WO2008125823A1

WO2008125823A1 - Apparatus for analysing a biological substance

Info

Publication number: WO2008125823A1
Application number: PCT/GB2008/001280
Authority: WO
Inventors: Ifor Daniel William Samuel; James Ferguson; Paul Nigel Marsh; Miles Padgett; Martin O'dwyer
Original assignee: The University Court Of The University Of St. Andrews; Tayside Health Board; The University Court Of The University Of Glasgow
Priority date: 2007-04-13
Filing date: 2008-04-11
Publication date: 2008-10-23
Also published as: GB0707199D0

Abstract

Apparatus for analysing a biological substance comprises a probe (1) for directing light emitted by a light source onto an area of the substance to cause fluorescence at that area. The fluorescent light is received by receiving means in the probe and is conveyed to spectral analysis means (4; 1-8; 2-8) for use in analysing the spectrum of the received fluorescent light and temporal analysis means (8, 10; 118) for analysing temporal characteristics of the received fluorescent light. The use of common receiving means ensures that the temporal and spectral analyses are conducted on the same area, and both types of analysis can, in combination, provide more information on the area being analysed than each individual type of analysis. Apparatus for analysing a biological substance by causing a substance to fluoresce and measuring characteristics of the resultant fluorescent light may have spectral analysis means and temporal analysis means, wherein the spectral analysis means is connected to the temporal analysis means so as to select the wavelength of the received light to be analysed by the temporal analysis means.

Description

Title: Apparatus for Analysing a Biological Substance

Field of the Invention

This invention relates to apparatus for analysing a biological substance, and more particularly to apparatus which irradiates the substance with excitation light to cause the substance to fluoresce and which analyses the resultant fluorescent light.

Background to the Invention

The invention is particularly, but not exclusively, applicable to apparatus for in-vivo monitoring or measurement of tissues and exogenous compounds therein. Data regarding the levels of drugs in tissue, for example, can be more important than blood levels if this is where the pharmacological action occurs. By better monitoring, individual drug dosing could be- realised in order to optimise a treatment regime. Fluorescence measuring and monitoring techniques are also increasingly used in the early detection of various types of cancers and pre-cancerous conditions. One approach utilises the auto-fluorescence properties of the tissues under investigation. These properties differ between healthy and cancerous or pre-cancerous tissues such as to produce a visible or measurable difference in the fluorescent light emitted by these tissues. Another type of fluorescence which may be used in cancer detection is that produced by a photosensitising drug which exhibits preferential accumulation in the cancerous tissues. Such drugs are often also used in photodynamic therapy to treat cancerous tissues. The drugs are preferentially absorbed into those tissues and then exposed to light which activates the drugs. As well as using such drugs as markers for cancer detection, fluorescence measurement techniques can also help to determine how much of the drug has been absorbed by the cancerous tissues, in the course of a PDT treatment.

Indeed, accurate determination of the timing and quantification of delivery of these agents is one of the major challenges facing research today.

Typically, the fluorescent light received from the tissue under investigation is subjected to a spectral analysis or an analysis of the decay rate of the fluorescence. In either case, however, it can be difficult to distinguish between different sources of the fluorescence. Thus, for example, a given drug may fluoresce at a wavelength which is one of the wavelengths at which autofluorescence of the tissue occurs.

Summary of the Invention

According to a first aspect of the invention, there is provided apparatus for analysing a biological substance by causing an area of the substance to fluoresce and measuring characteristics of the resultant fluorescent light, the apparatus comprising a source of excitation light, a probe for directing light emitted by the source onto an area of the substance to cause fluorescence at said area, receiving means, in the probe, for receiving fluorescent light from said area, spectral analysis means for use in analysing the spectrum of the received fluorescent light and temporal analysis means for analysing the temporal characteristics of received fluorescent light.

Preferably, the apparatus is operable to perform simultaneous temporal and spectral measurements on the same area of the substance.

Preferably, the apparatus is for use in analysing biological tissue samples. In this context, a tissue sample may, for example, be tissue extracted from the body. The apparatus is preferably, however, adapted for use for in-vivo analysis, the tissue sample being a selected area of tissue of the body.

In the latter case, the probe is preferably adapted for making measurements at the surface of the tissue or the insertion into a body cavity or interstice or an incision in the body.

For the purposes of the present specification, the term light (including the excitation light and the fluorescent light) includes within its scope infra-red and ultraviolet radiation, as well as visible light.

A given source of fluorescence can emit fluorescent light spectral analysis of which can relatively easily be used to identify the source, if the detected spectrum is not contaminated by light from other sources. The spectrum provides averaged steady state information on the substance and, possibly, its concentration. In addition, it is possible to integrate the fluorescence signal over a time interval so that even weak signals may be detected. However, where another source of fluorescence is present, the resultant spectrum is a composite of the spectra from the individual sources, and may not provide clearly interpretable information on the source of interest.

It has been found that different sources of fluorescence (for example, a drug or the tissue itself) which may emit fluorescent light in the same band of wavelengths can have measurably different temporal characteristics, in particular decay rates. Accordingly, data from the spectral analysis means can be combined with that obtained by the temporal analysis means so as to separate the contributions of these different sources to the spectrum of received fluorescent light.

The receiving means preferably comprises a light guide, preferably an optical fibre, which extends along the probe and connecting means for connecting the light guide to the spectral and temporal analysis means.

The light guide may also be arranged also to convey light from the source to said area.

Accordingly, the use of a light guide for both excitation and fluorescent light ensures that the light received by the apparatus comes from the area irradiated by the excitation light.

Alternatively the light source may be mounted on the probe in a position in which light from the source illuminates the area being analysed without passing along a light guide.

Preferably, the fibre is a multi mode fibre.

Such a fibre can convey all of the light mentioned above with relatively little loss.

Since the light guide conveys fluorescent light for analysis by both the spectral and temporal analysis means, this feature ensures that the two different types of analysis are conducted on fluorescent light emitted from exactly the same area of the sample, thus avoiding problems arising from local variations in the constitution of the sample.

In one embodiment, the spectral and temporal analysis means are separate. To that end, the spectral and temporal analysis means may have separate optical-electrical transducer elements for converting the received fluorescent light into electrical outputs, the connecting means comprising splitting means for splitting light received from the light guide along two paths, one to the temporal analysis means, the other to the spectral analysis means.

In that case, the connecting means may comprise two optical fibres connected together at a junction, the junction constituting the splitting means, and each fibre extending from the junction to a respective one of the spectral analysis means and the temporal analysis means.

Preferably, the connecting means comprises a "Y" fibre coupler, said optical fibres comprising the arms of the coupler, the stem of the coupler relaying received fluorescent light to the junction with the arms.

Preferably, the connecting means further comprises a further splitting means connected between the end of the probe light guide and the first said splitter, whereby the light guide conveys both excitation light and received fluorescence light.

Preferably, the light guide is arranged also to convey light from the source to said area.

Preferably, the temporal analysis means is also operable to analyse a temporal characteristic of reflected excitation light, from said area, received by the light guide. The reflected and fluorescent light will have travelled similar optical and electrical distances through the apparatus, so that a signal derived from the reflected light can be used as a reference against which a temporal characteristic of the signal from the fluorescent light is measured.

Preferably, the further splitter means comprises a further "Y" fibre coupler.

Preferably, the spectral analyser comprises a dispersive spectrometer. Preferably, the temporal analyser comprises a high bandwidth photodetector and a selection element for passing received light of a selected wavelength or range of wavelengths to the photodetector.

The selection element may be a filter or diffraction grating.

The apparatus may to advantage be operable to modulate the intensity of the light emitted by the source, the temporal analyser being operable to detect the phase lag between the received reflected excitation light and the received fluorescent light.

In this case, the apparatus may to advantage include means for modulating the power supply to the light source.

The apparatus may be operable to detect said phase lag by means of Fourier transforms of the fluorescent received light and the power supply to the light source, or received reflected excitation light.

Preferably, the temporal analysis means is operable preferentially to amplify received signals of a particular, determined phase and modulation frequency.

Preferably, the temporal analysis means is operable to amplify only said signals, signals of a differing phase or frequency making no substantial contribution to the output of the temporal analysis means. Thus, since said particular phase and frequency are known , the phase and frequency of the received signal can be deduced from the presence of a sufficiently strong signal at the output of the analysis means.

The temporal analysis means may to advantage be connected to the output of the spectral analysis means.

This provides a convenient way in which the spectra detected by the spectral analysis means can be modified by the temporal analysis means, since the spectral analysis means, in effect also functions as a wavelength selection device for controlling which wavelength of light is analysed by the temporal analysis means.

Such temporal analysis means may conveniently comprise a lock-in amplifier.

The lock-in amplifier may take the form of an analogue device or may comprise a computer programmed to perform lock-in amplification.

The lock-in amplifier also rejects signals not at the frequency at which the light source is modulated, and thus, for example, spurious signals caused by scattered light (from for example a computer display) entering the probe.

Preferably, the means for modulating the power supply to the light source comprises a digital frequency synthesizer which also acts a source of a reference signal for the lock-in amplifier. A digital frequency synthesizer has the potential advantage of being able to provide almost instantaneous frequency and phase shifts with lower setting times than analogue circuits with high phase locking stability. Such a synthesizer has other desirable characteristics, including the ring functionality at a relatively low cost and in a physically small package.

Preferably, the spectral analysis means and temporal analysis means have a common sensor for converting the received light into an electrical signal for subsequent analysis. This reduces the number of components of the apparatus and helps the apparatus to be of a relatively compact construction and cheap nature. In that connection, the spectral analysis means, preferably includes a selection element for directing only light of the selected wavelength or range of wavelengths onto the common sensor.

Thus, the temporal analysis means then analyses a signal attributable to the received light of the selected wavelength or range.

Preferably, the selector element comprises a refraction or diffraction element for sending the received light in different directions (relative to the element), related to the wavelength of the received light, the selection element being moveable relative to the sensor in order to enable the wavelength of received light incident on the sensor to be selected.

This can be achieved by having a moveable sensor, but more conveniently the selector is moveable by, for example, a stepper motor. Preferably, the sensor comprises a photomultiplier tube.

The invention also lies in apparatus for measuring characteristics of fluorescent light, the apparatus comprising spectral analysis means for analysing the spectrum of the received fluorescent light and temporal analysis means for analysing temporal characteristics of the received fluorescent light, wherein the spectral analysis means is connected to the temporal analysis means, so as to select the wavelength of the received fluorescent light to be analysed by the temporal analysis means.

Brief Description of the Drawings

The invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 is a schematic diagram of a first embodiment apparatus in accordance with the invention; Figure 2 is an example of a composite spectrum which could be detected by the apparatus, the spectrum being formed from fluorescent light resulting from Protoporphyrin IX ("PPIX") and from skin autofluorescence;

Figure 3 illustrates a sample signal received by the temporal analyser forming part of the apparatus to Figure 1 ;

Figure 4 is a graph of phase difference (between detected fluorescent light and the excitation light) in respect of a number of skin samples analysed by the apparatus some of the skin samples bearing PPIX, other having been marked with fluorescent ink;

Figures 5 and 6 are graphs showing fluorescent decay characteristics and spectra respectively of fluorescent materials when mixed with water or ethanol (Figure 5) or combined with other proteins (Figure 6);

Figure 7 shows the fluorescent decay characteristics of one side of an ex vivo skin sample, to the other side of which Dipyridamole has been applied, at various different times after the application of the drug;

Figures 8A-C and F-H are graphic representations which illustrate the effects that fluorescent lifetime (8 A-C) and concentration of active fluorescent compound (8F-H) have on phase difference between received fluorescent light and the excitation light.

Figures 9A and 9B show a second embodiment of apparatus in accordance with the invention, the apparatus being operable using phase information to resolve at least some component contributions to composite fluorescence spectra;

Figures 1OA and 1OB correspond to Figures 9A and 9B respectively, and show a modified version of the second embodiment.

Figure 11 graphically illustrates the spectrum of light emitted by skin autofluorescence, Trimovate® cream (when not applied to the skin) and skin to which the cream has been applied; Figure 12 shows spectra obtained from skin autofluoresence alone, and the fluorescent signal from skin to which Trimovate® cream has been applied, the spectra having been obtained by the apparatus of Figures 1OA and 1OB when operated at different phases relative to the modulation frequency of the excitation light;

Figure 13 shows the phase resolved spectrum for skin Trimovate® cream alone; and

Figure 14 illustrates the way in which the apparatus of the second embodiment conducts temporal analysis of received fluorescent light.

Detailed Description

With reference to Figure 1, the first embodiment of apparatus in accordance with the invention comprises a probe 1 that contains a single length of 600 micron core diameter multi mode fibre (Ocean Optics BIF 600-UV/VIS). The fibre conveys excitation light emitted by an LED source 2 to an area under investigation (denoted A), whilst simultaneously conveying fluorescent light received from that area back to the apparatus for separate analysis by a dispersive spectrometer unit 4 and temporal analysis means 6. The temporal analysis means 6 comprises an avalanche photodide (e.g. a Hamamatsu C5460) 8, the output of which is converted into a digital signal by an analogue to digital converter 10 connected, in turn, to an embedded computer 12.

The temporal analysis means 6 further comprises a second avalanche photodiode 9 (of the same type as the photodiode 8) and associated ADC 11. Excitation light reflected from the site A is relayed, via the probe 1 to the photodiode 9, the output of which is converted into a digital signal by the converter 11. This signal is used as a reference for measuring the phase difference between excitation and fluorescent light, as described below.

The spectrometer unit 4 has a spectral coverage of 320-1 lOOnm with a spectral resolution 2-3 nm (achievable with an Ocean Optics USB2000 grating#3-L2 diffusion grating). This spectral information is read via a USB interface, denoted by reference 14, to the computer 12. Similarly, the output of the ADC 10 is fed to the computer 12 via a USB interface 16, while a further USB interface 17 supplies the output from the ADC 11 to the computer 12.

In the present embodiment, the LED 2 is a Bivar LED 5-UV-400-30 which emits light at 405 nm, and is powered by a source 18 which provides a modulated driving current for the LED 2. The modulation is a sinusoidal amplitude modulation at frequencies of up to tens of MHz. A further ADC 20 converts the modulated current signal into a digital output fed to the computer 12 through a USB interface 22. Since the drive current supplied to the LED 2 is sinusoidally modulated, the intensity of light emitted by the diode is similarly modulated. That light is coupled into a 600 micron core diameter multi mode fibre (Ocean Optics BIF600UV/VIS) 24 which conveys the emitted light to the focal point of a lens 26 that directs the light onto a band pass filter 28 operable to remove any out of band emission from the source and enhance its spectral purity. The light passes through the filter 28 then enters a lens 30 via which it is coupled into a Y fibre Ocean Optics QBIF 600-VIS/NIR-BX) 32. A mechanical shutter (not shown) is interposed between the lens 26 and filter 28 or the filter 28 and lens 30, and is operable in response to user inputs via a suitable control such as a foot pedal to enable the user to interrupt the supply of light to the probe 1 in a controlled manner.

The Y fibre 32 couples the excitation light to an input/output connector 34 to which the fibre for the probe 1 is releasably attached (so that the probe may be interchanged with other probes). In the present example, the foot pedal activates the shutter which causes the area A to be exposed to the excitation light for approximately 0.5 seconds. At least some of the resultant fluorescent light is received by the same fibre probe 1 and is coupled back to the instrument (through the connector 34) and emerges from the other arm, denoted by reference numeral 36, of the Y fibre 32. The end of this arm 36 is connected to a further, similar Y fibre 37, the two arms of which are denoted by reference numerals 39 and 41 the arm 39 is connected to a further similar Y fibre 38 , the two arms of which are denoted by reference numerals 40 and 42. Light from the arm 40 is supplied via a lens 44 to a band pass filter 46 and then to a further lens 48 from which the light is fed to the spectrometer unit via a fibre optic connector 50. The lenses 44 and 48 and filter 46 form part of an Ocean Optics SHS- UV filter module which blocks the excitation wave length. The bands of the filters are 370-460nm for the excitation light channel and 470- 11 OOnm for the fluorescence channel

Light conveyed along the arm 42 is filtered by a similar in line filter module (comprising lenses 52 and 54 and filter 56) to select a wave length of interest. The filter's light is fed through a fibre connector 58 to the avalanche photodiode 8.

Light conveyed along the arm 41 is filtered by an in line filtering module comprising lenses 59 and 61 and a band pass filter 63 interposed between these lenses. In this case the filter 63 has a different bank from the filters 46 and 56, selected to filter out the fluorescent light and pass the excitation light, which is conveyed to the photodiode 9 along a fibre connector 65.

Since the excitation light is modulated, the output of the photodiode 8 will be similarly modulated, but the phase of the output, relative to that of the excitation light (and hence the output of photodiodes) signal, will be affected by the temporal decay of the fluorescent light emitted by the area A. Since this is related to the nature of the source/sources of the fluorescence, the phase difference between the signals from ADC 10 and ADC 11 will provide information on the nature of the sources, in addition to the information that can be gleaned from the spectrum detected by the spectrometer 4.

In order to ensure a proper comparison of the phases of the output of the converters 10 and 11, the converters are synchronised to a common clock such as a Picoscope 3206 (not shown).

The computer 12 applies a Fourier Transform to the frequency domain of the signals received from the converters 10 and 11 and deduces the relative phase of the signals from the arguments of their complex amplitudes. A modulation frequency of 65 kHz, a sampling rate of 500 kS/s and a record length of 2048 samples gives a phase accuracy corresponding to a relative time delay of better than 0.1ns. This makes it possible to deduce changes in the fluorescence lifetime with the emitted light of less than Ins. The results of the analysis conducted by the computer (i.e. the phase information at a selected wavelength and the spectrum detected by the spectrometer 4) can be displayed on a display screen (e.g. an LCD display) 60.

If the area A is a cancerous area of skin in which PPIX is also present, an example of the spectrum which may be detected by the spectrometer 4 is shown in Figure 2. Although the excitation light is modulated, the spectrometer integrates its signal over a period of between 0.1 and 1 second so that this modulation does not affect the output of the spectrometer 4. The spectrum has a peak at 630nm attributable to the drug, whilst skin autofluorescence gives a spectrum with a peak at around 500nm. The autofluorescence spectrum is spread over a wide range of wavelengths, and only gradually tapers with increasing wavelengths, thus providing a tail which can lead to some confusion as to the extent of contribution to the spectrum by the light emitted by the PPDC.

However, in addition to resolving the fluorescence emission spectrally, the apparatus according to the invention also offers temporal information regarding the fluorescence lifetime of the emission. The temporal information is deduced, not from precise temporal data, but from a phase technique as discussed below.

Since the excitation light, emitted by the diode 2 is modulated with a precisely defined frequency, the phase lag between the excitation light and the received fluorescence light will be related to the lifetime of the fluorescence compound or drug.

The traces in Figure 3 include a sine waveform 40 corresponding to the modulation signal fed by the source 18 to the LED 2, and detected/monitored by the computer 12 via the ADC 20. The power signal to the LED 2 will correspond, in phase, frequency and amplitude to the modulated excitation light emitted by the diode 2. The fluorescent light supplied through the probe 1 to the avalanche photodiode 8 (via the filter) gives rise to the trace 42 shown in Figure 3. This trace shows a similar modulation of exactly the same frequency as the power source, but the temporal exponential decay of the fluorescence results in a phase lag between the excitation light and the resulting fluorescence, and this phase lag is related to the lifetime of the fluorescence of the drug. Since the light released by the fluorescence of the drug and by skin autofluorescence will be of the same frequency, the detected phase will, in effect, be the vector sum of these two contributions (the length of each vector representing the peak intensity of each contribution, while the angle between the two vectors will represent their relative phases), and the phase difference will thus also provide data on the relative magnitude of the two different types of contribution.

In Figure 3, the trace 42 is of light filtered by the filter 56 to pass light only of a wavelength of 630nm, i.e. corresponding to the peak attributable to the PPEX. The filter 56 is interchangeable with other filters for passing other wavelengths, the requisite filter being selected with reference to the position of the peak in the output of the spectrometer 4 corresponding to the drug being investigated. In this case, the phase lag of the fluorescence arising from the PPIX differs from that of autofluorescence. Consequently, this technique can be used to differentiate between two different sources of fluorescence at exactly the same wavelength, whereas those sources would be indistinguishable from their spectral emissions alone.

In the present case, the computer 12 determines the phase difference by means of a Fourier analysis as discussed below with reference to Figure 4, which shows the detected phase for a number of different samples of skin some of which carry a dose of PPIX, whilst others have been marked with fluorescent ink of a colour similar to that of the fluorescent light emitted by the PPEX. These fluorescent materials produce modulated signals similar to those shown in Figure 3. The relative phase of the signals is calculated from a Fourier analysis, using a Fast Fourier Transform algorithm numerically to calculate the transform.

The algorithm used by the computer is pre-written and forms part of a lab view software package. An example of the types of algorithms that can be used can be found in, for example, the publication entitled "Numerical Recipes in C" by Press, et al. En fact, this book even provides the C code for FFT algorithms.

From the transform, the phase of the signals is represented by the argument of the complex part of the transformed signal. The phase relationship between the two signals (excitation power source and received fluorescence) is constant and can be represented graphically as shown in Figure 4. In order to obtain the data for Figure 4, a relatively low modulation frequency of 70-300 KHz was used, but it can be seen that the skin samples carrying PPIX give noticeably different phase information (in general) from the samples which were merely marked with ink.

The following table gives an indication of how differing drugs have differing fluorescence lifetimes:

It will be appreciated that much of the information that can be inferred from the spectral and/or phase data obtained by the apparatus will depend upon experimental investigations into the spectral and temporal characteristics of the fluorescence produced by different substances or combinations of such substances. For example, a significant problem that surgeons encounter during patient examinations is distinguishing between dysplasia/carcinoma and inflammation. These conditions appear spectrally similar, but are produced by significantly different underlying causes. Thus, by analysing samples of each type of tissue for spectral and temporal data information similar to that found in Figure 4, might be experimentally obtained. From this information, it should be possible to determine a threshold phase difference for identifying the source of a particular type of fluorescence (at a given wavelength) in the subsequent use of the apparatus. This is analogous to the data shown in Figure 4 since PPFX and fluorescent ink are spectrally similar, but are caused by fluorophores that still have different decay times, and therefore give rise to different phase delays relative to the driving modulation signal. The apparatus of the present invention can measure phase delay accurately, and thus distinguish between two different chemicals which emit spectrally similar fluorescent light.

The applicants also believe that it may be possible to produce other information about a substance (typically a drug) under investigation. For example, Figure 5 shows an example of changes in the lifetime and characteristics of the fluorescent light emitted by Dipyidamole dissolved in different solvents. The horizontal axis of Figure 5 represents time, whilst the vertical axis represents intensity of light detected. The trace denoted with reference to numeral 44 provides a lifetime for the substance in water, whilst trace 46 is for the substance when dissolved in ethanol. It can be seen that the two different types of solvent have a bearing on the lifetime. The data from Figure 5 was made by taking direct decay measurements by suitable equipment (other than the apparatus of Figure 1). However, the decay time will manifest itself as a phase difference between the detected fluorescence and the modulated output of the source 18.

Other information can be inferred spectrally, from the output of the apparatus. For example, Figure 6 demonstrates how the spectrum PPIX fluorescence is affected by the presence of different proteins as set out below:

Figure 7 illustrates the fact that the concentration of a drug related source of fluorescence in a skin sample (which exhibits autofluorescence) will affect the decay time of the composite fluorescent signal, and hence the detected phase difference between such fluorescent light and the excitation light (or the power source for the source of the excitation light). Figure 7 shows fluorescence of ex-vivo skin as a function of time after excitation by a light pulse of short duration. The curves were taken at different time points over a period of several days during which time the drug Dipyridamole was diffusing through the tissue from a base layer to the upper surface where the measurements were taking place. The increasing drug concentration can be deduced from the change of shape of the curves at different time points, irrespective of native fluorophores (i.e. the sources of autofluorescence having similar spectral, but differing temporal, characteristics). The curves after 207 and 528 minutes overlap and show little or no drug presence, both resulting mainly from the autofluorescence background.

With increasing time, the drug diffuses into the measurement volume and the characteristic fluorescence lifetime of the drug changes the shape of the fluorescent decay curves, almost completely dominating after 2,621 minutes. In an in- vivo case, such curves can be used to monitor the changes in drug concentrations over time. Alternatively, a single measurement could be performed to monitor patient compliance either by comparing the decay curve produced to a standard autofluorescence decay curve or by measuring the lifetime of the drug, thus ensuring its presence.

The instrument control logging and analysis of the spectral and temporal (phase) data in the apparatus of Figure 1 is achieved from a common software package within the Lab View programming environment (National Instruments Lab View v 7.1). The software can be configured to give full information output aimed at the experienced researcher or a simple diagnostic indicator based on a chosen algorithm aimed at the medical end user.

Where two spectra overlap, it may not be possible to identify either spectra definitively simply from spectral data, although the spectral data still indicate the likely candidate compounds being examined. The apparatus of Figure 1 also measures the phase delay, which will still allow drugs/compounds to be distinguished from the background. It is not invariably necessary to resolve the spectral data into the component spectra in this instance. Thus, with reference to Figure 7 if measurements were made of the tissue fluorescence of the sample used for Figure 7, by the apparatus of Figure 1, it will be noticed that there is a change in the phase delay between untreated tissue and when some drug had diffused into the tissue, hence indicating the presence of the drug. Increasing phase delay would be indicative of increasing drug concentration.

A similar effect is illustrated in Figures 8A-C, which show, in theory, how differing fluorescence lifetimes of differing drugs will affect phase delay (between excitation and fluorescent light), compared with background fluorescence attributable solely to skin autofluorescence.

The graphs show phase difference against the reciprocal of the angular frequency of excitation light, over a range of frequencies.

The overall fluorescence is shown by the dotted line, and the fluorescence of the background (i.e., if the drug had not been applied) is shown in solid white. Figure 8A shows an example of a drug and background with the same fluorescence lifetime, they are indistinguishable. Figure 8B shows an example where the fluorescence lifetime of the drug is ten times that of the background. In this case it would possible be conclude that the drug is present even if the spectral data overlapped. Figure 8C shows the increase in total phase delay if the fluorescence lifetime of the drug was 20 times that of the background.

In addition the phase difference is affected by the intensity of the drug fluorescence (the composite detected signal being the vector sum of the contributing components autofluorescence and drug related fluorescence).

This is illustrated in Figures 8F -H which show the effect a phase of differing concentrations of PPIX (phase being plotted against the reciprocal of frequency). Again the solid white curves represent the autofluorescence background, the dotted lines the composite fluorescent signal. Aminolaevulic acid is applied to somebody's arm, that person's tissue will convert this compound into Protophorphyrin IX (PPIX), which is fluorescent. Immediately after application there will not have been enough time for any PPIX effect on the overall fluorescence lifetime of the tissue will be negligible (equivalent to the situation in Figure 8F). After some time there will be build up of PPFX. This will affect the overall fluorescence lifetime of the tissue at the site of application of the drug (equivalent to the situation in Figure 8G). As the concentration of PPIX increases, the contribution to the overall fluorescence lifetime increases (equivalent to the situation of Figure 8H). The change in overall lifetime in itself indicates the presence of the drug. This may be useful information in its own right in some applications. The apparatus of Figure 1 simultaneously implements two different analysis techniques (temporal and spectral) on exactly the same tissue site in a single step, in order to yield different but complementary information on the tissue. Thus, where one technique is not useful, for example where the spectra overlap, the other technique can still provide useful information. It is possible that both techniques will provide useful information; for example, the spectral analysis could give an indication of the type of sources of fluorescent light present, whilst the combination of the two techniques could provide an indication of relative concentrations of the differing sources of fluorescence. Alternatively, it is believed, that one of the types of analysis may be used to interpret the results of the other type of analysis even in situations where there is no drug related fluorescence. Thus a spectrum of an area under investigation might suggest to the possibility of, for example, early stage dyplasia and the temporal analysis may help to confirm that the spectrum has been caused by this condition.

An alternative technique is to use the temporal characteristics of the fluorophore's decay to weight the detected spectrum thus simultaneously combining spectral and temporal information. This can be achieved in practice in numerous ways. In principle, if the wavelength is scanned across the detector (or by using multiple detectors and a dispersive element, such as the diffraction grating) and by using digital signal processing, phase spectra such as are shown in Figure 12 can be reconstructed at any phase difference. Figure 9a and 9b show one example of apparatus for achieving this.

The electronics of the apparatus will be described first with reference to figure 9A, then the optical paths with reference to figure 9B and the main components will then be listed.

As depicted in figure 9 A, two phase locked RF oscillators 100, 102 are both power split by power splitters 104 and 106 respectively. One of the outputs from power splitter 104 is fed into a bias tee 108 where it is combined with a dc bias voltage to provide a modulated signal at frequency f to drive the light emitting diode (LED) 110. The other output is mixed with the first output of power splitter 106 in frequency mixer 112 and low pass filtered by filter 114 to provide a reference signal at frequency df. This is fed into the reference input 116 of a lock-in amplifier 118. The second output of power splitter 106 is fed into frequency mixer 120 and combined with the electrical amplified output of a photomultiplier tube (PMT) 122. This is then low pass filtered by filter 124 to obtain a signal at frequency df and fed into the signal input 126 of the lock-in amplifier.

One feature of the lock-in amplifier 118 is its ability to detect a signal as a function of the phase difference between the reference signal and the signal to be measured, i.e. the signal at the input 126. By choosing appropriate excitation wavelengths, modulation frequencies and phase differences between the excitation light and the detector phase, the autofluorescence signal from skin can be substantially reduced allowing other signals, such as the fluorescence produced by a drug, to be detected.

The signal from the lock-in amplifier is recorded as a function of the wavelength selected using a monochromator 128 (described below) in order to create spectra such as are shown in figures 12 and 13.

The optical paths are shown in figure 9B. The bias Tee 108 provides a biased if driving signal which is used to produce modulated light output from an LED 130. Due to the broad spectrum of that light, a bandpass filter 132 is used to narrow this spectrum to lOnm, centred in this case at 365nm. A first lens 134 is used to collimate the output from the LED 130 . A dichroic mirror 126 is used to redirect the beam to a second dichroic mirror 138 which in turn reflects the beam towards the skin of a subject through a focussing lens 140. This lens also collects both backscattered excitation light and the generated fluorescence. The dichroic mirror 138 removes a large percentage of the backscattered excitation light whilst the fluorescence passes through. The remaining backscattered excitation light and the fluorescence are then coupled into a fibre optic bundle 142 via second lens 139. Light emerging from the bundle is coupled into the monochromator 128 and detected by use of the photomultiplier tube (PMT) 122. The grating in the monochromator is rotated by a steppermotor (not shown) and the output from the PMT 122 recorded, by the computer, for each associated angular position of the grating so as to obtain a spectrum of the received light for a given phase difference. In addition the apparatus can make non phase dependant measurements of intensity of light of a selected wavelength, so as to additionally enable steady state spectra to be taken.

The LED 130; filter 132; lenses 134, 139 and 140; mirrors 136 and 138 and fibre bundles 142 are housed in a probe 144.

Since the spectral data obtained from the monochromator 128 and PMT 122 also contains phase information, sampling the output data obtained from the PMT 122 (using the computer) at a selected phase can discriminate between contributions to the measured spectra, as discussed below.

Parts List

RF Oscillators 1 & 2 Hewlet Packard 8648C

Power Splitters 1& 2 Minicircuits ZSC-2-1

RF Mixers 1 & 2 Minicircuits ZP-3

Bias Tee Minicircuits ZFBT-4R2GW

Low pass filters 1 & 2 Minicircuits BLP -2.5

Monochromator Bentham M300

PMT Hamamatsu 928

Dichroic Comar 41 Onm

LED Nichia 365nm TOP

The method of operation of this apparatus is similar to that of the modified version of the second embodiment, shown in Figures 1OA and 1OB.

The modified version of the second embodiment, as shown in Figures 1OA and 1OB has many components which correspond to components of the embodiment shown in Figures 9A and 9B, and these are denoted by the reference numerals of those figures, raised by 100.

However, in place of oscillators and power splitters, the apparatus has a four channel direct digital frequency synthesizer 250 which provides four phased locked outputs denoted channels 0-3. Channels 0 and 2 provide sinusoidal outputs at a frequency of f+df, while channels 1 and 3 provide sinusoidal modulation at frequency f. The output of channel 0 is amplified by amplifier 252 and then fed into the bias tee 208. This signal is combined with a dc bias tee voltage and the output is used to drive the LED 30 so that the latter continuously emits light modulated at a frequency of f+df. Channels 1 and 2 are used to generate a reference signal for the lock-in amplifier, which in this embodiment is implemented in computer software. Channel 1 is amplified by amplifier 254 to provide the necessary power to the mixer diodes in the frequency mixer 212 and is applied to the Io port of the mixer. Channel 2 is fed into rf port of the mixer 212 and the output of that mixer is filtered by the low pass filter 214 to provide a reference signal at a frequency of df. This signal is supplied to data acquisition apparatus that digitises the signal and passes it to the computer. The channel 3 of the synthesizer 250 is amplified to 256 in order to drive the mixing diodes of the frequency mixer 220.' The other input to the mixer 220 is connected to the rf output of bias tee 258, the input of which is connected to the output of the photomuliplier tube 222 via an amplifier 260. As is indicated in the diagram, the bias tee receives from the amplifier 260 a signal which has both sinusoidal and dc components. The sinusoidal component is fed into the rf port of the frequency mixer 220, whilst the dc component is digitalised by the data acquisition 255 hardware (also connected to the bias tee 258) and fed to the computer.

The output of the mixer 220 is amplified by amplifier 262 and filtered by low pass filter 224 to yield the signal at a frequency df. This signal is also digitalised by the data acquisition apparatus 255.

The optical paths are shown in Figure 1OB. The method of delivering modulated light to the site under investigation and relaying the resultant fluorescence and scattered excitation light to the detector is likely to have different optimal geometries dependent upon the situation. The first embodiment used a bifurcated fibre bundle, a percentage of the individual fibres being used to transmit the light to the specimen and a percentage to receive the light and couple it into a monochromator. However, when using LEDs as excitation sources for fluorescence, coupling losses into the fibres used for excitation can be a limiting factor. In this case, it can be advantageous to incorporate the LEDs into the probe head and simply fibre couple the scattered light and fluorescence to the monochromator. Figure 1OB shows a geometry used to excite surfaces. As with the other embodiments the bias tee provides a biased rf driving signal which is used to produce modulated light output from the LED 250. Due to the broad spectrum of light, a bandpass filter is 232 used to narrow this spectrum to around IOnm, centred in this case at 363nm. A lens 240 (or combination of lenses) is used to focus the output from the LED onto the sample. The reflected excitation light and fluorescence is collected by the fibre optic bundle 242 in close proximity to the excited surfaces. Light from the bundle 242 is relayed to the monochromator 228 via a dichroic mirror. The fibre bundle 242 and monochromator 228 are chosen to have similar numerical apertures to improve optical coupling, the fibres in the bundle 242 being arranged in a slit pattern to further maximise this coupling. The output from the monochromator 228 is relayed to the detector 222, in this case a photomultiplier tube, using a lens 264 or lens combination. The grating in the monochromator is rotated using a steppermotor and the output from the PMT is recorded, by the computer, for each associated angular position of the grating so as to obtain a spectrum of the receive light. The dichroic mirror removes a large percentage of the backscattered excitation light whilst the fluorescence passes through. If this was not done, then the backscattered light might cause the detector to become saturated.

Parts List

Frequency Synthesiser Analog Devices AD9959 DDS board

Amplifier 1 Minicircuits Gali51+

Amplifier 2 & 3 Minicircuits ZFL-500B

Amplifier 4 Hamamatsu M8879

Amplifier 5 Operational Amplifier

RF Mixer 1 & 2 Minicircuits ZP-3

Bias Tee Minicircuits ZFBT-4R2GW

Low pass filters 1 & 2 Minicircuits BLP-2.5

Data Acquisition National Instruments Nl USB 6211

Monochromator Horiba Jobin Yvon Hl 034-B-500-OS

PMT Hamamatsu PMT Module H7732- 10

Filter Comar 365nm plus Semrock FF01-377/50

LED Nichia 365nm TOP

Dichroic Comar 41 Onm

Optical Detection of Drugs — Phase Discrimination The skin containing the drug will be illuminated with light, the fluorescence signal will be collected and analysed for the presence of the drug. If only one fluorescent species were present, then steady state spectra could be taken and the known drug emission spectra matched with the collected data to confirm the presence of the drug. However, skin contains endogenous fluorophores which give rise to the so-called autofluorescence spectra which will contribute to the measured spectra. The fluorescence lifetime of the drug and skin fluorophores can be used to aid in detecting the drug if they have different lifetimes but overlapping steady state spectra. This property can be exploited in either the time or phase domain.

When excited by modulated light (e.g. sinusoidal), the phase of the emitted fluorescence depends on the fluorescence lifetime of the fluorophore. This lifetime can be measured by, for example, comparing the phase shift between the reflected excitation light from the sample and the fluorescence from the fluorophore.

Measuring a lifetime component of the correct duration is one way of establishing the presence of the drug in the skin. A more powerful approach in certain circumstances is to combine spectral and temporal techniques in one measurement. By placing the detector out of phase with the fluorescence from one of the fluorescent species, the spectra of a second fluorophore can be uncovered or the background fluorescence greatly reduced. This latter technique is used by the apparatus of Figures 9A and 9B and 1OA and 1OB.

In the apparatus shown in Figures 9 A and 9B₃ the lock-in amplifier 118 has at least two modes of operation. In one such mode, the amplifier compares the phase of the signal at input 126 with that of the reference signal 116 as the wavelength under analysis is scanned by the monochromator 128. An example of this analysis is graphically illustrated in Figure 14 which shows the phase versus wavelength characteristics of Trimovate ® cream (in isolation). As can be seen, a first phase reading is obtained as the monochromator scans the wavelength through that of the scattered excitation light, whilst a second phase reading (at a range of longer wavelengths) is obtained as the wavelength is scanned through the range of wavelengths at which fluorescent light is emitted by Trimovate ® in response to wavelengths at which fluorescent light is emitted by Trimovate ® in response to excitation by the excitation light. The difference between these two readings gives an indication of phase lag and hence fluorescent lifetime.

In a second mode of operation, the lock-in amplifier 118 extracts a spectrum which is weighted by the fluorescent liftetime of the fluorophore(s) contributing the spectrum. In other words, the amplifier will preferentially amplify those signals which are at a certain phase relative to a reference signal, and will discriminate against the signals not in phase, to the extent of nullifying signals which are 90 ° out of phase with the selected phase.

Accordingly, when one contributor to the fluorescent signal is nulled by placing the detection phase at 90° out of phase with the signal attributable to that component, signals attributable to other components may be more clearly seen.

In a third mode of operation, the lock-in amplifier 118 can obtain steady state spectra (regardless of phase).

In the modified version shown in Figure 1OA and 1OB, the lock-in amplification is performed in software written in a commercially available LabView environment, but still provides the same modes of operation.

The use of synthesizer 250 in the apparatus shown in Figures 1OA and 1OB has a number of potential advantages. Being digitally generated, the four channels are inherently phase locked. The mixing of two channels to provide a reference signal for the lock-in amplifier and the mixing of the remaining channel with PMT signal reduces the sampling rate necessary for the analogue to digital conversion performed by the acquisition apparatus 255, and thus reduces the costs of the data acquisition hardware. Furthermore, the light source can be modulated at more than one frequency simultaneously, and the use of lock-in detection allows the different phase spectra (corresponding to the different modulation frequencies) to be collected at the same time. It is believed that the data thus obtained can provide more accurate results than the data obtained when only one modulation frequency is used for the light source. When using multiple outputs from a direct digital frequency synthesizer broad, the relative phases between the channels can be set or sweep digitally. This gives a large degree of flexibility to the instrument, whether it is simply to adjust the relative phases to compensate for different electrical path lengths to synchronise channels or to use the synthesizer to sweep the phase of one channel relative to the other, hi phase fluorimetry, this would be another way to perform the measurement: i.e. the lock-in detector looks at a fixed phase and the source is swept.

In this worked example, Trimovate® Cream (a cream containing a drug) was applied to skin in-vivo. The objective was then to detect and identify the drug in- vivo by using the temporal characteristics of the drug and skin fluorescence to modify the collected fluorescence spectra thus generating so-called phase spectra. An untreated area of skin was also analysed to obtain phase data for use in selecting the subsequent phase at which the phase spectrum mentioned above is to be obtained.

Steady State Spectra

The spectra obtained in the steady state for skin, the Triomovate® Cream and the skin with some Triomovate® Cream applied, appears in Figure 11.

In that figure, it can be seen that the "Trimovate on Skin" spectrum is probably a composite of the autofluorescence (curve labelled "SKIN") and the Trimovate Cream (curve labelled "Trimovate") spectra. A curve-fitting program could be used to try to fit the two component spectra to the composite spectra in order to obtain the best fit. Assuming a good fit, some confidence in establishing the presence of the drug could be attained. However, this requires prior knowledge of both the autofluorescence spectrum and the drug spectrum in-vivo. This will rarely be the case in practice. Thus, another source of information would be useful to confirm the presence of the drug and to determine its spectrum in-vivo (which may be different to its ex-vivo spectrum since the spectrum can be modified by the drug's environment). By using the temporal characteristics of both the drug and the autofiuorescence, so-called phase resolved spectra can be obtained. Phase Resolved Spectra

For the purpose of this example, the Triomovate® Cream was only applied on part of the skin of the chest. Thus the contralateral portion of the skin was available for comparisons to be made. The method in this case was to establish the phase angle between the modulated source and the detector which resulted in the largest signal at 486nm from skin when measuring on the contralateral side. This phase was denoted phase 2. Measurements of the contralateral position and the position at which the drug was applied both at this phase angle and when the phase angle had been advanced by 90 degrees, this being denoted phase 1, appear in Figure 12. Also in Figure 13 is a spectrum of the drug taken at phase 1.

It can be seen that in Figure 12 the autofluorescence spectrum has been reduced in amplitude, if not removed, by using the equipment to detect fluorescence at phase angle 1. The skin on the contralateral side generates little signal at this phase as can be seen from the curve denoted "Contralateral (Phase I)". In contrast, the area of the skin treated with the Triomovate® Cream generates a spectrum "Skin and Drug (Phase 1)" which substantially overlaps that produced by the drug alone, denoted "Trimovate ® (Phase 1)" shown in Figure 13. Small shifts in the drug spectrum would be expected in-vivo as compared to isolated drug since the drug environment has changed. The extent of this shift for different drugs is part of the ongoing research. Detecting at phase 2 essentially yields the autofluorescence spectrum in the case of the contralateral side, the curve being denoted "Contralateral (Phase 2)", and components of both the drug and the autofluorescence when measuring in the treated area, "Skin and Drug (Phase 2)".

hi principle, having obtained the drug spectra, a curve fitting routine could be used to establish the proportions of the "skin and drug (Phase 2)" curve that is attributable to the autofluorescence and the drug separately. From this (and more accurately if more phase angles were used) the phase giving the maximum drug fluorescence can be calculated. If a measurement of the phase angle giving maximum signal for the scattered excitation light from the skin is also taken, the drug lifetime can be calculated. Having obtained the spectra and the fluorescence lifetime of the drug through the use of phase resolved spectra, a higher confidence level in the discovery and identification of the drug can be attained as when compared to that obtained by looking at steady state spectra alone. In the simplest case, by nulling the autofluorescence component, the presence of a drug in skin can easily be detected and it's spectrum obtained.

Claims

1. Apparatus for analysing a biological substance by causing an area of the substance to fluoresce and measuring characteristics of the resultant fluorescent light, the apparatus comprising a source of excitation light, a probe for directing light emitted by the source onto an area of the substance to cause fluorescence at said area, receiving means, in the probe, for receiving fluorescent light from said area, spectral analysis means for use in analysing the spectrum of the received fluorescent light and temporal analysis means for analysing the temporal characteristics of received fluorescent light.

2. Apparatus according to claim 1 , in which the apparatus is operable to perform simultaneous temporal and spectral measurements on the same area of the substance.

3. Apparatus according to claim 1 or claim 2, in which the apparatus is for use in analysing biological tissue samples.

4. Apparatus according to claim 3, in which the apparatus is adapted for use for in-vivo analysis, the tissue sample being a selected area of tissue of the body.

5. Apparatus according to claim 4, in which the probe is adapted for making measurements at the surface of the tissue or the insertion into a body cavity or interstice or an incision in the body.

6. Apparatus according to any of the preceding claims, in which the receiving means comprises a light guide which extends along the probe and connecting means for connecting the light guide to the spectral and temporal analysis means.

7. Apparatus according to claim 6, in which the light guide is a multi mode optical fibre.

8. Apparatus according to claim 6 or claim 7,in which the spectral and temporal analysis means are separate, and have separate optical-electrical transducer elements for converting the received fluorescent light into electrical outputs, the connecting means comprising splitting means for splitting light received from the light guide along two paths, one to the temporal analysis means, the other to the spectral analysis means.

9. Apparatus according to claim 8, in which the connecting means comprises two optical fibres connected together at a junction, the junction constituting the splitting means, and each fibre extending from the junction to a respective one of the spectral analysis means and the temporal analysis means.

10. Apparatus according to claim 9, in which the connecting means comprises a "Y" fibre coupler, said optical fibres comprising the arms of the coupler, the stem of the coupler relaying received fluorescent light to the junction with the arms.

11. Apparatus according to any of claims 8 to 10, in which the connecting means further comprises a further splitting means connected between the end of the probe light guide and the first said splitter, whereby the light guide conveys both excitation light and received fluorescent light.

12. Apparatus according to any of claims 5 to 11, in which the light guide is arranged also to convey light from the source to said area.

13. Apparatus according to any of claims 5 to 12, in which the temporal analysis means is also operable to analyse a temporal characteristic of reflected excitation light, from said area, received by the light guide.

14. Apparatus according to claim 11, in which the further splitting means comprises a further "Y" fibre coupler.

15. Apparatus according to any of the preceding claims, in which spectral analyser comprises a dispersive spectrometer.

16. Apparatus according to any of the preceding claims, in which the temporal analyser comprises a high bandwidth photodetector and selection element for passing received light of a selected wavelength or range of wavelengths to the photodetector.

17. Apparatus according to any of the preceding claims, in which the apparatus is operable to modulate the intensity of the intensity of the light emitted by the source, the temporal analyser being operable to detect the phase lag between the reflected excitation light and the received fluorescent light.

18. Apparatus according to claim 17, in which the apparatus includes means for modulating the power supply to the light source,

19. Apparatus according to any of preceding claims, in which the temporal analysis means is operable preferentially to amplify only received signals of a particular, determined phase and frequency.

20. Apparatus according to claim 19, in which the temporal analysis means is operable to amplify only said signals, signals of a differing phase or frequency making no substantial contribution to the output of the temporal analysis means.

21. Apparatus according to claim 19 or claim 20, in which the temporal analysis means is connected to output of the spectral analysis means.

22. Apparatus according to any of claims 19 — 21, in which the temporal analysis means comprises a lock-in amplifier.

23. Apparatus according to claim 22, in which the apparatus includes means for modulating the power supply to the light source, and hence to modulate the intensity of light emitted by the latter, said means for modulating the power supply comprising a digital frequency synthesiser, which also acts a source of a reference signal for the lock-in amplifier.

24. Apparatus according to any of claims 19-23 in which the spectral analysis means and temporal analysis means have a common sensor for converting the received light into an electrical signal.

25. Apparatus according to claim 24, in which the spectral analysis means includes a selection elements for directing only light of a selected wavelength, or range of wavelengths, onto the common sensor.

26. Apparatus according to claim 25, in which the selection element comprises a refraction or diffraction element, for directing the received light in different direction, related to the wavelength of the received light, the selection element being moveable relative to the sensor in order to enable to wavelength component of received light incident on the sensor to be selected.

27. Apparatus according to claim 26, in which the apparatus includes a motor for moving a selection element in a controlled manner, so as to select said wavelength(s).

28. Apparatus according to any of claims 24 to 27 in which the sensor comprises a photomultiplier tube.

29. Apparatus for analysing a biological substance by causing substance to fluoresce and measuring characteristics of the resultant fluorescent light, the apparatus comprising spectral analysis means for analysing spectrum of the received fluorescent light and temporal analysis means for analysing the temporal characteristics of the received fluorescent light, wherein the spectral analysis means is connected to the temporal analysis means, so as to select the wavelength of the received fluorescent light to be analysed by the temporal analysis means.

30. Apparatus according to claim 29, in which the temporal analysis means comprises a lock-in amplifier, or computer programmed to function as such an amplifier.