger Steuer- und Meßelektronik 8 zu erkennen.Fig. 4 shows schematically an FTIR measurement setup. This shows the arrangement of an IR measuring radiation source 5 that can be adjusted with respect to the energy via an interferometer, a laser 6, an IR detector 7 and the associated control and measuring electronics 8.
Die dargestellte Vorrichtung stellt im wesentlichen eine bekannte Anlage zur Durchführung von FTIR-Messungen dar, die durch einen steuerbaren Kreuztisch zur Durchführung des erfindungsgemäßen Verfahrens erweitert ist. Dadurch lassen sich die bereits dargelegten Vorteile des erfindungsgemäßen Verfahrens realisieren. The device shown essentially represents a known system for carrying out FTIR measurements, which is expanded by a controllable cross table for carrying out the method according to the invention. The advantages of the method according to the invention which have already been explained can thereby be realized.

Claims

10Patentansprüche 10 patent claims
1 . Verfahren zur zeitaufgelösten Messung von Energiespektren molekularer Zustände bei extern angeregten Reaktionen und Änderungen innerhalb von Molekülen, insbesondere bei nicht zyklisch arbeitenden1 . Process for the time-resolved measurement of energy spectra of molecular states in externally excited reactions and changes within molecules, especially in non-cyclical ones
Systemen, durch Messung der Wechselwirkung von Infrarotstrahlung mit den Molekülen des Systems, wobeiSystems, by measuring the interaction of infrared radiation with the molecules of the system, whereby
• innerhalb einer flächenhaft ausgedehnten Probe eine Vielzahl von räumlich getrennten, lokalisierten Probensegmenten definiert wird,A multiplicity of spatially separated, localized sample segments is defined within an area-wide sample,
• eine Mehrzahl der Probensegmente sequentiell nacheinander lokal angeregt wird und dabei als Meßsignal jeweils die Intensität der Infrarotstrahlung nach der Wechselwirkung mit der Probe für einen jeweils voreingestellten spektralen Scan-Parameter gemessen wird,A plurality of the sample segments are sequentially locally excited one after the other, and the intensity of the infrared radiation after the interaction with the sample is measured as a measurement signal for a respectively preset spectral scan parameter,
• der Scan-Parameter schrittweise zwischen der Messung einzelner Probensegmente so variiert wird, daß mit einer vorgegebenen Anzahl von Messungen alle Meßwerte innerhalb des Scan- Meßbereichs durchgefahren werden.• The scan parameter is varied step by step between the measurement of individual sample segments in such a way that, with a predetermined number of measurements, all measurement values within the scan measurement range are carried out.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die2. The method according to claim 1, characterized in that the
Messungen mit zeitaufgelöster FTIR-Spektroskopie erfolgen. 11Measurements are carried out with time-resolved FTIR spectroscopy. 11
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß als Scan-Parameter der Energiewert der absorbierten Infrarotstrahlung vorgegeben wird.3. The method according to claim 2, characterized in that the energy value of the absorbed infrared radiation is specified as the scan parameter.
4. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die Anregung der Probe mit einem jeweils in ein Probensegment eingestrahlten4. The method according to claim 1, characterized in that the excitation of the sample with one irradiated in each case in a sample segment
Laserblitz erfolgt.Laser flash occurs.
5. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß der Meßkopf, über den die Anregungsstrahlung angekoppelt und das Infrarot- Meßsignal ausgekoppelt wird, und die Proben zwischen zwei Messungen relativ zueinander verfahren werden, so daß die Probensegmente nach einer vorgebbaren Meßsequenz sequentiell nacheinander gemessen werden.5. The method according to claim 1, characterized in that the measuring head, via which the excitation radiation is coupled and the infrared measuring signal is coupled out, and the samples are moved relative to one another between two measurements, so that the sample segments are measured sequentially in succession after a predeterminable measuring sequence .
6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß die in einer Meßsequenz aufeinanderfolgenden Messungen jeweils nicht unmittelbar in der Probenfläche benachbarte Probensegmente betref fen.6. The method according to claim 5, characterized in that the successive measurements in a measurement sequence each not directly in the sample area adjacent sample segments fen.
7. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, daß die7. The method according to claim 1, characterized in that the
Messungen mit Raman-Spektroskopie erfolgen.Measurements are made using Raman spectroscopy.
8. Vorrichtung zur Durchführung des Verfahrens nach Anspruch 1 mit einer Anregungsstrahlungsquelle, optischen Elementen zur Einstrahlung der Anregungsstrahlung auf ein Meßfeld einer Probe, einer Infrarot- Strahlungsquelle, optischen Elementen zur Erfassung eines aus dem Meßfeld abgegebenen Infrarot-Meßsignals, einem Infrarot-Detektor sowie einem verstellbaren Energiefilter, der im Strahlengang vor dem Infrarot-Detektor angeordnet ist und an dem die jeweilige Infrarot-Meßenergie einstellbar ist, sowie Speicher-, Meß- und Auswerteeinheiten, dadurch gekennzeichnet, daß die Probe ( 1 ) flächenhaft ausgedehnt ist, die Probenfläche ein Vielfaches des Meßfeldes beträgt und daß die Probe (1 ) in einer Scan- Einrichtung (4) angeordnet ist, in der das Meßfeld auf verschiedene Probensegmente innerhalb der Probenfläche verfahrbar ist. 128. An apparatus for performing the method according to claim 1 with an excitation radiation source, optical elements for irradiating the excitation radiation onto a measuring field of a sample, an infrared radiation source, optical elements for detecting an infrared measuring signal emitted from the measuring field, an infrared detector and one adjustable energy filter, which is arranged in the beam path in front of the infrared detector and on which the respective infrared measurement energy can be set, as well as storage, measuring and evaluation units, characterized in that the sample (1) is expanded over a large area, the sample area is a multiple of the measuring field and that the sample (1) is arranged in a scanning device (4) in which the measuring field can be moved to different sample segments within the sample area. 12
9. Vorrichtung nach Anspruch 8, dadurch gekennzeichnet, daß die Probe ( 1 ) auf einem motorisch in der Ebene der Probenfläche verfahrbaren Probenhalter (4) angeordnet ist.9. The device according to claim 8, characterized in that the sample (1) is arranged on a motor-driven in the plane of the sample surface sample holder (4).
10. Vorrichtung nach Anspruch 8, dadurch gekennzeichnet, daß die Vorrichtung als FTIR-Spektrometer ausgebildet ist.10. The device according to claim 8, characterized in that the device is designed as an FTIR spectrometer.
1 1 . Vorrichtung nach Anspruch 8, dadurch gekennzeichnet, daß die Vorrichtung als Raman-Spektrometer ausgebildet ist.1 1. Device according to claim 8, characterized in that the device is designed as a Raman spectrometer.
1 2. Vorrichtung nach Anspruch 8, dadurch gekennzeichnet, daß als Umlenkelement zur Einkopplung der Anregungsstrahlung ein Quarzprisma (3) eingesetzt wird.1 2. Device according to claim 8, characterized in that a quartz prism (3) is used as a deflecting element for coupling the excitation radiation.
1 3. Vorrichtung nach Anspruch 8, dadurch gekennzeichnet, daß der Meßkopf (2) als Cassegrain-Objektiv ausgebildet ist.1 3. Device according to claim 8, characterized in that the measuring head (2) is designed as a Cassegrain lens.
1 4. Vorrichtung nach Anspruch 1 3, dadurch gekennzeichnet, daß das Umlenkelement (3) vor dem Fangspiegel der Cassegrain-Optik (2) angebracht ist. 1 4. Device according to claim 1 3, characterized in that the deflecting element (3) is attached in front of the secondary mirror of the Cassegrain optics (2).
PCT/EP1999/000688 1998-02-04 1999-02-03 Method for time-resolved measurement of energy spectra of molecular states and device for carrying out said method WO1999040413A1 (en)

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