GB2531998A

GB2531998A - Diagnostic Apparatus

Info

Publication number: GB2531998A
Application number: GB1418803.1A
Authority: GB
Inventors: Prydal Jeremy; Wakefield Matthew; Bannister Nigel; Molyneux Philippa
Original assignee: HOSPITALS OF LEICESTER NHS TRUST, University of; University of Leicester
Current assignee: HOSPITALS OF LEICESTER NHS TRUST, University of; University of Leicester
Priority date: 2014-10-22
Filing date: 2014-10-22
Publication date: 2016-05-11
Also published as: WO2016063063A1; GB201418803D0

Abstract

A diagnostic apparatus for in vivo diagnosis of disease, optionally an ophthalmological disease and/or a tumour. The apparatus comprises: a monochromator 6 configured to receive light containing a range of wavelengths and create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target 14; a direction means configured to focus the light created by the monochromator on the target in vivo; a detection means 24 configured to detect autofluorescence emitted by the target upon exposure to each bandwidth; and a processing means 26 configured to diagnose disease based upon the detected autofluorescence. The apparatus may be configured to diagnosis a tumour situated on the skin or in a lung. The direction means may focus the monochromator light on the eye or eyelid for diagnosing an ophthalmological disease based upon detected autofluorescence. The apparatus may be configured to detect one or more micro-organisms such as amoeba, bacterium such as Staphylococcuss, Escherichia, Pseudomonas, or fungus such as Fusarium, Candida. The detection- means may disperse the autofluorescence into a spectrum used by the processing-means to diagnose the disease, where the processing-means may provide the autofluorescent spectra as Excitation-Emission Matrix (EEM) measurements. Also disclosed a bronchoscope.

Description

Intellectual Property Office Application No. GII1418803.1 RTM Date:8 April 2015 The following terms are registered trade marks and should be read as such wherever they occur in this document: Ocean Optics (registered) Intellectual Property Office is an operating name of the Patent Office www.gov.uk /ipo -1 -Diagnostic Apparatus The present invention relates to diagnostic apparatus, and particularly, although not exclusively, diagnostic apparatus for use in detecting autofluorescence in biological samples for detecting microorganisms and/or for diagnosing disease. The invention extends to apparatus and methods of detection and diagnosis, for example ophthalmological conditions, skin cancer or lung cancer.

Autofluorescence is the natural emission of light from certain substances as a result of absorption of incident radiation of a shorter wavelength. In biological structures, the most common autofluorescence molecules are NADPH and flavins. The extracellular matrix can also contribute to autofluorescence due to intrinsic properties of collagen and elastin. Hence, autofluorescence can be used to provide important information relating to the nature of biological samples. The potential of fluorescence spectroscopy to characterize the autofluorescence signatures and provide a useful diagnostic tool in /5 medicine has been recognized for decades (see e.g. Margarot & Deveze 1925, Ward et al. 1967, and more recently, VVagnieres et al. 1998).

Infections of the cornea, or infectious keratitis, may lead to profound loss of vision and in aggressive cases complete destruction of the cornea within 24-48 hours. it is most common in soft contact lens wearers with an incidence of approximately 1 in 10,000 per year (Palmer et al., 1993). infections may be caused by bacteria of various types, fungi, viruses and amoebae. Identification of the responsible organism is crucial to selection of appropriate treatment, but techniques have progressed little since those developed by Pasteur and earlier workers in the 18th and 19th century (Foster, 1970; Collard, 1976). Samples are taken from the infected area and attempts made to culture the organism on agar plates. Growth rates under laboratory conditions vary and it may be several days before organisms can be identified. This contrasts with the ocular environment where the bacterial population doubling time is as short as 20 minutes. in addition, only in about 5o% of cases can laboratory tests identify any growth at all.

Various molecular tests have been attempted (utilising the Polymerase Chain Reaction) and although these show promise, the results to date are of limited use in a clinical setting (Gaudio et al., 2002).

Initial treatment is crucial, while the number of organisms is still relatively small.

However, with the methodology currently available, this choice has to be empirical using broad-spectrum antibiotics. Modifications may be made later depending on subsequent laboratory results or the response to treatment, but by this time the infection may have spread at an exponential rate.

Wood's light was first described in 1903, and consisted of a high pressure mercury arc lamp fitted with a filter made of barium silicate and nickel oxide. This narrow band filter ("Wood's filter") passed light from 32o to 400 nm, peaking at 365 nm (Wood, 1919). It was first used in dermatology in 1925 (Margarot, 1925) for the diagnosis of various skin conditions, including fungal and bacterial infections. The ability to identify fungal elements in corneal infections using a modification of this technique would be of considerable value as the usual first-line broad spectrum antibiotics are inactive against fungi. It may be several days, or in some cases weeks, before it becomes apparent that this is why the patient's condition has continued to deteriorate (Tuft 2009).

Certain bacteria exhibit specific fluorescence and this may enable accurate early in vivo identification of the causative organism. Infections with Pseudomonas bacteria are amongst the most destructive as they can spread rapidly though tissue and can permeate the whole eye within two days. This species produces the pigment pyoverdin or fluorescein, which gives a green fluorescence under Wood's light (Ward et al, 1967). Use of micro-spectroscopy to identify the presence of pseudomonas at the time of presentation would be of considerable value in alerting clinicians. Optical techniques also have the advantage of being less invasive. In cases where the infection does not respond to treatment and in which no organisms can be identified, patients are taken to theatre for excision of infected tissue. It is hoped that laboratory tests are then more likely to be positive with the larger sample. However, this carries significant additional risks and may mark the start of rapid deterioration in the clinical condition. Diagnosis using non-contact and non-invasive optical methodology would clearly be preferable.

While there is a need to develop a system to detect microorganisms, thereby allowing a user to diagnose ophthalmological conditions, a system to detect and accurately diagnose cancer is also needed. in ophthalmological applications, malignant tumours inside the eye are challenging to diagnose due to the difficulty in obtaining biopsy tissue from areas such as the retina. Such procedures carry significant risks and are carried out by only a very small number of specialist centres. In some cases, no biopsy option is available, and the only course of action is removal of the eye. In some cases, subsequent analysis of the eye post-excision reveals the tissue to be benign. -3 -

Accordingly, the ability to detect and identify malignant tissue using a non-invasive method is highly desirable.

it will be appreciated that such a system would also have application identifying further tumours in vivo, such as those caused by lung or skin cancer, thereby removing the need for patients to undergo painful biopsies. Alternatively, if a biopsy was unavoidable, it would be beneficial to have a system that allowed a clinician to map the abnormality. This would allow a clinician to identify the abnormal tissue and could aid them in removing all of the abnormality and/or in biopsying the most abnormal tissue, thus increasing the likelihood of an accurate diagnosis by standard histopathological examination.

There is therefore a need to provide an alternative and/or improved apparatus for use in detecting and diagnosing ophthalmological conditions, and cancer.

The inventors have developed a novel diagnostic apparatus which detects in vivo autofluorescence of a biological sample (e.g. a microorganism or a tumour) upon exposure to monochromatic light.

Thus, in accordance with a first aspect of the invention, there is provided a diagnostic apparatus for the in vivo diagnosis of disease, optionally an ophthalmological disease and/or a tumour, the apparatus comprising:- - a monochromator configured to receive light containing a range of wavelengths, and create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; - direction means configured to focus the light created by the monochromator on the biological target in vivo; - detection means configured to detect autofluorescence emitted by the biological target upon exposure to each bandwidth; and -processing means configured to diagnose disease based on the detected autofluorescence.

The inventors have also developed a novel method for diagnosing disease in vivo. -4 -

Hence, in second aspect, there is provided an in vivo method for diagnosing disease in a subject, optionally an ophthalmological disease and/or a tumour, the method comprising:- - passing light containing a range of wavelengths to a monochromator, and then using the light to create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; focusing the light created by the monochromator on the biological target in vivo; - detecting autofluorescence emitted by the biological target upon exposure to each bandwidth; and - diagnosing disease based on the detected autofluorescence. Advantageously, the apparatus and method of the invention enable significantly improved resolution and hence better discrimination and sensitivity, as they offer the ability to examine the autofluorescence emitted by the target at any wavelength within the excitation range rather than a few discrete emission lines driven by available optoelectronics (e.g. LED's or laser diodes) instead of clinical requirements. As such, it allows a clinician to more accurately diagnose an infection or identify cancerous tissue in a subject. Additionally, the ability to use the apparatus and method in vivo prevents the need for samples to be taken from the subject, which could otherwise be painful and potentially harmful. Additionally, this prevents the need to culture the detected organism which is time-consuming. Accordingly, the subject may be diagnosed quickly with minimal risk of harm.

It will be appreciated that by varying the exact location of the biological target it will be possible for a user to build up a 2D or 3D image of the abnormal tissue.

Hence, in accordance with a third aspect, there is provided an in vivo method for mapping an abnormality in a subject, optionally where the abnormality is an ophthalmological disease and/or a tumour, the method comprising:- - passing light containing a range of wavelengths to a monochromator, and then using the light to create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; - focusing the light created by the monochromator at multiple locations within the biological target in vivo; -5 - - detecting autofluorescence emitted by the biological target upon exposure to each bandwidth at each location; and - constructing a map of the abnormality based on the detected autofluorescence.

The map may be a 2D map, e.g. it may be of the surface of the subject's skin. Alternatively, the map may be a 3D map, e.g. it may be of a given volume of the subject's lung.

o Advantageously, the method may enable clinicians to identify and biopsy the most abnormal tissue, thus increasing the likelihood of an accurate diagnosis by standard histopathological examination.

Preferably, the apparatus and method are configured or used to diagnose a tumour, which may be situated on the skin (e.g. basal cell cancer, squa mous cell cancer or melanoma) or in the lung (i.e. lung cancer).

Thus, in a fourth aspect, there is provided a bronchoscope comprising the apparatus of the first aspect.

Preferably, the bronchoscope comprises a UV-transparent optical element (e.g. an optical flat) configured to separate the apparatus of the first aspect from a biological target and direct light appropriately.

In a fifth aspect, there is provided an in vivo method for detecting a tumour in a subject's lung, the method comprising:- - passing light containing a range of wavelengths to a monochromator, and then using the light to create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; -focusing the light created by the monochromator on a region of a subject's lung in vivo; - detecting autofluorescence emitted by the region of the lung upon exposure to each bandwidth; and - diagnosing a lung tumour based on the detected autofluorescence. -6 -

Accordingly, in a sixth aspect, there is provided apparatus for the diagnosis of a skin cancer on a subject in vivo, the apparatus comprising:- - a monochromator configured to receive light containing a range of wavelengths, and create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; - direction means configured to focus the light created by the monochromator on a region of the subject's skin in vivo; - detection means configured to detect autofluorescence emitted by the region of the skin upon exposure to each bandwidth; and -processing means configured to diagnose a skin cancer based on the detected autofluorescence.

In a seventh aspect, there is provided an in vivo method for a diagnosis of a skin cancer on a subject, the method comprising:- -passing light containing a range of wavelengths to a monochromator, and then using the light to create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; focusing the light created by the monochromator on a region of the subject's skin in vivo; -detecting autofluorescence emitted by the region of the skin upon exposure to each bandwidth; and - diagnosing a skin cancer based on the detected autofluorescence. The skin cancer which is diagnosed may be basal cell cancer, squamous cell cancer or -0or melanoma. Preferably, the skin cancer is a melanoma.

Most preferably, however, the apparatus and method are configured or used to diagnose ophthalmological diseases (e.g. a tumour), which may be in or on the eye or eyelid. Using the apparatus and methodology described above, diagnosis of various eye infections is also possible using non-contact and non-invasive optical methodology. Ocular oncology is an especially preferred application for the apparatus and method of the invention. For example, the light created by the monochromator may be focused on to the cornea or retina of the eye, and autofluorescence is then detected therefrom. -7 -

Accordingly, in an eighth aspect, there is provided an ophthalmological diagnostic apparatus for the in vivo diagnosis of an ophthalmological disease in a subject, the apparatus comprising:- - a monochromator configured to receive light containing a range of wavelengths, and create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; - direction means configured to focus the light created by the monochromator on or in a region of a subject's eye in vivo; - detection means configured to detect autofluorescence emitted by the region of the eye upon exposure to each bandwidth; and - processing means configured to diagnose an ophthalmological disease based on the detected autofluorescence. In a ninth aspect, there is provided an in vivo method for diagnosing an ophthalmological disease in a subject, the method comprising:- - passing light containing a range of wavelengths to a monochromator, and then using the light to create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; focusing the light created by the monochromator on or in a region of a subject's eye in vivo; - detecting autofluorescence emitted by the region of the eye upon exposure to each bandwidth; and - diagnosing disease based on the detected autofluorescence.

-0or The direction means is preferably configured to focus the light created by the monochromator on or in the eye or eyelid.

The apparatuses and methods of the invention are preferably configured or used to detect one or more micro-organism via autofluorescence. The micro-organism may be a bacterium, amoeba or fungus. The bacterium may be Gram positive or Gram negative. Examples of bacteria which may be detected include Staphylococcus spp., Escherichia spp. and/or Pseudomonas spp.. Examples of fungus which may be detected include Fusarium spp. and/or Candida spp.

Preferably, S. aureus, E. coli, P. aeruginosa and/or C. albicans or Fusarium species are detected via autofluorescence. -8 -

The diagnostic apparatus preferably comprises a light source selected from any number of suitable light sources as known to those skilled in the art. Any portion of the electromagnetic spectrum that produces usable light can be used. Light sources capable of emission in the ultraviolet, visible and/or near-infrared spectra, as well as other portions of the electromagnetic spectrum, can be utilized and are known to those skilled in the art. For example, light sources may be continuum lamps such as a deuterium or xenon arc lamp for generation of ultraviolet light and a tungsten halogen lamp for generation of visible/near-infrared excitation.

The light source may be configured to emit light in the range ioonm to 2000nm, more preferably in the range of 12onm to 800 nm, more preferably isonm to 600 nm, and even most preferably 2oonm to 450 nm.

It will be understood that the excitation range will vary depending on the biological target. The excitation range may be in the range of 15onm to 500 nm, more preferably 2oonm to 450 nm, and even more preferably 25onm to 40o nm, and most preferably 25onm to 350 nm.

The light source may be functionally linked to the monochromator (e.g., using fiber optics). Preferably, the light source is connected to the monochromator via an optical fibre. The monochromator may reduce the broad emission range produced by the light source to produce the plurality of substantially continuous bandwidths. The monochromator may comprise optical interference filters, prisms, optical gratings, a scanning monochromator, an acousto-optic tunable filter, liquid crystal tunable filter, an array of optical interference filters and/or a prism spectrograph.

Preferably, the monochromator is configured to create the plurality of substantially continuous bandwidths of light in a sequential or step-wise manner along the excitation range of the biological target.

It will be appreciated that the monochromator is configured to create a plurality of substantially continuous bandwidths of light, wherein each bandwidth comprises substantially monochromatic light. It may be understood that each bandwidth will be considered to comprise substantially monochromatic light as the bandwidth comprises light with wavelengths with a selectivity of about to nm, more preferably with a -9 -selectivity of about 5 nm, 4 nm, 3 nm or 2 nm and most preferably with a selectivity of about 1 nm. Hence, each bandwidth created by the monochromator is approximately o.5nm-2nm in wavelength, preferably approximately mm in wavelength.

The term "substantially continuous bandwidths of light across an excitation range of a biological target" can mean that at least 85%, 9o%, 95%, 98%, 99% or r00% of the excitation range is covered. Advantageously, the prior art does not enable exposure of a biological target to such a large amount of its excitation range.

ro Preferably, the light created by the monochromator results in the excitation of the sample, followed by measurement of the emission of fluorescence from the target at predetermined time points, or continuously. Similarly, the reflected light from interaction of the excitation source with the target may be measured to provide pertinent data for detection, characterization and/or calibration.

It will be appreciated therefore that the essentially monochromatic light created by the monochromator is used as the excitation source, which is directed onto the biological target via the direction means. The direction means may comprise at least one illumination fibre along which the bandwidth created by the monochromator is passed to the target.

Preferably, the direction means comprises focusing optics functionally linked to the detection means (e.g., using fiber optics). Preferably, the focusing optics is configured to focus the plurality of substantially continuous bandwidths of light created by the -0or monochromator on the biological target. The focusing optics may comprise a lens system or a microscope configured to permit observations in the ultraviolet, visible, and infrared range. Preferably, the direction means comprises a reflectance probe.

In one preferred embodiment, the apparatus and method of the invention involves the application of excitation wavelength-resolved fluorescence spectrometry to ophthalmology diagnostics, for example for diagnosing a corneal ulcer, as shown in Figure 10. The apparatus therefore preferably comprises a short focal length converging lens and/or increases the probe-eye distance during the diagnostic method. Since this application relates to surface features of the eye, no problems of UV absorption inside the eye occur. The apparatus may be mounted on a slit lamp and targeting may be achieved effectively by eye, since the clinician can see the illumination spot on the -10 -surface of the cornea. However, the apparatus may be incorporated as a co-aligned channel in the slit lamp assembly, and comprise a reticle that indicates the precise measurement point in the field of view observed by the clinician.

In another preferred embodiment, the apparatus and method of the invention involves the application of excitation wavelength-resolved fluorescence spectrometry to ophthalmology diagnostics, for example for diagnosing a tumour inside the eye, as shown in Figure 14. This embodiment of the apparatus is similar to the embodiment shown in Figure 10, except that the converging lens has a different focal length, such that the light field is focused on a region inside the eye, such as the retina or choroid, rather than on the cornea.

Preferably, the direction means comprises at least one read fibre along which reflected light and autofluorescence from the target is passed to the detection means, preferably along a sensing waveguide.

Preferably, the detection means comprises a spectrometer. The spectrometer may be a scanning monochromator that detects specific emission wavelengths whereby the output from the scanning monochromator is detected by a photomultiplier tube and/or the spectrometer may be configured as an imaging spectrograph whereby the output is detected by an imaging detector array such as a charge-coupled device (CCD) detector array. In one embodiment, a discriminator allows the observation of the fluorescence and/or scattering signal by a photodetection means (such as a photomultiplier tube, avalanche photodiode, CCD detector array, and/or electron multiplying charge coupled or device (EMCCD) detector array). Preferably the spectrometer is sensitive in the range of loo to 800 nm, zoo to 70o nm, and/or 30o to 600 nm.

Preferably, the detection means is configured to disperse the autofluorescence into a spectrum, which is used by the processing means to diagnose the disease. Preferably, 3o the processing means is configured to provide the autofluorescent spectra as Excitation-Emission Matrix (EEM) measurements.

As used herein, EEM is defined as the luminescent spectral emission intensity of fluorescent substances as a function of both excitation and emission wavelength, and 35 includes a full spectrum or a subset thereof, where a subset may contain a single or multiple excitation/emission pairs. Preferably, the EEM comprises the excitation data for at least 10 continuous bandwidths of light, more preferably at least 20, 30, 40, 50, 60, 70, 80, 90 continuous bandwidths of light, and most preferably at least 100 bandwidths of light. Preferably, each illumination has an exposure time of less than about to seconds, more preferably less than about 5, 4, 3 or 2 seconds and most preferably less than about 1 second per wavelength. Preferably, each illumination is repeated at least 2, 3, 4 or 5 times. Preferably, the apparatus and method do not comprise any contact between the direction means and the target.

Additionally, a cross section of the EEM with a fixed excitation wavelength may be used /0 to show the emission spectra for a specific excitation bandwidth.

In one embodiment, emission spectra for a plurality of excitation bandwidths may be analysed. A plurality of excitation bandwidths may comprise the spectra of at least at 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 5o excitation bandwidths. In certain embodiments, the number of spectra of excitation bandwidths analysed is sufficient to determine the exact species of the microorganism or diagnose a tumour, e.g. about 1 to about 10 spectra, e.g., about 1 to about 5 wavelength pairs. In a preferred embodiment, 3 spectra of excitation bandwidths are analysed.

According to the invention, control measurements may be taken for colonies of known microorganisms and/or tumours, thus allowing for correlation of measured test data with characterization of the microorganisms or tumours of interest using various mathematical methods known to those skilled in the art. For example, the data from samples may be compared with the baseline or control measurements utilizing software systems known to one skilled in the art. More particularly, the data may be analyzed by a number of multivariate analysis methods, such as, for example, General Discriminant Analysis (GDA), Partial Least Squares Discriminant Analysis (PLSDA), Partial Least Squares regression, Principal Component Analysis (PCA), Parallel Factor Analysis (PARAFAC), Neural Network Analysis (NNA) and/or Support Vector Machine (SVM).

These methods may be used to classify unknown microorganisms or tumours into relevant groups based on existing nomenclature, and/or, for microorganisms, into naturally occurring groups based on the organism's metabolism, pathogenicity and/or virulence in designing the system for monitoring, detecting and/or characterizing the organism as described previously.

Most preferably, the data are analyzed by a PCA analysis, as described in the Examples.

-12 -All features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, maybe combined with any of the above aspects in any combination, except combinations 5 where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-Figure 1 is a schematic side view of a first embodiment of a diagnostic apparatus of the invention showing its key components required for general diagnostic applications (e.g. in ophthamology, skin cancer diagnostics and bronchoscopy), including a light source, monochromater, reflectance probe and spectrometer. The inset is an end view of the reflectance probe showing the arrangement of fibres therein. There are six illuminator (T) fibres and a central read (R) fibre; Figure 2 is a histogram of wavelength versus normalised intensity; Figure 3 shows various surface plots of emission wavelength (x-axis) versus intensity (z, or vertical, axis) as a function of excitation emission wavelength (y-axis) from Shelly et al. 1980 showing the difference in spectral response between a number of species of 20 Pseudomonas specises; Figure 4a is a 2D colour plot showing the response of E. Coil to analysis using the diagnostic apparatus shown in Figure 1. The plot shows the data after calibration and filtering to reduce noise and removal of intrinsic instrumentation characteristics; Figure 4b is a 2D colour plot showing the response of P. aeruginosa to analysis using the apparatus shown in Figure 1. The plot shows the data after calibration and filtering to reduce noise and removal of intrinsic instrumentation characteristics; Figure 5 shows two Excitation-Emission Matrices (EEMs) for four different microorganisms, namely S. aureus, E. coli, P. aeruginosa and C. albicans; Figure 6 shows histograms of the spectra acquired for the four microorganisms, with three histograms per axis (corresponding to the three selected excitation wavelengths used in the Principal Component Analysis, i.e. PCA); Figure 7 shows the results of PCA analysis of the samples, showing clear groupings which uniquely identify the microorganisms on the basis of their fluorescent spectra; Figure 8a shows two microscope slides the first being stained and containing skin slices comprising healthy tissue and melanoma, and the second slide having the same skin samples but being unstained; -13 -Figure 8b shows PCA analysis of the EEM showing dear grouping of normal tissue and a separate population of melanoma; Figure 8c shows EEM for normal skin; Figure 8d shows EEM for melanoma, showing a clear feature not visible in the normal skin data; Figure 9 is a schematic side view of the human eye; Figure lo shows a second embodiment of the diagnostic apparatus, for use in the detection of corneal ulcers; Figure n is a schematic side view of a third embodiment of the diagnostic apparatus, for use in the laboratory showing an adjustable converging lens and a precision X-Y-Z translation table under computer control; Figure 122 is a schematic side view of a fourth embodiment of the diagnostic apparatus, for incorporation in a bronchoscope, for use in detecting lung cancer; Figure 12b is a schematic side view of the tip of a bronchoscope, as depicted in Figure 13c, in use; Figure 12C is a schematic side view of the tip of a bronchoscope, as depicted in Figure 13d, in use; Figure 13a is a side view image of a standard bronchoscope; Figure 13b is an end view of the tip of the standard bronchoscope of Figure 13a; Figure 13c is an end view of the tip of a first embodiment of a modified bronchoscope according to the invention. In the modified bronchoscope, one light source has been replaced with a reflectance probe of the diagnostic apparatus of the invention, as shown in Figure 1; Figure 13d is an image of the tip of a second embodiment of a modified bronchoscope according to the invention. In this embodiment, both light sources have been replaced, one becoming the illuminator fibre and the other becoming the read fibre for the spectrometer; and Figure 14 shows a fifth embodiment of the diagnostic apparatus of the invention, for use in ocular oncology in which the excitation of fluorescence and recording the response from the back of the eye is required.

Examples

The original motivation for the inventors' work was the application of excitation wavelength-resolved fluorescence spectrometry to ophthalmology. As such, Examples 2 35 and 3 describe embodiments of a diagnostic apparatus 1 of the invention for use in detecting corneal ulcers and in ocular oncology. However, the inventors have also shown that the apparatus 1 can also be used to detect skin cancer and lung cancer, as described in Example 4. Example 1 discusses the background to the invention.

Example 1-Obtaining and comparing Excitation -Emission Matrices (EEMs) for S. 5 aureus, E. coil P. aeruginosa and C. albicans The characterization of a sample using excitation wavelength-resolved fluorescence spectroscopy involves:- 1. Setting the excitation wavelength to a starting value.

Jo Typically the starting value will be at the long or short wavelength extremes of the overall excitation bandwidth to be explored. The excitation bandwidth will typically include wavelengths which are at the short wavelength end of the visible spectrum to the ultraviolet, i.e. approximately zoo nm to 450 nm. These wavelengths act as the excitation stimulus.

2. Illumination of the sample at the excitation wavelength.

A portion of the excitation photons are reflected from the excited sample, but a significant fraction are absorbed and cause the sample to emit photons over a range of longer wavelengths via the process of fluorescence.

3. Detection of the reflected and fluorescence photons from the sample, and dispersal of these photons into a spectrum.

An individual spectrum corresponding to a single excitation wavelength is typically presented as wavelength on the abscissa, and intensity on the ordinate axis. -0or

4. Change of excitation wavelength by a small increment.

5. Repetition of steps 2-4 until the complete excitation wavelength space has been covered.

6. Presentation of the data in an appropriate form to enable extraction of the information contained in the spectra.

To facilitate this measurement, and with reference to Figure 1, a first embodiment of a diagnostic apparatus 1 has been designed. It comprises a light source 2 (e.g. an Ocean Optics HPX-2000 Xenon arc lamp (power 35W)), which emits light in the range 185 - -15 - 2000 nm. The light source 2 is connected via an optical fibre 4 to a monochromator 6 (e.g. a Newport Cornerstone 130 motorised monochromator), which allows a small (-1 nm) portion of the emitted light to be selected. The monochromator 6 is connected by an illumination waveguide 8 to a reflectance probe 10 (e.g. an Ocean Optics QR2oo-7 UV reflectance probe). The essentially monochromatic light 12 is used as the excitation source, which is directed onto a sample 14 using the reflectance probe 10. The probe 10 consists of a circumferential ring of six illumination fibres 16 along which the monochromatic light 12 is passed to the sample 14. The probe 14 includes a centrally aligned fibre 18 which branches away from the illumination fibres 16 to direct reflected to light and autofluorescence 20 from the sample 14 along a sensing waveguide 22 to a spectrometer 24, in this case an Ocean Optics Maya spectrometer which is sensitive in the range 25o -700 nm). A computer 26 is connected to the rest of the apparatus 1 to control it, and to analyse the results.

Typically, each sample 14 is illuminated at up to loo illumination wavelengths, with an exposure time of less than 1 second per wavelength. Measurements are repeated at least 5 times. This process is analogous to taking multiple photographs and involves light wavelengths and intensities no greater than those used in standard autofluorescence bronchoscopy. Energy levels are insufficient to cause damage to the tissues or other effects on subsequent processing or testing, and there is no contact between the reflectance probe 10 and sample 14.

The output of the spectrometer 24 is a histogram showing the intensity (ordinate) versus wavelength (abscissa) of the light which is produced by the apparatus 1. One 25 histogram is produced for each excitation wavelength, and an example of this type of output is shown in Figure 2.

As described below, a number of investigations published in the literature use a limited number of excitation wavelengths (typically between two and six) to attempt discrimination between tissue types or microbiology samples. In such cases it is possible to use the ratio of two plots of the kind shown in Figure 2 to quantify the difference in spectral characteristics. It may also be possible to use the ratio of two specific wavelengths in the plot, rather than produce another histogram representing the ratio of each wavelength bin. However, in accordance with the present invention, a large number of excitation wavelengths are used, generating more than 100 spectra of the type shown in Figure 2, and so an alternative method of plotting is required. One approach is to use a surface plot, an example of which is shown in Figure 3.

However, the inventors have adopted an alternative approach, which is to produce a 2D colour plot of the data in which the X-axis represents wavelength of the measured (response) light, the Y-axis represents the excitation wavelength, and the intensity is colour-coded so that red = 1 and black = o. An example of this approach is shown in Figure 4. Each row of these plots can be interpreted as a histogram like that shown in Figure 2, for a given excitation wavelength. The differences in the appearance of the two plots shown in Figure 4 are statistically significant and form the basis of the discrimination method used in this work.

This type of plot, which is referred to as an "Exitation-Emission Matrix" (EEM) provides a very comprehensive characterization of the fluorescent response of the sample. Some excitation wavelengths may lead to spectral responses that are quite similar in two different targets but the method relies on there being two or more excitation wavelengths that generate usefully different responses that provide discrimination between the samples. These more discriminating wavelengths can be identified, approximately, by eye from plots such as those in Figure 3 and Figure 4.

As described above, in some existing implementations of the fluorescence technique the light source comprises a series of LEDs and laser diodes which offer a small number of specific excitation wavelengths, and it is the availability of specific semiconductor devices which dictate the precise excitation wavelengths that can be used. However, in the case of instruments (such as that described here) which use continuum light sources and monochromating systems to generate excitation photons, considerable freedom exists to choose useful wavelengths with which to discriminate between tissue types of microbiology samples. In this case, the technique of Principal Component Analysis (PCA) can be thought of as identifying linear projections of a multi-variate data set that show the maximum disorder (i.e. which maximize the variance of the data points) and hence can be used to identify the most useful elements of large data sets such as the scans shown in Figure 4.

Principal Component Analysis (PCA) As explained in at ftp://statgen.ncsu.eduipub/thorne/molevoclass/AtchleyOctis.pdf, principal component analysis (PCA) is a mathematical procedure that transforms a -17 -number of (possibly) correlated variables into a (smaller) number of uncorrelated variables called principal components. The first principal component accounts for as much of the variability in the data as possible, and each succeeding component accounts for as much of the remaining variability as possible.

PCA is performed using matrix algebra and involves finding the eigenvalues of the data set: the magnitude of these eigenvalues is a measure of how much of the difference between the data sets can be explained by the corresponding eigenvector. The vector with the largest eigenvalue is called the first principal component of the differences and explains most of the differences in the data; the vector with the next largest eigenvalue is called the second principal component and explains most of the differences not explained by the first vector, and so on.

The inventors carried out PCA using a bespoke data processing pipeline written in the 15 IDE language, which contained some dedicated PCA algorithms. The process was as follows: 1. Perform scans of the microbiology targets. In this case, there are four microorganisms to be investigated, S. aureus, E. coli, P. aeruginosa and C. albicans, and each one is scanned twice. This generates a series of high resolution Excitation-Emission Matrices as shown in Figure 5.

2. Manually identify three excitation wavelengths which can be used to define the shape of the peak emission region seen in the EEMs These wavelengths define the -0or sample points which will be used to characterize the differences in the shape of the fluorescence response. In the current system, the three spectra corresponding to the three selected excitation wavelengths are appended and plotted on a single axis, as shown in Figure 6.

3. Run the data through the PCA algorithm. Data are input in the form of a table, with each row representing one spectrum, and each column representing the intensity of a given point in a spectrum. The algorithm performs the following functions: Calculates covariance of columns i.e. calculates which parts of the spectra vary together and which vary independently of each other. -i8-

Uses the covariance to find linear combinations of variables which maximise the differences in the dataset, these are the Principal Components (PCs).

Assigns weights to every intensity value based on how important it is for differentiating between spectra. The combination of weights that explains the most variation is the first principal component (PC1). The process is repeated to find other combinations of weights which explain different aspects of the variation; PC2, PC3 etc. it) 4. Plot the data in terms of PC1 vs PC2, PC( vs PC3 etc. This generates scatter plots which show distinct groupings of samples and permit unambiguous identification of the microorganism based on location within the plot. In the two plots shown in Figure 7, the left figure is PC) vs PC2, the right is PC) vs PC3.

In the PC). vs PC2 plot, the P. aeruginosa samples exclusively occupy the top right quadrant; the S. aureus in the lower left quadrant, and the E. coil and C. albicans are found in close proximity in the upper left quadrant. However, in the PC! vs PC3 data, the E. coli and C. albicans are widely separated in the left side, while P. aeruginosa now occupies two regions, one of which is close to the location of S. aureus. Hence, using the location of the data point in two PC plots uniquely identifies the microorganism.

The work described above involved scanning bacterial and fungal colonies on agar plates. However, the invention can clearly extend to microorganisms suspended in water instead. This technique has the advantage of allowing a direct comparison -0or between fungal and bacterial autofluorescence signatures and the autofluorescence of protozoa such as acanthamoeba, which is grown in a nutrient-rich broth rather than on agar.

While the apparatus 1 can vary depending upon its intended use (e.g. eye, skin or lung detection), each embodiment described below contains the light source 2, monochromator 6, spectrometer 24, and computer 26 for control and analysis. The principal differences between the embodiments of the apparatus are in the packaging of the fibre optic probe 10 and the optical elements which allow the instrument to access the target in the specific environment of that application, i.e. eye (surface cornea or retina), lung or skin.

Example 2 -0m Ulcers The original motivation for the inventor's work was the application of excitation wavelength-resolved fluorescence spectrometry to ophthalmology. Using the apparatus and methodology described above, diagnosis of various eye infections is possible using 5 non-contact and non-invasive optical methodology.

Referring to Figure 9, there is shown the general anatomy of the human eye. Detecting microbiological signatures in corneal ulcers requires that the illumination and readout spot is located on the cornea 34, which forms part of the outer surface of the eye 36.

The second embodiment of the apparatus 86 used for ocular oncology is shown in Figure 10. The apparatus 86 uses a short focal length converging lens 38 and/or increases the probe-eye distance. Since this application relates to surface features of the eye, no problems of UV absorption inside the eye 36 occur. The apparatus 86 can be mounted on a slit lamp and targeting can be achieved effectively by eye, since the clinician can see the illumination spot on the surface of the cornea.

However, it may be desirable or necessary to incorporate the apparatus 86 as a co-aligned channel in the slit lamp assembly, and provide a reticle that indicates the precise measurement point in the field of view observed by the clinician.

Example 3 -Ocular Oncology This further ophthalmological application combines the ability to detect malignant tissue with the ophthalmological motivation for the inventor's work discussed in -0or Example 2.

Malignant tumours inside the eye 36 are challenging to diagnose due to the difficulty in obtaining biopsy tissue from areas such as the retina. Such procedures carry significant risks and arc carried out by only a very small number of specialist centres. In some cases, no biopsy option is available, and the only course of action is removal of the eye 36. In some cases, subsequent analysis of the eye post-excision reveals the tissue to be benign. The ability to detect and identify malignant tissue using a non-invasive method is highly desirable. Work carried out by the inventors into the detection of scleritis, has shown that it is possible to obtain reflectance spectra from the retina. If this approach can be combined with fluorescence spectroscopy, this opens the possibility to the detection of malignant tissue through use of an appropriate excitation wavelength, and subsequent detection of a characteristic fluorescence spectrum in response.

Referring to Figure 14, there is shown another embodiment of the apparatus 88, for use in ocular oncology. The extreme proximity (-few microns) of the illuminator fibres 16 to the read fibre 18 in the reflectance probe 10 lends itself well to the task of measuring fluorescence from tissue inside the eye (i.e. on the retina). This is due to the closely shared light-paths of the output and input beams. The presence, in the measured signal, of reflections from the surface of the eye 36 do not pose any difficulty for the instrument since the reflection will be in the narrow excitation waveband which is filtered from the measured signal during data processing. Consequently, coupling the spectrometer 24 to the target tissue can, in principle, be achieved using only a single optic of the type found in the final stage of a generic fundus camera, as shown in Figure 14. This embodiment 88 of the apparatus is very similar to the embodiment 86 shown in Figure 10, except that the converging lens 82 has a different focal length such that the light field is focused on the retina 84 rather than on the cornea 34.

Three practical challenges face the implementation of the apparatus 88: 1. The UV opacity of the structures inside the eye 36 along the input and output light paths.

2. Enabling the clinician to control precisely the location of the measurement.

3. The surface of the eye 36 will also fluoresce.

In the first case, it is noted that fluorescence measurements are already used to detect lesions on the fundus, albeit using broadband techniques and excitation at a single wavelength (e.g. Shields et al., 2008 and Singh et al., 2010). In both of these papers, the excitation wavelength is longer than the range considered in the current project (580 nm and 488 nm respectively, compared to the 260-40o nm range used in the current work). Laboratory work will be required to establish the UV transmissivity of the eye 36 and hence the predicted sensitivity and illumination power requirements of the application.

In the second case, one approach to precise guidance and selection of the measurement 35 would be to integrate and co-align the instrument with the optical axis of a fundus -21 -camera, such that the measurement area is coincident with the centre of the field of view (which could be marked with a suitable reticle).

In the third case, there are a number of ways to overcome this problem. For instance, it 5 will be to take a measurement with the excitation source at 90° to the optical path of the read measurement using a modification in which the illuminator and read fibres 16 and 18 are in different fibre optic assemblies.

Example 4 -Melanoma and Bronchoscopy Autofluorescence spectra provide a potentially rich source of information on tissue state, in terms of component protein expression, overall tissue architecture and metabolic activity, which may be used to improve current methods of disease detection and treatment. Hurzeler (1975) described the use of broadband autofluorescence signals to examine lung tissue and this led to the development of several commercial systems based on the observation that pre-cancerous lesions illuminated with violet light display a decrease of autofluorescence intensity in the green region of the spectrum and a relative increase in the red region. Current imaging devices rely on this principle and are used to indicate possible early cancerous lesions in the bronchi in autofluorescence bronchoscopy (AFB). However, the principle excitation and emission wavelengths have never been optimised or refined in the literature. The first randomised controlled in-vivo trial (1173 patients) reported by Haussinger et al (2005) did not demonstrate an advantage for detecting carcinoma in-situ, but was superior in detecting pre-cancerous dysplasia of grades II and III. One of the open issues regarding AFB is this question of its usability in lung cancer detection. Although currently not supported by the literature, "it is believed that software enhancements such as spectral analysis can improve the specificity of AFB" (Zaric et al, 2013).

The use of a tuneable light source combined with high spectral resolution enables autofluorescence signals of significantly higher resolution (1nm wavelength increments) and sensitivity to be detected. Measurements of histology slides provided by Dr Gerald Saldanha (Consultant Histopathologist and Honorary Senior Lecturer at the University of Leicester) have been shown to provide good discrimination between normal tissue and melanoma, based on visual inspection of the Excitation-Emission Matrices, and also on PCA analysis. The results are shown in Figure 8. It should be noted that while a stained slide 4o is shown for reference in Figure 8a, the measured samples were unstained 42. By comparing the EEM for normal skin, Figure 8c, with the EEM for melanoma, Figure 8d, it is apparent that a feature clearly visible in the melanoma is not present in normal skin. The PCA analysis of the EEMs confirms this and shows a clear difference between the melanoma and skin.

By incorporating the teachings of the current invention into a bronchoscope 44, another embodiment of the apparatus 90 is created, which is shown in Figure 12. This apparatus 90 permits a clinician to identify areas of malignant tissue in-vivo to inform surgical procedures and treatment approaches. In contrast to the apparatus for use in the ophthalmology applications described in Examples 2 and 3, an optical flat 46 has been provided on the end of the probe 10. The optical flat 46 comprises a thin UV-transparent material and is provided to separate the tissue 48 from the probe 10. This ensures that sufficient space is left between probe 10 and tissue 48 for the diverging cones of light from illuminators 16, to cover the area of interest and for this to be visible by the read fibre.

The first stage in this work is demonstration, ex-vivo, of the instrument's 90 capability to distinguish between malignant lesions and normal healthy tissue. The ex-vivo biopsy specimens of suspected malignant lesions are transferred to a pathology laboratory and prepared following standard histopathological protocols. Immediately before fixing the tissue in formalin, the autofluorescence spectra are recorded from a number of different sites on the specimen.

Referring to Figure 13, there is shown a standard bronchoscope 44 as well as embodiments of a modified bronchoscope 94 according to the invention. The standard bronchoscope 44 comprises an eye piece 5o, which may be attached to a camera (not shown), a diapter ring 52 for focusing, and a control lever (not shown) which controls the tip 53 and only allows movement in a vertical plane. The bronchoscope 44 also has a working channel port and suction valve and port 54 for suction, instillation of local aesthetic and oxygen delivery, a body 56, an insertion cord 58 and a light source 6o.

Figure 13b shows an end view of the tip 53 used in the standard bronchoscope 44. The tip 53 comprises a suction channel 62, a lens 64 with an optic centre 66 and two lights 68 arranged around the geometric centre 70 and held in place due to a metal housing 72 which is surrounded by a rubber sleeve 74.

In the embodiment the apparatus 90, the tip 53 is modified by replacing one of the lights 68 with a reflectance probe 76, while leaving the other light 68 in place, as shown in Figure 13c. How the light would be reflected and emitted in practice is shown schematically in Figure 12b. Alternatively, one of the lights 68 may be replaced with an illuminator fibre 78 and the other light 68 with a read fibre 80. How this would work in practice is shown schematically at Figure 12c. By mounting the fibres 78, 80 at an angle, it is possible to illuminate and read a portion of tissue 48 along the central axis of the probe, and this may offer the advantage of being able to "paint" the target in the centre of the field of view of the camera which the clinician uses to conduct the bronchoscopic investigation.

Example s -General Laboratory instrument In the embodiment shown in Figure 1, no focusing optics, such as a lens, is used to control the size of the illumination/read area. Referring to Figure 11, there is shown a further embodiment of the apparatus 92, for use as a general laboratory instrument. In this embodiment, a converging lens 28 is mounted on an adjustable lens mount 30 which allows more precise selectivity of the sample area, thereby allowing highly specific or area-averaged results to be obtained. The versatility and control of the instrument would be enhanced by mounting the reflectance probe + optic on a motorised X-Y-Z stage 32 to allow precise, computer controlled positioning and systematic scanning of samples in a range of forms.

Conclusions

By offering the ability to examine autofluorescence at any wavelength within the excitation range rather than a few discrete emission lines driven by available optoelectronics rather than clinical requirements, the inventors anticipate that the apparatus 1 will offer improved resolution and hence better discrimination and sensitivity (by allowing a truly optimized set of diagnostic lines to be selected). This will allow a clinician to more accurately diagnose an infection or identify cancerous tissue.

Additionally, the ability to use the apparatus 1 in vivo will prevent the need for samples to be taken from a patient, which could otherwise be painful and potentially harmful.

Additionally, this method prevents the need to culture the organism which is time-consuming. Accordingly, patient may be diagnosed quickly with minimal risk of harm.

References Collard P. The Development of Microbiology. Cambridge: Cambridge University Press, 1976 Foster WD. A History of Medical Bacteriology and Immunology. London: Heinemann, 1970 Gaudio PA, Gopinathan U, Sangwan V, Hughes 1E. Polymerase chain reaction based detection of fungi in infected corneas. Br J Ophthalmol 2002;86:755-76o Haussinger K, Becker H, Stanzel F, Kreuzer A, Schmidt B, Strausz J, Cavaliere S, Herth F, Kohlhaufl M, Muller KM, Huber R1\4, Pichlmeier U, Bolliger ChT. Autofluorescence bronchoscopy with white light bronchoscopy compared with white light bronchoscopy alone for the detection of precancerous lesions: a European randomised controlled multicentre trial. Thorax. 2005 Jun;60(6):496-5o3 Margarot J, Deveze P. Aspect de quelques dermatoses lumiere ultraparaviolette. Note preliminaire. Bull Soc Sci Med Biol Montpellier 1925;6:375-8. Quoted from: Asawanonda P, Charles TR. Wood's light in dermatology. Int J Dermatol 1999;38:8o1-7.

Palmer ML, Hyndiuk RA. Contact lens-related infectious keratitis. Int Ophthalmol Clin. 1993; 33(1):23-49.

Shelly, D. C., Quarles, J. M., & Warner, I. M. (1980). Identification of fluorescent Pseudomonas species. Clinical chemistry, 26(8), 1127-1132.

Shields, C. L., Bianciotto, C., Pirondini, C., Materin, M. A., Harmon, S. A., & Shields, J. A. (2008). Autofluorescence of choroidal melanoma in 51 cases. British Journal of Ophthalmology, 92(5), 617-622.

Singh, A. D., Belfort, R. N., Sayanagi, K., & Kaiser, P. K. (2010). Fourier domain optical coherence tomographic and auto-fluorescence findings in indeterminate choroidal melanocytic lesions. British Journal of Ophthalmology, 94(4), 474-478.

Tuft SJ, Tullo AB. Fungal keratitis in the United Kingdom 2003-2005. Eye (2009) 23, 1308-1313.

Wagnieres, G. A., Star, W. M., & Wilson, B. C. (1998). In vivo fluorescence spectroscopy 5 and imaging for oncological applications. Photochemistry and photobiology, 68(5), 603-632.

Ward CG, Clarkson JG, Taplin D, Polk HC. Wood's light fluorescence and pseudomonas burn wound infection. JAMA 1967;202:27-8.

Zaric B, Stojsic V, Sarcev T, Stojanovic G, Carapic V, Perin B, Zarogoulidis P, Darwiche K, Tsakiridis K, Karapantzos I, Kesisis G, Kougioumtzi I, Katsikogiannis N, Machairiotis N, Stylianaki A, Foroulis CN, Zarogoulidis K. Advanced bronchoscopic techniques in diagnosis and staging of lung cancer. J Thorac Dis. 2013 Sep;5(Suppl 4):S359-S370

Claims

Claims 1. A diagnostic apparatus for the in vivo diagnosis of disease, optionally an ophthalmological disease and/or a tumour, the apparatus comprising:- - a monochromator configured to receive light containing a range of wavelengths, and create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; - direction means configured to focus the light created by the monochromator on the biological target in vivo; - detection means configured to detect autofluorescence emitted by the biological target upon exposure to each bandwidth; and - processing means configured to diagnose disease based on the detected autofluorescence.
2. The diagnostic apparatus according to claim 1, wherein the apparatus is configured to diagnose a tumour, which is situated on the skin or in the lung.
3. The diagnostic apparatus according to claim 1, wherein the direction means is configured to focus the light created by the monochromator on or in the eye, or eyelid.
4. An ophthalmological diagnostic apparatus for the in vivo diagnosis of an ophthalmological disease in a subject, the apparatus comprising:- - a monochromator configured to receive light containing a range of wavelengths, and create a plurality of substantially continuous bandwidths of light across an excitation range of a biological target; -0or -direction means configured to focus the light created by the monochromator on or in a region of a subject's eye in vivo; - detection means configured to detect autofluorescence emitted by the region of the eye upon exposure to each bandwidth; and - processing means configured to diagnose an ophthalmological disease based on the detected autofluorescence.
5. The diagnostic apparatus according to any preceding claim, wherein the apparatus is configured to detect one or more micro-organism via autofluorescence.
6. The diagnostic apparatus according to claim 5, wherein the micro-organism is a bacterium, amoeba or fungus.
7. The diagnostic apparatus according to claim 6, wherein the bacteria which is detected is Staphylococcus spp., Escherichia spp. and/or Pseudomonas spp., and the fungus which is detected is Fusarium spp. and/or Candida spp.
8. The diagnostic apparatus according to any preceding claim, wherein S. aureus, E. colt, P. aeruginosa and/or C. albicans or Fusarium species are detected via autofluorescence.
9. The diagnostic apparatus according to any preceding claim, wherein the apparatus comprises a light source which is capable of emission in the ultraviolet, visible and/or near-infrared spectra.
10. The diagnostic apparatus according to claim 9, wherein the light source is a continuum lamp, such as a deuterium or xenon arc lamp, for generation of ultraviolet light, or a tungsten halogen lamp for generation of visible/near-infrared excitation.
11. The diagnostic apparatus according to either claim 9 or 10, wherein the light source is configured to emit light in the range of loonm to 2000nm, or 12onm to 800 20 nm, or 15onm to 600 nm.
12. The diagnostic apparatus according to any one of claims 9-11, wherein the light source is configured to emit light in the range of 2oonm to 450 nm.
13. The diagnostic apparatus according to any preceding claim, wherein the excitation range is in the range of i5onm to 500 nm, or 2oonm to 450 nm, or 25onm to 400 nm.
14. The diagnostic apparatus according to any preceding claim, wherein the excitation range is in the range of 25onm to 350 nm.
15. The diagnostic apparatus according to any preceding claim, wherein the monochromator comprises optical interference filters, prisms, optical gratings, a scanning monochromator, an acousto-optic tunable filter, liquid crystal tunable filter, an array of optical interference filters and/or a prism spectrograph.
16. The diagnostic apparatus according to any preceding claim, wherein the monochromator is configured to create the plurality of substantially continuous bandwidths of light in a sequential or step-wise manner along the excitation range of the biological target.
17. The diagnostic apparatus according to any preceding claim, wherein the monochromator is configured to create a plurality of substantially continuous bandwidths of light, wherein each bandwidth comprises substantially monochromatic light.
18. The diagnostic apparatus according to any preceding claim, wherein each bandwidth comprises light with wavelengths with a selectivity of about to nm, or about 5 nm, 4 nm, 3 nm or 2 0M.
19. The diagnostic apparatus according to any preceding claim, wherein each bandwidth created by the monochromator is approximately o.5nm-2nm in wavelength, or approximately mm in wavelength.
20. The diagnostic apparatus according to any preceding claim, wherein the light created by the monochromator results in the excitation of the sample, followed by measurement of the emission of fluorescence from the target at predetermined time points, or continuously.
21. The diagnostic apparatus according to any preceding claim, wherein the -0or direction means comprises at least one illumination fibre along which the bandwidth created by the monochromator is passed to the target.
22. The diagnostic apparatus according to any preceding claim, wherein the direction means comprises focusing optics functionally linked to the detection means.
23. The diagnostic apparatus according to claim 22, wherein the focusing optics is configured to focus the plurality of substantially continuous bandwidths of light created by the monochromator on the biological target.
24. The diagnostic apparatus according to claim 22 or 23, wherein the focusing optics comprises a lens system or a microscope configured to permit observations in the ultraviolet, visible, and infrared range.
25. The diagnostic apparatus according to any one of claims 22-24, wherein the focusing optics comprises a short focal length converging lens, such that the light field is focused on the cornea.
26. The diagnostic apparatus according to any preceding claim, wherein the apparatus is mounted on a slit lamp assembly and targeting is achieved effectively by eye.
27. The diagnostic apparatus according to claim 26, wherein the apparatus is incorporated as a co-aligned channel in the slit lamp assembly, and comprises a reticle that indicates the precise measurement point in the field of view observed by the clinician.
28. The diagnostic apparatus according to any one of claims 22-24, wherein the focusing optics a converging lens, such that the light field is focused on a region inside the eye, such as the retina or choroid.
29. The diagnostic apparatus according to any preceding claim, wherein the direction means comprises at least one read fibre along which reflected light and autofluorescence from the target is passed to the detection means, preferably along a 25 sensing waveguide.
3o. The diagnostic apparatus according to any preceding claim, wherein the detection means comprises a spectrometer.
31. The diagnostic apparatus according to any preceding claim, wherein the spectrometer is a scanning monochromator that detects specific emission wavelengths whereby the output from the scanning monochromator is detected by a photomultiplier tube and/or the spectrometer is configured as an imaging spectrograph whereby the output is detected by an imaging detector array, such as a charge-coupled device (CCD) detector array.

-3o -
32. The diagnostic apparatus according to any preceding claim, wherein the apparatus comprises a discriminator which allows the observation of the fluorescence and/or scattering signal by a photodetection means.
33. The diagnostic apparatus according to any one of claims 30-32, wherein the spectrometer is sensitive in the range of 100 to 800 nm, 200 to 700 nm, and/or 300 to 60o nm.
34. The diagnostic apparatus according to any preceding claim, wherein the /o detection means is configured to disperse the autofluorescence into a spectrum, which is used by the processing means to diagnose the disease.
35. The diagnostic apparatus according to claim 34, wherein the processing means is configured to provide the autofluorescent spectra as Excitation-Emission Matrix (EEM) measurements.
36. The diagnostic apparatus according to claim 35, wherein the EEM comprises the excitation data for at least 10 continuous bandwidths of light, or at least 20, 30, 40, 50, continuous bandwidths of light.
37. The diagnostic apparatus according to claim 35, wherein the EEM comprises the excitation data for at least 60, 70, 80, 90 or 100 bandwidths of light.
38. The diagnostic apparatus according to any preceding claim, wherein each -0or illumination has an exposure time of less than about 10 seconds, or less than about 5, 4, 3, 2 or 1 second per wavelength.
39. The diagnostic apparatus according to any preceding claim, wherein each illumination is repeated at least 2, 3, 4 or 5 times.
4o. The diagnostic apparatus according to any one of claims 35-39, wherein a cross section of the EEM with a fixed excitation wavelength is used to show the emission spectra for a specific excitation bandwidth.
41. The diagnostic apparatus according to any one of claims 35-40, wherein emission spectra for a plurality of excitation bandwidths is analysed.

-31 -
42. The diagnostic apparatus acording to claim 41, wherein a plurality of excitation bandwidths comprises the spectra of at least at 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 5o excitation bandwidths.
43. The diagnostic apparatus according to any preceding claim, wherein the apparatus is configured to take control measurements for colonies of known microorganisms and/or tumours, thereby allowing for correlation of measured test data with characterization of the microorganisms or tumours of interest.
44. The diagnostic apparatus according to any preceding claim, wherein the processing means is configured to analyse the data by Principal Component Analysis (PCA) analysis.
45. A bronchoscope comprising the apparatus according to any one of claims 1-44.
46. A bronchoscope according to claim 45, wherein the bronchoscope comprises a UV-transparent optical element configured to separate the apparatus from a biological target and direct light appropriately.
47. Apparatus for the diagnosis of a skin cancer on a subject in vivo, the apparatus comprising:- - a monochromator configured to receive light containing a range of wavelengths, and create a plurality of substantially continuous bandwidths -0or of light across an excitation range of a biological target; - direction means configured to focus the light created by the monochromator on a region of the subject's skin in vivo; - detection means configured to detect autofluorescence emitted by the region of the skin upon exposure to each bandwidth; and -processing means configured to diagnose a skin cancer based on the detected autofluorescence.
48. Apparatus according to claim 47, wherein the skin cancer which is diagnosed is a melanoma.