WO2008144831A1

WO2008144831A1 - Method and apparatus for inspecting tissue

Info

Publication number: WO2008144831A1
Application number: PCT/AU2008/000768
Authority: WO
Inventors: Damian Keith Bird
Original assignee: Invision Medical Technologies Pty Ltd
Priority date: 2007-05-30
Filing date: 2008-05-30
Publication date: 2008-12-04

Abstract

An apparatus for inspecting tissue, comprising a light source of coherent light, a needle with a bore and a tip, a delivery light transmitter for transmitting light from the light source to an exit tip of the delivery light transmitter, a collection light transmitter for collecting return light, and a photodetector. A portion of the delivery light transmitter is located in the bore so that the exit tip can be introduced into a sample by inserting the tip of the needle into the sample and illuminating a volume of the sample with light from the light source, and a portion of the collection light transmitter is located in the bore to collect return light from the sample and transmit the collected return light to the photodetector.

Description

METHOD AND APPARATUS FOR INSPECTING TISSUE

RELATED APPLICATION

This application is based on and claims the benefit of the filing date of US application no. 60/940,933 filed 30 May 2007, the content of which as filed is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method and apparatus for inspecting tissue samples, such as in or from suspicious breast masses, and in particular to a fibre optic device for multiphoton excitation of fluorescence and collection of emitted fluorescence at a lesion site for indication of disease therein by time- resolved fluorescence measurements, such as prior to tissue biopsy.

BACKGROUND OF THE INVENTION

Escalating health care costs have encouraged the development of minimally- invasive surgery. Diagnosis-related (rather than treatment-related) insurance reimbursements have led to less traumatic surgical procedures, requiring shorter hospital stays and faster recuperation. Patients have become more educated as to available treatment options and demand treatments that minimise pain and recovery time. Endoscopic procedures often play a pivotal role in minimally-invasive surgery and have been encouraged by insurance companies and hospital administrators who are anxious to reduce treatment costs and provide the most effective choices obtainable. As an example, a common orthopaedic surgery is the meniscectomy, in which torn cartilage is removed from the knee. Until 1974 all meniscectomies performed in the United States were open procedures requiring a large incision, a full leg cast and a recovery period of several months.

Current arthroscopic meniscectomies require only a few puncture wounds in the area of the knee for insertion of a 4 mm diameter arthroscope and the instrumentation for irrigation and surgery. Patients begin physical rehabilitation therapy on the day of the surgery and can often be released the following day. The first practical arthroscope was developed for knee surgery in 1931 [1]. The instrument was 3.5 mm in diameter and contained a right-angle reflecting prism at the distal tip. Optical fibre was first used for flexible endoscopy in 1958 by Hirchowitz, Curtis, Peters, and Pollard [1]. Rigid endoscope optics also improved in this time period with the introduction of the rod lens designs of H. H. Hopkins [1]. A typical rigid endoscope can contain as many as 60 lens components for relaying the image from the inside of the body. Diameters of optical components can be 1 mm or less. Quantities sold by any one manufacturer range only between a few hundred and a few thousand endoscopes per year but production techniques have always contained an element of craft. Accelerating interest in the last decade in advanced endoscopes with superior brightness and resolution and smaller diameter have advanced the limits of technology in producing these lens systems cost- effectively. Most medical endoscopes are manufactured in Germany or Japan. Selfoc gradient-index rod lenses, which are manufactured primarily for table-top copiers, have proved to be important in the development of small diameter endoscopes. Two years after the development of Selfoc, Watanabe in Japan built a 1.7 mm diameter gradient-index endoscope [2], but these were of limited use owing to large amounts of chromatic aberration in the Selfoc material until improvements were made in the Selfoc material and hence in chromatic aberration.

One of the principal advantages of two-photon excitation of fluorescence (TPEF) in microscopy over conventional single-photon techniques is the confinement of the fluorescence excitation to within the focal volume of the imaging objective. This is because, in TPEF, two-photon induced absorption is most probable in the focal volume, that is, where the photon energy density is at its highest. As a consequence, the out-of-focus fluorophores are safeguarded from effects like photobleaching, as the energy density is not sufficient to induce fluorescence at these points. The third-order nonlinear excitation probability makes it possible to access a single point of the specimen, preserving the fluorophores at other axial depths for subsequent observation. The use of infrared beams for TPEF provides additional advantages. Biological tissue of considerable optical thickness and density (such as the breast) can be observed owing to a reduction in the scattering of incident photons, as the absorption and scattering coefficients for infrared light are much lower than those for shorter wavelengths [3].

Furthermore, most tissues absorb less in the near infrared (NIR), so TPEF can effectively exploit the tissue optical window between 700 and 1000 nm. The tissue absorbance of NIR light in this range is orders of magnitude less than the absorption in the ultraviolet (UV). In addition, the individual photon energy is halved in two-photon absorption, so thermal damage to sensitive biological material is reduced. TPEF delivers a high photon density to a sample to increase absorption probability. This is typically achieved with the use of ultrashort-pulsed laser excitation. Femtosecond, picosecond and continuous wave laser sources have all been used for TPEF. Currently, the most commonly used laser sources are pulsed femtosecond titanium-sapphire (Ti- Sapphire) systems, which are capable of generating sub 100-fs pulse trains at a repetition rate of between approximately 75 and 82 MHz. The tuning range of these systems typically extends from 700 to 1000 nm.

A technique of more recent years is time-resolved fluorescence analysis (or 'fluorescence lifetime imaging' (FLIM)), which is essentially independent of local intensity variations and fluorophore concentration. FLIM provides an additional data dimension with opportunities for functional imaging and is a direct approach to all effects that involve energy transfer. Typical examples are the mapping of cell parameters such as pH, ion concentrations or oxygen saturation by fluorescence quenching, and fluorescence resonance energy transfer between different fluorophores in the cell [4]. The technique also allows the difficulties associated with different fluorescent biomarkers in multi- stained samples (or with different natural fluorophores within the cells themselves) to be overcome by clearly distinguishing individual emitting species via their characteristic fluorescence lifetime. The potential value of FLIM as a diagnostic tool for functional biomedicine has been well documented [4]. It has proved to be a useful method for oncological applications [5], studies of tissue constituents [6], cell cultures [7], histopathology [8], human skin [9] and as a practical way of deriving functional in vivo metabolic maps in human breast cells [10]. Acquisition of FLIM data can be achieved in either the frequency domain, where a high-frequency modulated laser beam excites the sample and the lifetime is determined from the demodulation and phase shift of the fluorescence signal, or in the time domain where the fluorescence decay is measured directly after a laser excitation pulse. One time-domain technique is time-correlated single photon counting (TCSPC) [11]. This has the advantage that it can be combined with multiple detectors for simultaneous multi- wavelength detection [12] on the basis that the detection of several photons in different detector channels in one laser period is unlikely [12]. Hence, the single-photon pulses from all detector channels can be combined into a common photon pulse line and sent, together with routing information identifying the detector channel that generated the pulse, through the normal time measurement procedure of the photon counting electronics. This is a particularly attractive feature of TCSPC and allows all of the intrinsic features of the fluorescence signal (intensity, spectra and the excited state lifetime) to be simultaneously monitored.

Percutaneous, image-guided breast needle biopsy is a minimally invasive procedure, which is increasingly used for breast cancer diagnosis in preference to surgical biopsy. However, compared with surgical biopsy the sampling accuracy of the needle biopsy is severely limited because only a few small pieces of tissue are sampled in a suspicious mass, and it is particularly difficult to verify that the samples are removed from the cancerous tissue site because two-dimensional imaging (i.e. mammography) is used to guide the needle into a three-dimensional mass. Up to a 7% false negative rate can result [13] and up to 18% of patients have to endure repeat biopsies [14]. According to the Australian National Breast Cancer Centre, approximately 80% of all biopsies performed in Australia identify only benign tissue, so normal healthy tissue is being excised, which is traumatic for the patient, time consuming and costly.

One way to increase the sampling accuracy of needle biopsy is optically to probe and characterise breast cancer cells at the molecular level. Breast tissue (being composed of epithelial cells, extra-cellular matrix and fat) contains a number of endogenous fluorophores. Tryptophan, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are present in the epithelial cells [15]. Tryptophan accounts for most protein fluorescence and its emission (spectrally and temporally) is sensitive to the polarity of the environment. NADH and FAD are involved in the oxidation of fuel molecules and can be used to probe changes intracellular metabolism. The primary fluorophore in the extra-cellular matrix of the breast is type I collagen and its fluorescence is attributed to cross-links [15]. Analysis of fluorescence emission is particularly useful because the signal contains multiple molecule-specific sources of contrast, including the amount of fluorescence (intensity), the colour of the fluorescence (spectrum) and the time it takes for the molecule to fluoresce (lifetime). The fluorescence lifetime is extremely sensitive to the surrounding microenvironment, which makes it a useful property to measure in order to reveal subtle changes in the cell that may be indicative of disease progression.

Recently a two-photon fluorescence microscope based on a single-mode optical fibre was demonstrated as a new potential tool for the acquisition of high-resolution three-dimensional images in vivo [16]. This device (depicted in Fig. 1 of reference [16]) is small enough to be passed through a needle core, but it includes a large number of bulk components at the sample site, including the imaging objective, optics mounts and the scanning stage. This limits its use.

SUMMARY OF THE INVENTION

In a first broad aspect, the invention provides a method and apparatus for inspecting tissue. In one particular embodiment, the invention provides an apparatus for inspecting tissue, comprising: a light source of coherent light; a needle with a bore and a tip; a delivery light transmitter (typically comprising one — but possibly more — optical fibres) for transmitting light from the light source to an exit tip of the delivery light transmitter; a collection light transmitter (typically comprising one or more optical fibres) for collecting return light; and a photodetector; wherein a portion of the delivery light transmitter is located in the bore so that the exit tip can be introduced into a sample by inserting the tip of the needle into the sample and illuminating a volume of the sample with light from the light source, and a portion of the collection light transmitter is located in the bore to collect return light from the sample and transmit it to the photodetector.

The delivery light transmitter and the collection light transmitter will generally be flexible.

Thus, with the assistance of stains or otherwise, return light can be collected and used to assist in characterizing, for example, cells such as those in a sample comprising breast tissue, including in vivo. It is envisaged that the apparatus will also be useful with, for example, other organs that can be accessed by needle biopsy, such as the ovaries or kidneys, or tissues in other accessible locations, such as the mouth and skin. Such applications are of particular value in detecting or diagnosing cancers, but other conditions may also be detected or observed.

The collected data will typically not constitute an image, as it will generally comprise at most a few pixels, but in such applications imaging is not essential; characterizing the cells as benign or otherwise is generally sufficient.

The collection light transmitter and the delivery light transmitter may include a common portion located in the needle, or distinct portions in the needle. In one embodiment, the collection light transmitter comprises a plurality of optical fibres (such as photonic crystal fibres, including in particular zero-dispersion wavelength fibres). In a particular embodiment, the collection light transmitter comprises up to and preferably six fibres and the portion of the collection light transmitter in the needle at least partially surrounds the portion of the delivery light transmitter (which may also act as a collection light transmitter) that is in the needle.

In a particular embodiment, the collection light transmitter comprises a fibre bundle. In one particular embodiment, the collection light transmitter comprises a plurality of fibres with entry tips that surround the exit tip of the delivery light transmitter.

The light source may comprise a stand-alone laser source. In one embodiment, the light source comprises a fibre laser.

In one embodiment, the apparatus includes one or more optical elements (which may be integral with the delivery light transmitter) located at the exit tip of the delivery light transmitter, for focussing light emerging from the exit tip of the delivery light transmitter to an observational volume. The one or more optical elements may comprise a microlens or microlenses, such as gradient index rod lenses of numerical aperture matching closely (if not exactly) that of the delivery light transmitter. The optical focusing elements may be affixed rigidly to the delivery light transmitter, such as with a fibre optic resin of appropriate refractive index.

The apparatus may comprise an additional delivery light transmitter for transmitting light from the light source to an exit tip of the additional delivery light transmitter, and an additional collection light transmitter for collecting return light, wherein a portion of the additional delivery light transmitter is located in the bore so that the exit tip of the additional delivery light transmitter is introduced into the sample with the exit tip of the delivery light transmitter, to illuminate an additional volume of the sample with light from the light source, and a portion of the additional collection light transmitter is located in the bore to collect return light from the sample and transmit it to the photodetector.

In this embodiment, the light source may comprise a plurality of individual light sources (such as one for each delivery light transmitter). Also, still further delivery light transmitters and collection light transmitters may be provided, so that multiple volumes of the sample can be illuminated.

The apparatus may comprise an additional needle with a respective bore and tip, an additional delivery light transmitter for transmitting light from the light source to an exit tip of the additional delivery light transmitter, and an additional collection light transmitter for collecting return light, wherein a portion of the additional delivery light transmitter is located in the bore of the additional needle so that the exit tip of the additional delivery light transmitter can be introduced into a sample by inserting the tip of the additional needle into the sample and illuminating a volume of the sample with light from the light source, and a portion of the additional collection light transmitter is located in the bore of the additional needle to collect return light from the sample and transmit it to the photodetector.

According to another aspect of the invention, the invention provides a method for inspecting tissue, comprising employing the apparatus described above to inspect and characterize a sample. In one embodiment of this aspect, the method comprises: inserting a tip of a needle into a sample, the needle having a bore containing a portion of a (typically flexible) delivery light transmitter and a portion of a (typically flexible) collection light transmitter; transmitting light with the delivery light transmitter from a light source of coherent light to an exit tip of the delivery light transmitter to illuminate a volume of the sample with the light; collecting return light with the collection light transmitter; and transmitting the collected return light with the collection light transmitter to a photodetector.

According to still another aspect of the invention, the invention provides a kit comprising at least some of the components of the apparatus described above, for assembly into the apparatus or for use as replacement parts of such an apparatus, particularly when some of the components are provided in disposable form. For example, in one embodiment, the needle and portions of the light transmitters (such as a single delivery and two collection light transmitters) contained therein are detachable from the rest of the apparatus so that they can be replaced for prompt use of the apparatus with a further patient (without having to sterilize the needle, etc, that come into contact with the sample).

Thus, in one embodiment the invention provides a device for use in a system for inspecting tissue, comprising: a needle with a bore and a tip; a delivery light transmitter optically couplable (such as with a flexible light transmitter) to a light source, for transmitting light from the light source to an exit tip of the delivery light transmitter; and a collection light transmitter optically couplable (such as with a flexible light transmitter) to a photodetector, for collecting return light; wherein the delivery light transmitter and the collection light transmitter are located in the bore so that the exit tip can be introduced into a sample by inserting the tip of the needle into the sample and illuminating a volume of the sample with light from the light source, and the collection light transmitter is located in the bore to collect return light from the sample and transmit the collected return light to the photodetector.

As will be understood by those skilled in the art, each of the optional features of the above aspects of the invention may be employed where suitable in any combination with the other optional features and with any other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWING

In order that the present invention may be more clearly ascertained, embodiments will now be described, by way of example, with reference to the accompanying drawing, in which:

Figure 1 is a schematic diagram of an optical biopsy system according to an embodiment of the present invention;

Figure 2 is a schematic view of a detail of figure 1, showing the needle and a portion of the fibres of the system of figure 1 ;

Figure 3 is a cross sectional view of the needle and enclosed fibres of the system of figure 1 ;

Figure 4 is a schematic view of the exit ends of the collection fibres and photodetectors of the system of figure 1; Figure 5 is a schematic diagram of an optical biopsy system according to another embodiment of the present invention;

Figure 6 is a schematic diagram of an optical biopsy system according to yet another embodiment of the present invention;

Figure 7 is a schematic diagram of an optical biopsy system according to a further embodiment of the present invention;

Figure 8A is a cross sectional view of the needle and enclosed fibres of the system of figure 7;

Figure 8B is a cross sectional view of an alternative arrangement of the needle and enclosed fibres of the system of figure 7; and Figure 8C is a cross sectional view of an alternative needle (with enclosed fibres) of the system of figure 7.

DETAILED DESCRIPTION OF THE INVENTION

Figure 1 is a schematic diagram of an optical biopsy system 10 according to an embodiment of the present invention. The system 10 includes a pulsed coherent light source in the form of a femtosecond pulsed laser source 12 for providing illuminating light (which may be a single photon source, but in this embodiment is a two-photon source), neutral density filter 14 and a focussing lens 16 for focussing the illuminating light output by laser source 12.

System 10 is arranged for performing two-photon excitation but, in other embodiments, single-photon excitation may be employed. In single photon time correlated studies, for example, certain pulsed laser diode sources that are capable of single photon excitation only may be employed.

Laser source 12 emits light in the approximate range of 700 to 900 nm at a pulse repetition rate in the approximate range of 75 to 82 MHz. The neutral density filter 14 may be rotated to vary the power of illuminating light to be employed. The system 10 further includes a low dispersion delivery optical fibre in the form of a zero-dispersion wavelength delivery photonic crystal fibre (PCF) 18 for receiving the illuminating light from focussing lens 16 and transmitting it to a sample located at its distal or exit end 20, a focussing element (not shown) attached to the delivery PCF 18 at its exit end 20 for focussing the illuminating light onto or into the sample and six high numerical aperture multimode collection fibres 22 (which may be standard multimode fibres but, in this embodiment, are PCFs) for collecting return fluorescent light from the sample. The focussing element may comprise, for example, a single lens, multiple lenses, or an exit portion of the delivery PCF 18 (which may be, for example, convex). In this embodiment it comprises one or more microlenses, such as gradient index rod lenses of numerical aperture matching closely (if not exactly) that of the delivery PCF 18, affixed rigidly to the delivery PCF 18 with a fibre optic resin of appropriate refractive index.

The system 10 also includes a conventional biopsy needle 24 (such as a 16G needle) in which the delivery PCF 18 and collection PCFs 22 are located for insertion into the sample. Collectively, needle 24, the portions of delivery PCF 18, collection PCFs 22 enclosed within needle 24 and the focussing element constitute a fibre probe. Insertion of the needle 24 into the sample inserts the bundle 26 of delivery PCF 18 and collection PCFs 22. This arrangement is illustrated more clearly in figure 2, which is a schematic, enlarged view 28 of needle 24 and delivery PCF 18 and collection PCFs 22. Figure 3 is a cross section view 30 of the needle 24 and its contents: delivery PCF 18 and collection PCFs 22. It will be seen that the delivery PCF 18 is located within the six collection PCFs 22, which are hence closely packed. Emitted two-photon excited fluorescence is collected by the collection PCFs 22 (at their entry ends adjacent to and around the exit end 20 of delivery PCF 18). As is apparent from figure 3, the centre-to-centre spacing between adjacent collection PCFs 22 is the diameter d of an individual fibre. Collected fluorescence traverses the collection PCFs 22

Referring to figure 1 , system 10 includes a fibre chuck 32 at the end of the collection PCFs 22 (and, in some embodiments, a positioning device for adjusting the position of the fibre chuck). System 10 also includes one or more barrier filters 34 optically after the collection PCFs 22, for removing residual fundamental illumination, and then a photodetector 36 in the form of one or more photomultiplier tubes coupled to photon counting electronics 38 for counting or otherwise determining a measure of the number of returned photons. In some embodiments, system 10 includes six photomultiplier tubes (or other photodetectors), one for each of collection PCFs 22.

In another embodiment, photodetector 36 comprises one or more single photon avalanche diodes (SPAD) detectors and a time-correlated single photon counting module. This arrangement is particularly adapted to providing sensitive detection of endogenous fluorescence lifetime signals in, for example, breast cancer lesions.

It should be noted that system 10 does not employ an optical coupler, but rather comprises an arrangement of individual fibres (delivery PCF 18 and collection PCFs 22). Other embodiments may include a single mode 2 x 1 optical fibre coupler or a 2 x 1 (preferably double clad) PCF coupler, located at the point where the delivery PCF 18 and collection PCFs 22 meet in system 10. In such embodiments, needle 24 may contain only a single PCF for both delivering light to and collecting light from the sample, which would allow a needle with a smaller bore to be used. In such embodiments preferably a double clad PCF is provided from the coupler to the tip of the needle 24. A portion of the collected light is then directed by the coupler to the photodetector 36 upon its return from the sample.

It should also be noted that additional returned fluorescence, which enters the exit end 20 of delivery PCF 18, may be collected from delivery PCF 18 — such as with a beamsplitter, coupler or otherwise — and combined with that collected by collection PCFs 22 and passed to photodetector 36 (in some embodiments comprising one or more photomultiplier tubes or SPAD detectors), via barrier filter 34.

Figure 4 is a schematic view of the arrangement 40 of the exit ends 42 of collections PCFs 22 (i.e. as held by fibre chuck 32). The exit ends 42, in this embodiment, are arranged linearly for convenient delivery of collected light 44 to one or more photodetectors, and in this embodiment an array 46 of six photodetectors or spectrometers 37 (collectively constituting photodetector 36). It should be noted that barrier filter 34 may itself comprise a plurality of separate portions 35 of filter material, according to desired geometry or other constraints.

In one embodiment, electronic switching between individual photodetectors is employed to enable signals from separate collection PCFs 22 to be analysed. As may be seen in figure 3, the collection PCFs 22 are spaced either by of (the diameter of each PCF 22), V3 d (i.e. two collection PCFs 22 separated by a third) or 2d (i.e. two collection PCFs 22 on opposite sides of delivery PCF 18). In some cases this arrangement facilitates the positioning of the fibre probe (comprising needle 24 and enclosed portions of delivery and collection PCFs 18, 22), and hence a biopsy excision device introduced along the track of needle 24, closer to a suspicious site. For example, if a first of collection PCFs 22 detects a time-domain signal indicative of suspect tissue but that signal is not apparent in the light collected from a second of collection PCFs 22 at a distance V3 d or 2dfrom the first, the operator could move the biopsy excision device along the track of needle 24 but in the direction of the first of these collection PCFs 22 and hence, in all likelihood, closer to the lesion.

Referring to figure 5, in another embodiment the invention provides an optical biopsy system 50 that is similar to system 10 (and like references numerals have been used to indicate like features), but with a coherent light source in the form of a fibre laser 52. (Consequently, neutral density filter 14 and a focussing lens 16 of system 10 are omitted). Fibre laser 52 allows a less expensive and more compact system to be provided. Ultrashort-pulsed fibre lasers capable of producing sub 500 femtosecond pulses have been reported [17, 18] (though this is usually achieved through soliton generation around the 1.5 μm wavelength). Efficient frequency doubling techniques have been implemented that provide a more suitable operating wavelength of 768 nm for two-photon excitation [19]. Such an arrangement may have comparatively low peak power, but may be suitable for some applications.

According to another embodiment, a fibre bundle based collection system is provided. Fibre bundle based systems have been proposed [20, 21 , 22, 24] for in vivo applications as, in combination with galvanometric scanning mirrors, it is possible to scan the proximal end of a bundle of single-mode fibres to reconstruct an image from the detected signal at the distal end. However, the practical advantage comes at the expense of axial resolution [23] since, in general, fibre-bundle based imaging systems have a comparatively poor optical sectioning ability. There are methods however (slit scanning techniques, for example [23, 24]) that can limit the magnitude of this degradation.

According to another embodiment, a hybrid detection system is provided that provides the simultaneous operation of time-resolved multiphoton fluorescence endomicroscopy and another imaging modality. One potential imaging modality that would be suitable for such a hybrid is second harmonic generation (SHG). The combination of this technique with time-resolved multiphoton fluorescence endomicroscopy is envisaged to provide useful real-time acquisition of detailed sample information that, ordinarily, would require two separate and time consuming processes.

As will be appreciated by those skilled in the art, comparative data can be collected according to the above embodiments in a number of different ways. For example, the tip of biopsy needle 24 can be located in a tissue to be screened for abnormalities, and the collected data (indicative of the collected return light) compared with a library of comparable data from tissue known to have been normal. Alternatively, the tip of biopsy needle 24 can be located successively in comparable tissue at different sites (possibly in different subjects) where at least one of those sites is thought to be healthy.

In a variant of this approach, two optical biopsy systems 10 — or an optical biopsy system 10 modified by the addition of a second biopsy needle 24 — can be used to collect comparison data from different sites, simultaneously if desired. Figure 6, for example, is a schematic diagram of an optical biopsy system 60 according to an embodiment of the present invention. System 60 essentially comprises system 10 (and like references numerals have been used to indicate like features), but with the addition of a second low dispersion delivery optical fibre 18', a second biopsy needle 24', and a second set of multimode collection fibres 22' (which may be standard multimode fibres but, in this embodiment, are PCFs). Fibre chuck 32 thus — according to this embodiment — holds the exit ends of first and second sets of collection PCFs 22, 22', and photodetector 36 comprises an array of twelve photodetectors or spectrometers each corresponding to one of the collection fibres. The output of photodetector 36 is again input into photon counting electronics 38.

System 60 includes a optical coupler 62 for use as a beamsplitter, located optically after focussing lens 16. (Other beamsplitters may alternatively be employed, such as a right-angle partially reflecting prism.) Coupler 62 splits the light from pulsed laser source 12 equally into its two downstream arms, which are coupled respectively to (first) delivery optical fibre 18 and second delivery optical fibre 18'.

According to still another embodiment, first and second delivery optical fibres 18, 18' and first and second sets of collection PCFs 22, 22' are provided (cf. system 60 of figure 6), but with a single biopsy needle. Figure 7 is a schematic diagram of an optical biopsy system 70 according to an embodiment of the present invention. System 70 essentially comprises many of the features of system 60 and like references numerals have been used to indicate like features. System 70 thus has an optical coupler 62 for splitting the light from pulsed laser source 12 equally into its two downstream arms, which are coupled respectively to first delivery optical fibre 18 and second delivery optical fibre 18'. However, in system 70 the fibre bundle 72 comprising first delivery optical fibre 18, second delivery optical fibre 18', first collection PCFs 22 and second collection PCFs 22' are inserted into a single biopsy needle 74.

Figure 8A is a cross section view 80 of needle 74 and its contents: first delivery PCF 18, second delivery PCF 18', first collection multimode fibres 22 PCFs 22 and second collection multimode fibres 22'. In this embodiment, first collection fibres 22 and second collection fibres 22' are all PCFs, but in other embodiments may be standard multimode fibres.

It will be seen that first delivery PCF 18 is located within the first six collection PCFs 22, which are hence closely packed, and that second delivery PCF 18' is located within the second six collection PCFs 22', which are also closely packed. (As with needle 24 in figure 3, the wall thickness of needle 74 is not shown to scale.) Each delivery PCF 18,18' illuminates a respective volume of the sample, and emitted two-photon excited fluorescence is collected by the respective collection PCFs 22,22' (at their entry ends adjacent to and around the respective exit ends of delivery PCFs 18,18'). The centre-to-centre spacing between adjacent collection PCFs 22,22' is the diameter d of an individual fibre.

Thus, the respective illuminated volumes have centres separated by 3d. However, this separation can be increased — according to other embodiments — as desired, to increase collection efficiency. Referring to figure 8B, for example, one or more separating members can be located between the respective collection PCFs 22,22', such as in the form of one or more additional fibres. Figure 8B is a cross section view 90 of an alternative needle 92 and its contents; needle 92 is comparable to needle 74 of figure 8A, and is also for use with system 70 of figure 7. However, in this embodiment an intermediate, spacing fibre 94 is located between the respective collection PCFs 22,22' to increase the separate of delivery PCFs 18,18' and hence of the illuminated volumes. As will be appreciated, still further fibres could be employed to further separate delivery PCFs 18,18'; indeed, needle 92 may, in some embodiments, contain a portion of a fibre bundle of which seven adjacent fibres - generally located at or near the wall of needle 92 - constitute first delivery PCF 18 and first collection PCFs 22, and another seven adjacent fibres - generally located at or near a diametrically opposite wall of needle 92 - constitute second delivery PCF 18' and second collection PCFs 22'. Other fibres in the bundle (for example, fibres 76 shown in ghost in figure 8B) can either merely serve to securely locate the delivery and collection fibres, or - in some embodiments - constitute further sets of delivery and collection fibres.

In still another variant of needle 74, 92, for use with system 70 of figure 7, a needle is provided with two bores, each for receiving a respective bundle of delivery and collection fibres. Figure 8C is a cross section view 100 of an alternative needle 102 (for use with system 70 of figure 7) and its contents. Needle 102 has two bores 104, 104'; the first bore 104 encloses first delivery PCF 18 and first collection PCFs 22, while the second bore 104' encloses second delivery PCF 18' and second collection PCFs 22'. Optionally, the needle may be provided with additional bores, each enclosing respective delivery and collection fibres.

In the needle configurations of figures 3, 8A, 8B and 8C, other members may be located in the needle (such as a power cable for a cutting head, or a suction tube). In the configuration of figure 8C, this may require the provision of one or more additional bores for accommodating such members.

In system 70 of figure 7, collection PCFs 22, 22' may collect light from both volumes excited by delivery PCFs 18,18'. In such cases, the diagnostic signal may be said to be mixed. Thus, if— for example — delivery PCF 18 illuminates a volume at the boundary between normal and malignant tissue, and delivery PCF 18' illuminates only normal tissue, the user would be prompted to move needle 74, 92 towards the malignant tissue until the signals more closely matched or were indicative of malignant tissue in all collection fibres (particularly those collection fibres between delivery PCFs 18, 18'). Once this matching was achieved, the user would have located a suitable and possibly optimal location to take a biopsy, as he or she could have greater confidence that the biopsy would be made into malignant tissue.

Modifications within the scope of the invention may be readily effected by those skilled in the art. It is to be understood, therefore, that this invention is not limited to the particular embodiments described by way of example hereinabove.

In the claims that follow and in the preceding description of the invention, except where the context requires otherwise owing to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, that is, to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Further, any reference herein to prior art is not intended to imply that such prior art forms or formed a part of the common general knowledge.

REFERENCES

[1] G. Berci. Endoscopy. Prentice Hall, New York, 1976.

[2] M. Watanabe. Atlas of Arthroscopy. Springer Verlag, New York, 1979.

[3] P. T. C. So, H. Kim and I. E. Kochevar. Two-photon deep tissue ex vivo imaging of mouse dermal and subcutaneous structures. Opt. Exp., 3 (1998) 339-350.

[4] Joseph R. Lakowicz. Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum, New York, 1999.

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

[6] K. Dowling, M. J. Dayel, M. J. Lever, P. M. W. French, J. D. Hares and A. K. L. Dymoke-Bradshaw. Fluorescence lifetime imaging with picosecond resolution for biomedical applications. Opt. Lett., 23 (1998) 810- 812.

[7] P. I. Bastiaens and A. Squire. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends in Cell Biol., 9 (1999) 48-52.

[8] K. W. Eliceiri, C. H. Fan, G. A. Lyons and J. G. White. Analysis of histology specimens using lifetime multiphoton microscopy. J. Biomed. Opt., 8 (2003) 376-380.

[9] R. Cubeddu, A. Pi eri, P. Taroni, A. Torricelli, G. Valentini and E. Sorbellini. Fluorescence lifetime imaging: an application to the detection of skin tumors. IEEE J. SeI. Top. Quant. Electr, 5 (1999) 923-929.

[10] D. K. Bird, L. Yan, K. M. Vrotsos, K. W. Eliceiri, E. M. Vaughan, P. J. Keely, J. G. White and N. Ramanujam. Metabolic mapping of mcflOa human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme nadh. Cancer Res., 65 (2005) 8766-8773.

[11] W. Becker, A. Bergmann, , M. A. Hink, , K. Knig, K. Benndorf and C. Biskup. Fluorescence lifetime imaging by time-correlated single-photon counting. Microscopy Res. Tech., 63 (2004) 58-66.

[12] W. Becker, A. Bergmann, C. Biskup, T. Zimmer, N. Kicker and K. Benndorf. Multi-wavelength tcspc lifetime imaging. In Biomedical Optics, Proc. SPIE, 4620 (2002) 79-84.

[13] R. J. Jackman, K. W. Nowels, J. Rodriguez-Soto, F. A. Marzoni Jr, S. I. Finkelstein and M. J. Shepard. Stereotactic, automated, large- core needle biopsy of non-palpable breast lesions: false-negative and histologic underestimation rates after long-term follow-up. Radiology, 210 (1999) 799- 805. [14] D. D. Dershaw, E. A. Morris, L. Liberman and A. F. Abramson. Nondiagnostic stereotaxic core breast biopsy: results of re-biopsy. Radiology, 198 (1996) 323-325.

[15] K. Sokolov, J. Galvan , A. Myakov , A. Lacy , R. Lotan and R. Richards- Kortum. Realistic 3d epithelial tissue phantoms for biomedical optics. J. Biomed. Opt, 7 (2002) 148-156.

[16] Damian Bird and M. Gu. Compact two-photon fluorescence microscope using a single-mode optical fiber coupler. Opt. Lett., 27 (2002) 1031-1033.

[17] M. H. Ober, M. Hofer, R. Hofer, G. A. Reider, G. D. Sucha, M. E. Fermann, D. Harter, C. A. C. Mendonca and T. H. Chiu. Widely tunable femtosecond neodymium fiber laser. Opt. Lett., 20 (1995) 2303-2305.

[18] M. E. Fermann, D. Harter, J. D. Minelly and G. G. Vienne. Cladding- pumped passively mode-locked fiber laser generating femtosecond and picosecond pulses. Opt. Lett, 21 (1996) 967-969.

[19] V. Pruneri, G. Bonfrate, P. G. Kazansky, C. Simonneau, P. Vidakovic and J. A. Levenson. Efficient frequency doubling of 1.5 μm femtosecond laser pulses in quasi-phase-matched optical fibers. Appl. Phys. Lett., 72 (1998) 1007-1009.

[20] A. F. Gmitro and D. Aziz. Confocal microscopy through a fiber optic imaging bundle. Opt. Lett., 18 (1993) 565-567.

[21] R. Juskaitis, T. Wilson and T. F. Watson. Real-time white light reflection confocal microscopy using a fibre-optic bundle. Scanning, 19 (1997) 15-19.

[22] A. R. Rouse and A. F. Gmitro. Multispectral imaging with a confocal microendoscope. Opt. Lett, 25 (2000) 1708-1710.

[23] C. P. Un and R. H. Webb. Fiber-coupled multiplexed confocal microscope. Opt. Lett., 25 (2000) 954-956. [24] Y. S. Sabharwal, A. R. Rouse, L. Donaldson, M. F. Hopkins and A. F. Gmitro. Slit-scanning confocal microendoscope for high-resolution in vivo imaging. Appl. Opt, 38 (1999) 7133-7144.

Claims

CLAIMS:

1. An apparatus for inspecting tissue, comprising: a light source of coherent light; a needle with a bore and a tip; a delivery light transmitter for transmitting light from said light source to an exit tip of said delivery light transmitter; a collection light transmitter for collecting return light; and a photodetector; wherein a portion of the delivery light transmitter is located in said bore so that said exit tip can be introduced into a sample by inserting said tip of said needle into said sample and illuminating a volume of said sample with light from said light source, and a portion of the collection light transmitter is located in said bore to collect return light from said sample and transmit said collected return light to said photodetector.

2. An apparatus as claimed in claim 1 , wherein said delivery light transmitter comprises at least one optical fibre.

3. An apparatus as claimed in claim 1 , wherein collection light transmitter comprises an optical fibre.

4. An apparatus as claimed in claim 1 , wherein said collected data does not constitute an image.

5. An apparatus as claimed in claim 1 , wherein said collection light transmitter and said delivery light transmitter include a common portion located in said needle.

6. An apparatus as claimed in claim 1 , wherein said collection light transmitter and said delivery light transmitter include distinct portions in said needle.

7. An apparatus as claimed in claim 1 , wherein said collection light transmitter comprises a plurality of optical fibres.

8. An apparatus as claimed in claim 1 , wherein said collection light transmitter comprises a plurality of photonic crystal fibres.

9. An apparatus as claimed in claim 1 , wherein said collection light transmitter comprises six optical fibres and a portion of said collection light transmitter in said needle at least partially surrounds a portion of said delivery light transmitter in said needle.

10. An apparatus as claimed in claim 1 , wherein said collection light transmitter comprises a fibre bundle.

11. An apparatus as claimed in claim 1 , wherein said collection light transmitter comprises a plurality of fibres with entry tips that surround said exit tip of said delivery light transmitter.

12. An apparatus as claimed in claim 1 , including one or more optical elements located at said exit tip of said delivery light transmitter, adapted to focus light emerging from said exit tip to an observational volume.

13. An apparatus as claimed in claim 12, wherein said one or more optical elements comprise a microlens or microlenses.

14. An apparatus as claimed in claim 13, wherein said one or more optical elements comprise gradient index rod lenses of numerical aperture matching closely that of said delivery light transmitter.

15. An apparatus as claimed in claim 1, comprising a fibre bundle light collection system.

16. An apparatus as claimed in claim 1 , comprising a hybrid detection system configured to provide simultaneous time-resolved multiphoton fluorescence endomicroscopy and another imaging modality.

17. An apparatus as claimed in claim 16, wherein said other imaging modality comprises second harmonic generation.

18. An apparatus as claimed in claim 1 , comprising an additional delivery light transmitter for transmitting light from said light source to an exit tip of said additional delivery light transmitter, and an additional collection light transmitter for collecting return light, wherein a portion of said additional delivery light transmitter is located in said bore so that said exit tip of said additional delivery light transmitter is introduced into said sample with said exit tip of said delivery light transmitter, to illuminate an additional volume of said sample with light 5 from said light source, and a portion of said additional collection light transmitter is located in said bore to collect return light from said sample and transmit it to said photodetector.

19. An apparatus as claimed in claim 1, comprising an additional needle with ao respective bore and tip, an additional delivery light transmitter for transmitting light from the light source to an exit tip of the additional delivery light transmitter, and an additional collection light transmitter for collecting return light, wherein a portion of the additional delivery light transmitter is located in the bore of the additional needle so that the exit tip of the additional delivery light transmitter5 can be introduced into a sample by inserting the tip of the additional needle into the sample and illuminating a volume of the sample with light from the light source, and a portion of the additional collection light transmitter is located in the bore of the additional needle to collect return light from the sample and transmit it to the photodetector. 0

20. A method for inspecting tissue, comprising: employing an apparatus as claimed in any one of claims 1 to 19 to inspect and characterize a sample.

5 21. A method for inspecting tissue, comprising: inserting a tip of a needle into a sample, said needle having a bore containing a portion of a delivery light transmitter and a portion of a collection light transmitter; transmitting light with said delivery light transmitter from a light o source of coherent light to an exit tip of said delivery light transmitter to illuminate a volume of said sample with said light; collecting return light with said collection light transmitter; and transmitting said collected return light with said collection light transmitter to a photodetector. 5

22. A method as claimed in claim 21 , wherein said bore contains a portion of an additional delivery light transmitter and a portion of an additional collection light transmitter, and said method further comprises transmitting light with said additional delivery light transmitter from said light source to an exit tip of said additional delivery light transmitter to illuminate an additional volume of said sample, collecting return light with said additional collection light transmitter; and transmitting said return light collected by said additional collection light transmitter to said photodetector with said additional collection light transmitter.

23. A method as claimed in claim 21 , further comprising: inserting a tip of an additional needle into a sample, said needle having a bore containing a portion of an additional delivery light transmitter and a portion of an additional collection light transmitter; transmitting light with said additional delivery light transmitter from said light source to an exit tip of said additional delivery light transmitter to illuminate a volume of said sample; collecting return light with said additional collection light transmitter; and transmitting said return light collected by said additional collection light transmitter to said photodetector with said additional collection light transmitter.

24. A device for use in a system for inspecting tissue, comprising: a needle with a bore and a tip; a delivery light transmitter optically couplable to a light source, for transmitting light from said light source to an exit tip of said delivery light transmitter; and a collection light transmitter optically couplable to a photodetector, for collecting return light; wherein said delivery light transmitter and said collection light transmitter are located in said bore so that said exit tip can be introduced into a sample by inserting said tip of said needle into said sample and illuminating a volume of said sample with light from said light source, and said collection light transmitter is located in said bore to collect return light from said sample and transmit said collected return light to said photodetector.