Method and System for Spectroscopically Measuring Optical Properties of Samples

Abstract

In a method for the spectrally resolved measurement of optical properties of samples, a sample is arranged at a measurement position, and light is generated using a light source. Spectral components of the light are transmitted as excitation light in a first optical path to the sample. Light that has been emitted or transmitted by the sample is transmitted in a second optical path to a detector. A tunable monochromator is arranged in the first optical path and/or in the second optical path. A spectrum of the emitted or transmitted light is recorded over an effective spectral range by shifting a spectral passage range of the tunable monochromator. The method is characterized in that light in the form of light pulses with a specifiable pulse frequency is used. The spectral passage range of the tunable monochromator is shifted at a shifting speed continuously from an initial wavelength to an end wavelength for recording a spectrum. The pulse frequency of the light is synchronized with the shifting speed of the spectral passage range by way of a control such that a plurality of measurements of the emitted or transmitted light takes place within the effective spectral range at a corresponding plurality of spectral support points.

Claims

1. A method for the spectrally resolved measurement of optical properties of samples, the method comprising the acts of: arranging a sample at a measurement position; generating light using a light source; transmitting spectral components of the light as excitation light in a first optical path to the sample; and transmitting light that has been emitted or transmitted by the sample in a second optical path to a detector; wherein a tunable monochromator is arranged in the first optical path and/or in the second optical path; recording a spectrum of the emitted or transmitted light over an effective spectral range by shifting a spectral passage range of the tunable monochromator, wherein light in the form of light pulses with a specifiable pulse frequency is used; the spectral passage range of the tunable monochromator is shifted at a shifting speed continuously from an initial wavelength to an end wavelength for recording a spectrum; and the pulse frequency of the light is synchronized with the shifting speed of the spectral passage range by way of a controller such that a plurality of measurements of the emitted or transmitted light takes place within the effective spectral range at a corresponding plurality of spectral support points.

2. The method according to claim 1, wherein excitation light is radiated onto the sample in the form of light pulses with a specifiable pulse frequency, wherein excitation light in the form of light pulses with a specifiable pulse frequency is generated by way of a pulsed light source.

3. The method according to claim 2, wherein the spectral passage range is shifted continuously at a constant shifting speed from the starting position to the end position.

4. The method according to claim 1, wherein the spectral passage range is shifted continuously at a constant shifting speed from the starting position to the end position.

5. The method according to claim 1, wherein the spectral passage range is shifted at a varying shifting speed from the starting position to the end position, wherein the shifting speed is varied in dependence on at least one property of the spectrum.

6. The method according to claim 5, wherein the spectral passage range is shifted at a varying shifting speed from the starting position to the end position, wherein the shifting speed is varied in dependence on at least one property of the spectrum.

7. The method according to claim 5, wherein an intensity change in the detected light between successive spectral support points is ascertained during the shifting of the passage range, and the shifting speed of the passage range is changed in dependence on the intensity change.

8. The method according to claim 5, wherein, before recording of a spectrum begins, parameters of a speed variation function are preset, and the shifting speed is controlled in accordance with the speed variation function.

9. The method according to claim 5, wherein a control or regulation of the shifting speed is performed inversely proportionally to the intensity change between successive spectral support points such that spectral ranges with relatively strong intensity changes are travelled with a relatively smaller shifting speed and a correspondingly higher density of the support points, and spectral ranges having relatively weaker intensity changes are travelled with relatively greater shifting speed and a lower density of the support points.

10. The method according to claim 7, wherein a control or regulation of the shifting speed is performed inversely proportionally to the intensity change between successive spectral support points such that spectral ranges with relatively strong intensity changes are travelled with a relatively smaller shifting speed and a correspondingly higher density of the support points, and spectral ranges having relatively weaker intensity changes are travelled with relatively greater shifting speed and a lower density of the support points.

11. The method according to claim 8, wherein a control or regulation of the shifting speed is performed inversely proportionally to the intensity change between successive spectral support points such that spectral ranges with relatively strong intensity changes are travelled with a relatively smaller shifting speed and a correspondingly higher density of the support points, and spectral ranges having relatively weaker intensity changes are travelled with relatively greater shifting speed and a lower density of the support points.

12. The method according to claim 1, wherein the recording of a spectrum over the effective spectral range is repeated at least once with the same synchronization of the pulse frequency with the shifting speed of the spectral passage range, and the measurement values obtained for each of the recordings are added in wavelength-correct fashion.

13. A system for spectrally resolved measurement of optical properties of samples, comprising: a sample holding device for arranging a sample at a measurement position; a light source for generating light; a detector; a control unit; a first optical path for transmitting spectral components of the light as excitation light to the sample; a second optical path for transmitting light that has been emitted or transmitted by the sample to the detector; a tunable monochromator which is controllable by the control unit arranged in the first optical path and/or in the second optical path; wherein the system is configured to record a spectrum of the emitted or transmitted light over an effective spectral range by shifting a spectral passage range of the tunable monochromator, wherein the control unit has an operating mode for recording a spectrum in which the control unit is configured such that: the system is controlled such that light in the form of light pulses with specifiable pulse frequency is used; the spectral passage range of the tunable monochromator is shifted at a shifting speed continuously from an initial wavelength to an end wavelength for recording a spectrum; and the pulse frequency of the light is synchronized with the shifting speed of the spectral passage range by way of a control such that a plurality of measurements of the emitted or transmitted light takes place within the effective spectral range at a corresponding plurality of spectral support points.

14. The system according to claim 13, wherein during the operation mode, the light source is controlled such that excitation light in the form of light pulses with a specifiable pulse frequency is generated.

15. The system according to claim 14, wherein the tunable monochromator is a dispersive monochromator with an adjustable dispersive element, or a tunable filter monochromator, or a tunable interference monochromator.

16. The system according to claim 13, wherein the tunable monochromator is a dispersive monochromator with an adjustable dispersive element, or a tunable filter monochromator, or a tunable interference monochromator.

17. The system according to claim 13, wherein the system is integrated in a multitechnology reader.

18. The system according to claim 14, wherein the system is integrated in a multitechnology reader.

19. The system according to claim 15, wherein the system is integrated in a multitechnology reader.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Further advantages and aspects of the invention can be gathered from the claims and from the following description of preferred exemplary embodiments of the invention, which are explained below with reference to the figures.

[0039] FIG. 1 schematically shows an exemplary embodiment of a system according to the invention for spectrally resolved measurement of optical properties of samples;

[0040] FIG. 2 shows the signal flow between components of the system in a first operating mode;

[0041] FIG. 3 shows the signal flow between components of the system in a second operating mode;

[0042] FIG. 4 shows a diagram for the connection between the motor speed of an actuating motor that is responsible for adjusting a monochromator and the time or the wavelength corresponding to the time;

[0043] FIG. 5 shows a schematic diagram of the dependence of the travel distance of the actuating motor on time in a conventional system with start/stop operation;

[0044] FIG. 6 shows a schematic diagram of the dependence of the travel distance of the actuating motor on time in an exemplary embodiment with continuous change of the spectral location of the spectral passage range;

[0045] FIG. 7 shows an I() diagram with a hypothetical spectrum of a sample substance, wherein the spectrum is scanned with varying support point density by changing the shifting speed of the spectral passage range within the effective spectral range during the continuous shifting in dependence on the local gradient of the spectrum.

DETAILED DESCRIPTION OF THE DRAWINGS

[0046] FIG. 1 schematically shows an exemplary embodiment of a system SYS in accordance with the invention for spectrally resolved measurement of optical properties of samples. The system is a component part of a multitechnology reader, which, in addition to the measurement of fluorescence, also permits other measurements, for example the measurement of the absorption in a sample.

[0047] The system SYS has a primary light source LQ in the form of a xenon flash lamp. The light source has a broad emission spectrum in the visible spectral range (white light). The flash frequency of the light source is settable within specific limits, such that excitation light in the form of light pulses having a specifiable pulse frequency can be generated. The light source LQ is connected to the control unit SE of the system, by way of which the pulse frequency can be set.

[0048] A first optical path OP1, also referred to as the excitation path, leads from the light source LQ, via a tunable first monochromator MC1 and a beam splitter arrangement ST connected downstream, to a measurement position MP, at which a sample P is located during system use. The excitation light is radiated into the sample substantially perpendicularly from above. The sample is located in a depression (e.g. a well) of a microwell plate MPL having many wells.

[0049] The first monochromator MC1 is a tunable dispersive monochromator connected to the control unit SE. The location of the spectral passage range of the first monochromator MC1 can be adjusted continuously over a large spectral range as a reaction to control signals of the control unit SE.

[0050] The optical elements of the first optical path (excitation path) serve for the transmission of spectral components of light from the primary light source LQ as excitation light into the measurement position MP. Located in the sample is a substance that can be excited by the excitation light to emit fluorescent light. The fluorescent light is shifted toward lower energies, or larger wavelengths, with respect to the excitation light. The extent of the spectral red shift is specific to the substance and is referred to as Stokes shift. Information relating to the properties of the sample substance is contained in the spectrum of the emission light.

[0051] The emission light passes, via a second optical path OP2 (also referred to as the emission path), from the sample P to a detector DET, which generates electrical signals in dependence on incident light, which are fed to an evaluation unit in order to spectrally evaluate the emission light for characterizing the sample. The evaluation unit can be integrated in the control unit SE.

[0052] Located in the second optical path between the beam splitter arrangement ST and the detector is a tunable second monochromator MC2, which is provided for transmitting, from the spectrum of the emission light, only a relatively narrow portion, i.e. a spectral passage range with a specifiable spectral location, to the detector at any one time. The second monochromator MC2 is also connected to the control unit SE, with the result that the spectral location of the passage range can be specified at any time by way of control signals of the control unit.

[0053] Arranged below the measurement position MP is an absorption detector ABS, which is likewise connected to the control unit SE. The absorption detector can be used to measure the intensity of emission light which, after excitation of the sample, passes via the first optical path OP1 through the sample and a transparent bottom of the well to the absorption detector.

[0054] The first monochromator MC1 in the first optical path and the second monochromator MC2 are in the form of a dispersive double monochromator. A double monochromator of this type has three gaps overall, specifically an entry gap, a central gap, and an exit gap. The central gap is at once the exit gap of a first dispersive monochromator that is connected upstream and the entry gap of a second dispersive monochromator that is connected downstream. Each of the gaps is defined by a corresponding gap aperture in an associated gap plane. The gap width is able to be adjusted in each case in a stepless fashion. It is also possible in other embodiments, for example for cost reasons, for fixed gaps or gap widths to be provided at the entry and/or at the exit.

[0055] Provided within the double monochromators are in each case dispersive elements in the form of concave reflection gratings. The reflection gratings are rotatably mounted and can be rotated or pivoted synchronously with one another about mutually parallel rotational axes by way of a stepper motor M. As a result, the spectral passage range of each of the monochromators can be shifted continuously from a starting wavelength to an end wavelength at a shifting speed that is specifiable by the control unit SE for recording of a spectrum.

[0056] A synchronization module in the software of the control unit SE can be used to synchronize the pulse frequency of the light source LQ with the shifting speed of the spectral passage range of the first monochromator MC1 and/or of the second monochromator MC2 such that a large number of measurements of the emitted or transmitted light can take place within an effective spectral range of interest at a corresponding large number of spectral support points.

[0057] The system SYS can be operated in different operating modes. Two of the operating modes are explained by way of example with reference to FIGS. 2 and 3. These figures each show schematic diagrams for signal transmission between different components of the system. A microcontroller C of the control unit is connected, in signal-conducting fashion, to the motor controller MOTC of the stepper motor of a monochromator MC, which can be a monochromator in the first optical path or in the second optical path. The microcontroller C also controls the flash operation of the light source LQ, i.e. the emission of light pulses PU with a specifiable pulse frequency or at specifiable times t.sub.1, t.sub.2 etc. In the first operating mode (of FIG. 2), the microcontroller C triggers both the light source LQ (flash lamp) and the motor travel of the stepper motor, which controls the rotational movement of the dispersive grating in the monochromator MC and thus the shifting of the spectral passage range. In the second operating mode (of FIG. 3), the motor is operated at a specifiable motor travel speed, and the motor travel speed determines the flash frequency. It is also possible for the light source to be triggered directly via the motor controller. In this case, the motor controller is connected, in signal-conducting fashion, to the light source.

[0058] In both operating modes it is possible for the rotational movement of the dispersive grating or the dispersive gratings of a monochromator to be synchronized with the flash frequency of the flash lamp during the recording of an absorption spectrum, an excitation spectrum or an emission spectrum. The motor travel of the motor in this case in the monochromator is continuous (without stops in-between). Stopping ramps and braking ramps are dispensed with during the spectrum recording, and they are provided only at the beginning and at the end of a spectral scan. As a result, the spectral passband range of a monochromator shifts continuously, and spectral support points of the spectrum are recorded in sync with the flash lamp.

[0059] FIG. 4 shows, for illustrative purposes, a schematic diagram that shows the connection between the motor speed VM, plotted on the y-axis, of the stepper motor M responsible for adjusting the monochromator as a function of time t (solid line). At times t.sub.1, t.sub.2 etc., the light source LQ emits in each case one flash or light pulse. Since the spectral location of the spectral passage range of the monochromator is adjusted using the stepper motor M, the light pulses which are emitted or received at different times correspond to different wavelengths , with the result that the x-axis also acts as the axis for the wavelength .

[0060] During this pulsed operation, every light pulse gives a spectral support point ST1, ST2 etc. for the spectrally resolved measurement of the optical properties of the sample. The spectral range that extends from the first first support point ST1 used for the measurement (at time t.sub.1) to the last support point STn (at the time t.sub.n) is referred to here as the effective spectral range SPE. The wavelength associated with the first support point ST1 (or the associated spectral location of the passage range) is referred to as the initial wavelength, and the wavelength associated with the last support point STn (or the associated spectral location of the passage range) is referred to as the end wavelength. The start of the measurement is at the initial wavelength that corresponds to the first support point ST1 (here 500 nm, for example), and the end of the measurement is reached at the last support point STn (which corresponds to an end wavelength of 690 nm in the case of the example).

[0061] An important characteristic of the exemplary embodiment can be seen easily by way of the temporal profile of the motor speed VM. The motor travel of the stepper motor takes place over the entire effective spectral range SPE, i.e. from the initial wavelength to the end wavelength, continuously, specifically in the case of the example at a constant finite movement speed. Before measurement begins, a start-up ramp takes place, during which the motor is accelerated from a standstill (motor speed zero) to the moving speed VS for the spectrum recording. After completion of the measurement, i.e. in time terms after the last support point is reached, comes a braking ramp, during which the motor speed is reduced back to zero.

[0062] If, for example, a typical flash operation is assumed at 100 Hz, the recording for example of a fluorescence spectrum over an effective spectral range of 200 nm with a high spectral resolution of 1 nm only takes 2 seconds (2 s).

[0063] The spectral resolution can be set by varying the travel speed of the monochromator and/or correspondingly by adapting the flash frequency of the flash lamp. For example, if an overview spectrum is recorded with a spectral resolution of 4 nm, it is possible to record a spectrum over an available spectral range of 200 nm to 1000 nm in only 2 s. Depending on the flash lamp used, higher flash frequencies, for example of up to 500 Hz, can also be used, with the result that such a spectrum can then mathematically be recorded in approximately 400 ms. As can be seen in principle from FIG. 4, relatively short time intervals for the start-up ramp and the braking ramp, e.g. in the order of magnitude of 100 ms, should be added to these values for the effective measurement time.

[0064] The spectra recorded in this way theoretically have a slight spectral smear, since the adjustment movement of the dispersive element (one or more) in the monochromator is continued during flashing. However, since typical flash durations are frequently in the order of magnitude of 2 s, while the dead time at typical frequencies of 100 Hz is around 10 ms, then at a spectral resolution of a theoretical 1 nm, the smear is approximately 0.2 pm, which is negligibly small in most cases or in all practical cases. At larger spectral steps between the support points (and a correspondingly greater travel speed), the smear becomes greater and can increase, for example at a step width of 10 nm, to approximately 2 pm, which can still be considered negligible for most or all cases.

[0065] To illustrate differences with respect to the prior art, FIG. 5 shows a schematic diagram of the dependence of the travel path SM of the stepper motor of a tunable monochromator on time in a conventional system with start/stop operation (SdT=prior art). FIG. 6 shows a corresponding diagram in an exemplary embodiment with a continuous change in the spectral location of the spectral passage range due to a constant travel speed. The lightning symbols in each case characterize a flash or a light pulse. In the prior art, the stepper motor is paused in each case before a flash is triggered, and the flash falls into a stopping phase without motor movement (travel path does not change during the resting phase). In methods and systems in accordance with the present application, flashes are triggered with a moving stepper motor and thus a changing spectral location of the passage range.

[0066] It can occur in particular in fluorescence measurements that a single flash per spectral support point is not enough for sensitive measurements. In order to address this circumstance, the synchronized spectral scan of a sample can be repeated once or multiple times. The individual spectra obtained during each measurement can then be added accordingly. By adding up the signals of a plurality of flashes for each wavelength or each support point, the measurement statistics can be improved in a similar manner as if a longer measurement time per wavelength were used.

[0067] In the exemplary embodiment of FIG. 4, a constant shifting speed of the spectral passage range in the entire effective spectral range SPE of interest is present. This facilitates the synchronization, and in addition the spectra obtained in this manner are directly evaluable in the sense that no corrections with respect to different distances between the spectral support points need to be made.

[0068] However, it can also be advantageous to change the shifting speed during the continuous shifting within the effective spectral range, but without completely stopping the shifting movement. In this respect, FIG. 7 by way of example illustrates an I() diagram (intensity I as a function of the wavelength ) with a hypothetical spectrum SPK of a sample substance. In the present case, this can be a characteristic spectrum of a specific substance class, in which relatively strong changes in intensity per wavelength occur in certain spectral ranges B1, while the intensity hardly varies with a varying wavelength in other spectral ranges B2. The measurement can here be carried out such that, in the end, there is a variation in the spectral density of support points ST1, ST2 in dependence on properties of the spectrum for different spectral ranges. The dependence can be such that in ranges with relatively strong changes in intensity per wavelength interval, a relatively high spectral support point density occurs, while in other spectral ranges with relatively smaller changes in intensity (of type B2), a lower support point density or a greater wavelength distance between neighbouring support points is selected. The support point density can be approximately or directly proportional to the absolute value of the first derivation of the function I(). With a constant pulse frequency, this can be achieved by way of the travel speed of the stepper motor being controlled such that it is approximately or directly inversely proportional to the absolute value of the first derivation of the function I().

[0069] Control can be effected, for example, such that the intensity difference of two successive spectral support points ST(n) and ST(n+1) is ascertained, and the reciprocal value of it serves as a prefactor of a previously specified shifting speed. The change in the spectral passage range per unit time k/t is thus small for great intensity changes |I(ST(n))I(ST(n+1)|, and thus the number of spectral support points is directly proportional to the local spectral intensity dynamic of the spectrum.

[0070] Using a different number of support points per wavelength interval can also be advantageous for the non-continuous operation, i.e. for the classical move-stop-measure operating mode or start/stop operation. It is possible in this case for there to be a variation in the step width between immediately successive stopping positions in different wavelength ranges.

[0071] For example, the step width can be varied in dependence on a previously known or a measured property of the spectrum. In a variant with variable step width, an intensity change in the detected light between successive stop positions or spectral support points is ascertained during the shifting of the passage range to the next stop position, and the step width is varied in dependence on the intensity change. As a result, a regulation of the step width in dependence on a measured property of the spectrum can be realized. The regulation can be effected, for example, inversely proportionally such that spectral ranges with relatively strong intensity changes are travelled with relatively smaller step widths and a correspondingly higher support point density, while spectral ranges having fewer events are travelled more quickly, i.e. with greater step widths or lower support point density.

[0072] It is also possible that, before recording of a spectrum begins, parameters of a step width variation function are preset, and the step width is controlled in accordance with the step width variation function. Here, the change in the step width can be optimized based on previously known properties of an examined spectrum type.

[0073] The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.