The present invention relates to the field of optical spectrometry generally and, in particular fluorescence spectroscopy. To achieve higher luminosity and suppress stray light and unwanted grating orders automatically, the present invention uses tunable interference filters.
Monochromators are often used in optical spectroscopy. A monochromator generally comprises an entrance slit for admitting an incident beam of light having a range of wavelengths, a diffraction grating and an exit slit through which light is transmitted at a substantially monochromatic wavelength. The bandwidth of the light output is determined by the width of the exit slit. The wavelength of the monochromator can be tuned across a desired range by rotating the diffraction grating. A photodetector is used to record the optical power as a function of the wavelength.
Within a monochromator, particularly from the grating, a proportion of light is scattered and appears as a stray signal at the photodetector, see the article by T. N. Woods, R. T, Wrigley, G. J. Rottman & R. E. Haring App. Optics 33, 4273-4285. This stray signal can lead to unacceptable performance when measuring weak fluorescence spectra. To reduce this problem, the monochromator output can be improved using a second monochromator having a wavelength that is tuned to be substantially equal to the wavelength of the first monochromator, thereby reducing stray light. However, such an arrangement requires twice the number of optical components, has a reduced optical throughput and a larger footprint. Furthermore, at the extremes of a grating spectral range throughput drops off significantly. Gratings inevitably reflect second and higher diffraction orders through the exit slit giving unwanted stray shortwave signals.
According to a first aspect of the invention, there is provided a system for spectrally filtering light using a variable interference filter synchronously tuned to a wavelength selected by a device that has a variable wavelength output, for example a grating or a fabry perot filter. The interference filter can have a very high background wide band rejection to five or six orders of magnitude of rejection.
By using a variable wavelength selector, such as a single monochromator, in conjunction with such a tunable interference filter, there is provided an optical system that has the substantially same spectral performance as the known double monochromator, but a significantly improved background rejection and sensitivity. For many applications, such as fluorescence spectroscopy of biological samples in particular, this is very important. This superior performance can be provided in a cheaper, more compact spectrometer.
The system may include a controller for controlling the synchronously tuning of the variable interference filter and the wavelength selector. The controller may, for example, store calibration data for synchronous tuning which may, for example, take the form of a look-up table comprising output wavelength values and the corresponding filter settings that are required to substantially align the wavelength of the tunable interference filter with grating setting output. Interpolation may subsequently be used to control a filter setting corresponding to a desired monochromator wavelength, which is intermediate in value between two of the stored monochromator wavelength values.
The calibration data for synchronous tuning may, alternatively, take the form of an equation/expression and associated fitting parameters for a desired monochromator wavelength to calculate filter setting that is required to substantially align the wavelength of the tunable interference filter with the monochromator wavelength.
The tunable interference filter may comprise variable thickness interference layers along the lateral direction. This is known as a wedge filter. By masking part of the wedge the transmitted wavelength can then be caused to vary.
The tunable interference filter may utilise a transparent substrate such as glass, quartz or the like.
Additionally, the tunable interference filter may comprise a pair of reflective elements. The reflective elements may have reflective surfaces that are parallel. An intermediate air gap layer may be provided between the surfaces of the reflective elements. The interference filter may be tunable by changing the dimension of this air gap.
The tunable interference filter may have a pass band of wavelengths the width of which can be designable or can be a long wavelength pass (LWP) edge filter or a shortwave pass (SWP) as determined by the design of the multilayers, e.g. J. S. Seeley and S. D. Smith Applied Optics, 5, 81-85, 1966.
The tunable interference filter may comprise a filter actuator that is operable to tune the wavelength by translating the filter perpendicularly to the optical axis and to the line of the spectrometer slits.
The filter actuator may be automatically actuated. The filter actuator may, for example, comprise a worm and wheel and a stepper motor or an adaption of printer mechanics.
The monochromator variable wavelength selector may comprise a reflective diffraction grating.
The tunable interference filter may be positioned at various locations in the optical train, for example either side of entrance and exit slits. Positioning the filter at the exit slit, and in particular on the same side of the exit slit as the diffraction grating and contiguous with the light detector, is particularly advantageous because it results in the largest improvement in the suppression of stray light and, correspondingly the largest improvement in signal to noise ratio. This is important in fluorescence spectroscopy applications where the fluorescence may be weak.
The use of the synchronous filter significantly improves the signal to noise ratio of the industrial standard “Water Raman” Test of fluorescence spectrometer sensitivity.
The system may be an absorption or other form of spectrometer. The spectrometer may include a sample area; an excitation source for illuminating the sample area and an emission path that includes the variable wavelength selector and the tunable interference filter.
The source may comprise a narrow band light source such as a narrow band laser source and, in particular, a tunable laser source. The light source may be a broadband light source such as a white light source, supercontinuum laser, or a flash lamp i.e. a continuous wave source such as a Xenon lamp or the like combined with a fixed or tunable bandpass filter to determine excitation wavelengths.
The system may comprise two wedge interference filters moved synchronously for wavelength control and with respect to each other for band width control. This device would allow the spectral selection and triggering of a supercontinuum source. The device would prevent beam distortion in comparison with the use of a grating for wavelength selection. In addition the triggering mechanism would minimize temporal beam walk.
According to another aspect of the invention, there is provided a method of spectrally filtering light comprising selecting an output wavelength using a variable wavelength selector, and synchronously tuning a variable interference filter to the selected output wavelength, thereby to provide a spectrally filtered output. The method may involve scanning the output wavelength and the tunable filter across a wavelength range.
The method may involve using a wedge interference filter and using of off-axis illumination of a spherical mirror to provide a line focus to define the area of the filter wedge illuminated, and so the wavelength of the output.
The present invention will now be described further by way of example only with reference to the accompanying drawings, of which:
Fluorescence emitted from the sample 4 is collected by an emission lens 18 that couples an emission beam 19 along an emission portion 20 of the optical axis 5 into an emission monochromator 22. The emission portion 20 of the optical axis 5 is substantially perpendicular to the excitation portion 16 to limit the amount of excitation light on the emission path. In general, the fluorescence (and therefore the emission beam 19) has a wavelength that is different from a wavelength of the excitation beam 14. The emission monochromator 22 spectrally filters the emission beam 19 and the filtered fluorescence is coupled onto the PMT 8.
Each excitation and emission monochromator 12,22 has a reflective diffraction grating 24,26, for example a grating that has 1800 grooves/mm. Each grating 24,26 is rotatable via stepper motors 50,52 respectively so as to vary the wavelength of the light transmitted by the monochromators 12,22. To collimate and direct light, a collimating element is provided in the form of a concave mirror 28,30 between the entrance slit 32,34 and the diffraction grating 24,26. A focussing element, in the form of a further concave mirror 36,38, is also provided for focussing light towards the exit slit 40,42, which slit 40,42 determines the transmission bandwidth. To direct light from the concave mirror 36 towards the exit slit 40, the excitation monochromator 12 has an output steering mirror 44. Similarly, to direct light from the entrance slit 34 towards the concave mirror 30, the emission monochromator 22 has an input steering mirror 46 and for directing light from the further concave mirror 38 to the exit slit 42 an output steering mirror 48.
Within the emission monochromator 22 at a position adjacent the exit slit 42 between the output steering mirror 48 and the exit slit 4 is a tunable interference filter 60 that has a wedge-type variable interference filter 61 and a linear actuator 62 comprising a stepper motor. The tunable interference filter 61 provides additional spectral filtering and is tuned to light from the monochromator grating. Off-axis illumination of a spherical mirror (not shown) may be used provide a line focus to define the area of the filter wedge illuminated, and so the wavelength of the output.
Included in the spectrometer 2 is a controller 80 having a processor 82 and a memory 84. As indicated by the dotted lines in
Prior to using the spectrometer 2, the wedge-type variable interference filter 61 is removed and the emission monochromator grating 26 is calibrated in terms of absolute wavelength according to conventional methods. This is required because raw spectra, acquired by scanning the monochromator through a wavelength range and monitoring the signal on the detector, will not be a true representation of the sample being measured. The sensitivity of the detector, throughput of the monochromator and performance of the optics will all vary with wavelength and so will give a contribution to the measured spectra. The acquired spectra have to be corrected in order to obtain a true spectrum of the emission from the sample. Correction is applied by dividing the measured spectrum by a correction file.
To attain a correction file a calibrated light source (such a tungsten lamp), where the spectrum is precisely known, is placed above the sample chamber so that the light is incident on a piece of PTFE scatter located at the sample position 86. The spectrum of the lamp is measured using the emission arm of the spectrometer; this is divided by the known spectrum of the lamp to give a spectrum of the sensitivity of the instrument—the calibration file, which can then be used to correct measurements. The same correction regime can be used with a system containing a single monochromator and variable interference filter.
To synchronise the wavelength 72 of the interference filter 61 with the wavelength of the light transmitted by the emission monochromator exit slit 42 (hereinafter the emission monochromator wavelength), the sample 4 is removed from the spectrometer 2 and the light source 6 is switched off. A mercury lamp (not shown) is coupled into an optical fibre (not shown) and the distal end of the optical fibre is located at the sample position 86, so that the emission lens 18 collects light from the lamp. With the wedge-type variable interference filter 61 initially removed from the spectrometer 2, the PMT signal is measured for each emission monochromator wavelength, for example over the range of 200-870 nm, and stored in the controller memory 84 along with the corresponding emission monochromator wavelength value. The corresponding plot of PMT signal as a function of emission monochromator wavelength is shown in
The wedge-type variable interference filter 61 is subsequently calibrated against the emission monochromator grating 26 as follows. The filter 61 is replaced in the system and the emission monochromator grating 26 is set to a zero order position using the stepper motor 52 so that it functions as a non-dispersive plane mirror. The PMT signal is then monitored as the wedge-type variable interference filter 61 is translated through the mercury lamp light by the stepper motor of the filter actuator 62. At each position of the stepper motor of the filter linear actuator 62 (hereinafter the filter stepper motor), the PMT signal value and the corresponding position of the filter stepper motor are stored in the controller memory 84. The resulting plot of PMT signal as a function of the filter stepper motor position comprises a series of broad peaks with each peak corresponding to one of the mercury lamp lines, as shown in
Using the data of
An alternative method, using a broadband source, such as a Xenon lamp, can be used to calibrate the variable interference filter on its own, if the monochromator has already been calibrated. The monochromator is moved to a set of wavelengths. At each of these wavelengths the filter is moved over its range and the spectrum recorded. A peak-searching algorithm can be used to find the maximum value of the spectrum i.e. the motor position where the filter and monochromator wavelengths are the same. The set of filter positions and wavelengths can then be used to generate a polynomial fitting function. This method does not require a calibrated light source and so can be used if a filter is exchanged for one with a different wavelength range, or dimensions. In addition, once the selected wavelengths have been chosen, the calibration process can be automated in software.
Synchronous tuning of the monochromator and the interference filter may be achieved by software. In this case, in response to an instruction to move to a particular wavelength, the controller passes an instruction to the monochromator actuator to move to a position corresponding to a monochromator wavelength and at the same time passes an instruction to the filter actuator to move to a position corresponding to the a wavelength equal to the monochromator wavelength. Synchronous tuning may alternatively be achieved by hardware linking wherein the monochromator actuator is mechanically linked to the filter actuator, so that movement on the monochromator actuator automatically causes movement of the filter to a position at which it is tuned to the same wavelength as the monochromator. As another alternative, synchronous tuning may be achieved by firmware linking wherein a single instruction to move to a wavelength is passed to firmware that passes respective instructions to the monochromator and filter actuators to move to respective positions corresponding to the wavelength.
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The experimental set-up of
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Whilst the above systems have been described with reference to a wedge type filter, any suitable variable interference filter can be used.
Moving the fabry-perot filter 122 shifts the transmitted wavelength. In use, the fabry-perot filter 122 is moved to the position required to output the wavelength of interest and the wedge interference filter 122 is used to spectrally filter the resultant fabry-perot output. As with the previously described embodiments, the fabry-perot filter 122 and the wedge interference filter 124 are synchronously tuned. Synchronous tuning may be achieved by for example software or hardware or firmware linking wherein a single instruction to move to a wavelength is passed to firmware that passes respective instructions to the wedge filter 124 and the fabry-perot filter 122 actuators to move to respective positions corresponding to the desired wavelength.
A skilled person will appreciate that variations of the disclosed arrangements are possible without departing from the invention. For example, although the system of
Number | Date | Country | Kind |
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0818822.9 | Oct 2008 | GB | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/GB2009/002448 | 10/14/2009 | WO | 00 | 5/18/2011 |