AU2003204717B2 - Apparatus - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

Name of Applicant: Swinburne University of Technology Actual Inventor(s): Address for Service: Invention Title: GU, Min BIRD, Damien DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, Victoria 3000.

Apparatus Details of Associated Provisional Application No: 2002951841 The following statement is a full description of this invention, including the best method of performing it known to us: P.OPERMKRSFEClawinpp-AUdX- 1V"3 -1-

APPARATUS

FIELD OF THE INVENTION The present invention relates to a non-linear fluorescence, particularly two-photon fluorescence, microscope or endoscope, including an endomicroscope. The invention also relates to components of such apparatus and to processes for microscopy, endoscopy and/or endomicroscopy.

BACKGROUND OF THE INVENTION Since its inception i non-linear fluorescence microscopy (that is, where for a given power input there is non-linear fluorescence intensity), particularly two-photon fluorescence microscopy, has rapidly emerged as an important technique for three-dimensional imaging of biological specimens, and in particular for use in surgical biopsy and early cancer detection. For example in relation to two-photon fluorescence microscopy this rapid emergence can be attributed to advantages offered by two-photon excitation relative to single-photon excitation. These advantages include an inherent optical sectioning property, confinement of photo damage to the focal region and improved depth penetration into a sample'. The feature of confinement arises as two-photon induced absorption is most probable in the focal volume where the photon energy density is highest. In fact, the out-of-focus fluorophores are safeguarded from photobleaching since the energy density is not sufficient to induce fluorescence at these points. The third-order nonlinear excitation probability makes it possible to image a single point of the specimen, preserving the fluorophores at other axial depths for subsequent observation. The ability to discriminate against fluorescence originating from outside of the focal spot is a powerful property of the imaging modality that pirovides an inherent optical sectioning effect for the acquisition and reconstruction of 3-D images.

However, one of the greatest restrictions to the development of two-photon microscopy particularly in relation to in vivo applications, is that the known apparatus require the use P.OFERJMKR' SPECrinapp-AU. doc- I 06D3 -2of complicated pulse lasers and bulk optics. While the introduction of optical fibres and fibre components to coherent (single-photon) fluorescence imaging systems 2 5 has overcome some physical limitations, and offered an ability to image specimens in vivo by delivering excitation radiation to a remote sample, the present single-photon arrangements remain bulky, cumbersome to operate, expensive and limited in their functionality. In addition to these problems the application of fibre optics to two-photon fluorescence systems has been hindered by the perceived problem of dispersion of the short pulses required for two-photon fluorescence. The present inventors have now devised apparatus that may address or at least obviate to some extent a number of the problems outlined above.

In one aspect of the present invention the microscope or endoscope comprises single-mode fibre for delivery of an ultra short pulsed laser beam, and a multi-port fibre coupler replaces bulk optics for illumination delivery and signal collection. Although a multi-port fibre coupler has been used in scanning differential interference contrast microscopy 7 confocal reflection microscopy"' 8 and confocal interference microscopy 9 such arrangements have not successfully been adopted in relation to two-photon fluorescence microscopy or endoscopy. It was previously thought impractical to adopt such an arrangement due to the fact that the separation of the excitation and fluorescence wavelengths is so large that the fibre coupler would be unable to efficiently transmit light at these distinct wavelengths. However, the present inventors have found that it is possible in the present apparatus for the excitation and fluorescence wavelengths to be transmitted with satisfactory efficiency.

A further advantageous aspect of the present invention is that it exhibits a self-aligning nature, because the illumination delivery and signal collection beams utilise the same port.

In embodiments of the invention that utilise a small fibre aperture, image resolution of the new system may be significantly improved, and multiple scattering may be reduced, relative to conventional two-photon fluorescence endoscopes or microscopes (as for example described in UK patent applications 2,353,369 and 2,341,943).

r I P:OPERMKR\SPECI1\2003204717 2pa 366.d-I 1/0105 -3- UK patent number 2,338,568 discloses an endoscope/microscope apparatus where the laser light pulses are split by a beam splitter and are reflected back towards the beam splitter by anti-dispersive gratings in order to condense the light pulses. Within this document there is insufficient information provided to demonstrate effectiveness of the arrangement, which is not considered likely to be particularly suitable for use in two-photon fluorescence endoscopic applications due to component bulk and space separation between the dispersing means and sample.

In another aspect the present invention utilises a scanning head for delivery of illuminating light to the sample and for signal collection from the sample. Although a similar approach has previously been used for acquisition of optical coherence tomography (OCT) images of in vitro human saphenous vein it was previously considered that this type of microscanning head would result in unacceptable levels of temporal dispersion at the increased powers required for non-linear (especially two-photon) fluorescence, compared to those adopted in OTC.

SUMMARY OF THE INVENTION According to one embodiment of the present invention there is provided a two-photon fluorescence endoscope comprising: an ultrashort pulsed laser source of coherent light suitable to initiate twophoton fluorescence in a sample of interest located at a sample region; a first single mode fibre optic light conductor positioned to receive at its terminal light from said light source and direct said light to a three-port single mode fibre coupler; a second single mode fibre optic light conductor having at its sample end a scanning head comprising a gradient index lens located to receive light from said sample end, and an optical prism which directs light from said gradient index lens transverse to axis of said gradient index lens, the second conductor located to receive light from said first conductor and direct said light to said sample via the sample end, said sample end also located to receive fluoresced light from said sample that is collected by said optical prism, P:\OPERW1KRSPECI 003204717 2sp 366.doc-I 1/0105 -4passes through said gradient index lens and is thereby directed to said sample end; a third single mode fibre optic light conductor located to direct fluoresced light from said coupler to a light detection device via a terminal of said third light conductor.

In another preferred aspect a neutral density filter is provided, rotation of which allows power variation of the coherent light. Preferably the neutral density filter is located such that light emanating from the source of coherent light passes through it directly from the source. A lens is preferably provided to direct coherent light from the source to the terminal of the first conductor.

It is also preferred that a fibre chuck is located at the terminal of the first light conductor.

The position of the fibre chuck located at the terminal of the first light conductor may be controlled by a positioning device.

Preferably a first lens is provided to collimate light emanating from the sample end toward the sample. A second lens is preferably provided to collect said fluoresced light from said sample and a variable aperture is preferably provided between said first lens and said second lens. Preferably the first lens, the second lens, the variable aperture and the sample end are contained within a protective housing.

A fibre chuck is preferably located at the sample end of the second conductor. The fibre chuck located at the sample end may be controlled by a positioning device.

Preferably a filter is provided between the detection device and the terminal of the third light conductor which substantially prevents passage to the detection device of light other than the fluoresced light. Preferably this filter is a bandpass filter. A fibre chuck is preferably located at the terminal of the third light conductor. The position of the fibre chuck located at the terminal of the third light conductor may be controlled by a positioning device.

P:\OPER\MKR\SPECl\003204717 2sp, 366doc. 11/01/05 In a preferred embodiment of the invention the detection device is a photomultiplier tube.

Preferably the laser is a Ti:Sapphire laser. Preferably the coherent light is at a wavelength of between about 730nm to about 870nm.

In the case of an endoscope a suction mechanism for drawing the sample into the sample region is optionally provided. A camera and light source to assist in directing the sample region to a desired location may also be provided. Also in the case of an endoscope a heater for heating some or all components of the endoscope located internally during use of the endoscope, may be provided.

In a further preferred embodiment there is provided a scanning head at said sample end.

The scanning head may comprise a gradient index lens located to receive light from said sample end, and an optical prism which directs light from said gradient index lens transverse to axis of said gradient index lens. Preferably the optical prism directs light in a substantially perpendicular direction from said axis.

In another embodiment of the present invention there is provided a two-photon fluorescence microscope or endoscope comprising: an ultrashort pulsed Ti:Sapphire laser suitable to initiate two-photon fluorescence in a sample of interest to be located at a sample region; a first single mode fibre optic light conductor having at its terminal a fibre chuck under positional control of a positioning device, wherein light from said source passes through a neutral density filter and a lens which directs the light to the terminal of the first conductor, which in turn directs said light to a three-port single mode fibre coupler; a second single mode fibre optic light conductor having at its sample end a fibre chuck under positional control of a positioning device, the second conductor located to receive light from said first conductor and direct light to said sample via the sample end from which a first lens collimates light emanating from said sample end, which light is then directed to said sample through a variable aperture and a second lens, said sample end P:.OPERNMKR\SPECI2003204717 25p 366.do 11101105 -6also located to receive fluoresced light from said sample that is collected by said second lens, passes through said aperture and is directed to said sample end by said first lens; a third single mode fibre optic light conductor having at its terminal a fibre chuck under positional control of a positioning device, the third conductor located to direct light from said coupler to a photomultiplier tube light detection device via said terminal of said third conductor and a bandwidth filter that substantially prevents passage to said detection device of light other than said fluoresced light.

In another embodiment of the present invention there is provided a two-photon fluorescence endoscope comprising: an ultrashort pulsed laser suitable to initiate two-photon fluorescence in a sample of interest to be located at a sample region; a first single mode fibre optic light conductor having at its terminal a fibre chuck under positional control of a positioning device, wherein light from said source passes through a neutral density filter and a lens which directs the light to the terminal of the first conductor, which in turn directs said light to a three-port single mode fibre coupler; a second single mode fibre optic light conductor having at its sample end a scanning head comprising a gradient index lens located to receive light from said sample end, and an optical prism which directs light from said gradient index lens transverse to axis of said gradient index lens, the second conductor located to receive light from said first conductor and direct light to said sample via the sample end, said sample end also located to receive fluoresced light from said sample that is collected by said optical prism, passes through said gradient index lens and is thereby directed to said sample end; a third single mode fibre optic light conductor having at its terminal a fibre chuck under positional control of a positioning device, the third conductor located to direct light from said coupler to a photomultiplier tube light detection device via said terminal of said third conductor and a bandwidth filter that substantially prevents passage to said detection device of light other than said fluoresced light.

P. OPE RXIKRSPE Cowinpp-AUdo* 1310&03 -7- BRIEF DESCRIPTION OF THE FIGURES The present invention will be further described by way of example only with reference to the following figures, wherein: Fig. 1 shows a schematic diagram of a two-photon fluorescence microscope or endoscope according to the invention.

Fig. 2 shows a plot of two-photon excitation efficiency of the microscope or endoscope shown in Fig. 1 showing the log of fluorescence intensity on the Y axis verses the log of input power (mW) on the X axis. Insert shows axial responses to a thin fluorescent layer in the system using a coupler (solid) or a large-area detector without a pin hole (dashed) and insert shows the.dependence of the width of the axial response on the excitation power.

Fig. 3 shows images of 10m diameter fluorescent polymer microspheres obtained using the two-photon fluorescence microscope or endoscope as shown in Fig. 1. The size of slices is 60pm x 60pm and the slice spacing is 1lm. The excitation power adopted was 8mW at the focus.

Fig. 4 shows images of Griffithsia sea algae obtained using a two-photon fluorescence microscope or endoscope as shown in Fig. 1. The size of slices is 150pm x 150gm and the slice spacing is lym. The excitation power adopted was 8mW at the focus.

Fig. 5(a) shows a schematic diagram of a two-photon fluorescence endomicroscope of the invention using a single-mode optical fibre coupler. Oi:0.25 NA 10 x microscope objective, ND:Neutral density filter; BF:Bandpass filter; Ci,C 2 :Fibre chucks,

P

1

,P

2

,P

3 :Coupler ports and Fig. 5(b) shows an enlargement of the scanning head.

P.'FOPER1KR'SPCtrinj-AU doc- 1O6'03 -8- Fig. 6 shows a plot of output power at focus (mW) against input power (mW) measured at port 3 of the coupler, which demonstrates coupling efficiency of the endomicroscope as shown in Fig. Fig. 7 shows a plot of normalised intensity against axial position (pm) obtained using the endomicroscope shown in Fig. 5, which demonstrates axial response to a plane mirror.

Fig. 8 shows a plot of normalised intensity against axial position (pm) obtained using the endomicroscope shown in Fig. 5, which demonstrates axial response to a polymer sample.

Fig. 9 shows a schematic representation of a rigid rotation scanning geometry that may be achieved in use of the endomicroscope as shown in Fig. Fig. 10 Schematic diagram of the cylindrical sample holder used in Example 9.

Fig. 11 Plot of rotation angle against fluorescence intensity for a series of transverse cross-sectional images taken at 0.5 mm steps into a cylindrical channel having a 5 mm equilateral triangle fluorescent sheet attached to the internal surface. The initial series (No. 1) was acquired 1.0 mm into the cylinder relative to the base of the fluorescent triangle.

DETAILED DESCRIPTION OF THE INVENTION Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

P.OPER\MKR\PECrinqp.AU doc. 1 03 -9- The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

It should be understood that general reference to microscopes or endoscopes within this specification is also intended to encompass endomicroscopes.

In its simplest form the microscope or endoscope of the invention comprises a source of coherent light suitable to initiate non-linear and preferably two-photon fluorescence in a sample or specimen of interest, which is transmitted from the light source through a first light conductor to a coupler. The light is then directed from the coupler to a second light conductor, the sample end of which is located in the vicinity of the sample region where the sample or specimen is located for imaging. Located in this region the sample end not only transmits coherent light to the sample but is also in position to receive fluoresced light from the sample that is then directed back to the coupler, allowing a proportion of this fluoresced light to be transmitted through a third light conductor to a light detection device located at its terminal.

The nature of the light conductor adopted should be such that it allows contained passage of light therewithin, without substantial loss of power. Preferably the light conductor is comprised of optic fibre, either in the form of single fibre or as a bundle of closely associated fibres. The optic fibre is preferably single-mode optic fibre. For example, the optic fibre used may be of the conventional type, having GeO 2 doped cores with pure silica cladding, or of a type having pure silica cores with fluoride doped cladding.

In the case where the light conductors comprise optic fibre the coupler will take the form of a three-port optic fibre coupler, which is preferably also of the single-mode type (at the operational wavelength).

In-fibre Bragg gratings have emerged as a promising solution for dispersion compensation and have received much attention for application in optical communications [11-13]. This ?;'OPEMI MKSPECI'* inpp- A V 3'03 technology can be applied to a two-photon fibre optical microscope/endoscope to recompress pulses dispersed by the fibre and fibre coupler. This will allow for a greater twophoton efficiency and therefore increased quantities of two-photon excited fluorescence, leading to an improved signal-to-noise ratio.

So as to allow accurate positioning of the ends of the light conductors the terminals of the first and/or third light conductors and/or the sample end of the second light conductor may have a fibre chuck fitted thereto. These fibre chucks can then be placed in a chuck holder within a positioning device such as an X-Y-Z micro-positioner to allow precise positioning of the fibre tips in each case, so as to achieve the desired function.

The source of coherent light must be suitable to initiate non-linear and preferably twophoton fluorescence in the sample or specimen of interest, which will be located at the sample region for the purpose of imaging. By the phrase "suitable to initiate two-photon fluorescence" it is intended to convey that the coherent light must have a wavelength, pulse width, power and repetition rate that will give rise to two-photon fluorescence in the sample. As will be readily understood two-photon fluorescence involves the illumination of the sample (preferably with coherent light, ie. where waves have a constant phase relationship, preferably in the visible to infra-red wavelength range) and whereby the sample will on absorption of two photons fluoresce by emitting a single higher energy photon.

Preferably the source of coherent light utilised in the present invention is a pulse laser, more preferably a Ti:Sapphire pulse laser. The laser may for example emit coherent light at a wavelength of between about 730nm to about 870nm. The pulse width of illumination may for example be within the range of 80 to 120fs and the repetition rate may for example be within the range of 80 to 82 MHz. Femtosecond, picosecond and continuous wave laser sources may all be used in two-photon fluorescence microscopy.

Ultrashort-pulsed fibre lasers may be used, which demonstrate femtosecond passively mode-locked fibre oscillators by a variety of Kerr-type saturable absorbers. The P.'OPRmKRSEC..iapp-AU Jdoc- 13' 3 -llintegration of this highly integrated fibre-based ultra-fast technology to the endoscope system as the primary illumination source provides a cost-effective and even more compact arrangement, through gross reduction of thenumber of bulk optical components.

To vary power of illumination from the light source it is possible to pass the light through a neutral density filter, rotation of which allows for power variation. The neutral density filter may suitably be located adjacent to the light source. It may also be appropriate to utilise a lens to direct light emitted from the light source or passing through the neutral density filter to the terminal of the first conductor. In the case where the light conductor is or includes optic fibre, the light will preferably be directed to the core of the optic fibre.

For example, lenses with numerical aperture (NA) between 0.1 to 0.3 may be adopted for this purpose.

Similarly, it may be appropriate to collimate light emanating from the sample end of the second light conductor, also by utilising a lens, which will be referred to as the first lens.

Again, lenses suitable for this purpose may have NA values in the range of 0.1 to 0.3.

Preferably the light directed from the first lens will then pass through a variable aperture having a diameter in the range 0.5 to 10mm. From the variable aperture light is then directed to a second lens. If present this second lens may serve to focus light from the light source onto the sample or specimen of interest, as well as collecting fluoresced light from the sample or specimen that is travelling back towards the aperture, first lens and sample end of the second conductor. The characteristics of the second lens may for example be as follows: NA 0.85 1.2; 40x, -/170.

In a further preferred embodiment of the invention the fixed bulk objective lens or lenses and the variable aperture located at the sample end are replaced by a scanning head. The scanning head may comprise a gradient index (GRIN) lens located to receive light from the sample end, and an optical prism which directs light from the gradient index lens transverse to the axis of the gradient index lens. These components will preferably be micro-sized, especially in the case of use within an endomicroscope apparatus. Preferably the optical prism directs light in a substantially perpendicular direction from the axis. The ?.'OP'tlMKR'SflC1 di4.*, AU &C I 3i0603 -12scanning head arrangement will allow freedom of the sample end to be placed and manipulated in vivo. Adoption of the scanning head arrangement, as for example shown in Fig, 5(b) allows the adoption of a rigid rotation scanning geometry, as for example schematically illustrated in Fig. 9. The components of the scanning head are readily commercially available and may be connected by conventional means, such as by using UV curing optical adhesive.

After passing into the second conductor the fluoresced light will return to the coupler and at least a portion of this light will be transmitted to the third conductor, at the terminal of which is located the light detection device. Preferably fluoresced light passing from the terminal to the detection device will first pass through a filter that will substantially prevent passage to the detection device of light other than the fluoresced light. This filter will preferably be a bandpass filter having transmittance in the expected fluorescent light wavelength range. It may be appropriate for the filter to operate at wavelengths within the range of between about 450nm to about 650nm, preferably between about 500nm and about 600nm, most preferably between about 530nm and about 570nm.

While other devices may be utilised to detect the fluoresced light, such as for example photodiodes and split photodiodes, it is preferred that the light detection device comprises a photomultiplier tube. The signal from the photomultiplier tube (PMT) may then be manipulated in a conventional manner to produce an image of the sample. This may be achieved by scaling the relative PMT voltage in a suitable range (0 to 10 V, for example) and using a 256 bit greyscale colour map to digitally reconstruct images. In this way an analogue signal of OV corresponds to digital bit 0 (black) and IOV corresponds to digital bit 255 (white). All other values of the PMT voltage in this range are scaled over the remaining bits, i.e. 10/254 represents 0.03937007V per bit.

In preferred embodiments of the invention lenses, filters and the terminals of the light conductors will be located within protective housings. In particular it is preferred for the sample end of the second conductor and the optional first and/or second lenses and aperture to be located within a protective housing. This is of course especially important P.',OPER1)KR'?tPECI inpp-AU doc-I 3003 -13in the case where the apparatus of the invention is being utilised as an endoscope or endomicroscope. Also in the case of endoscopic use it may be appropriate for a camera such as a video or digital camera to be provided at or about the sample region, along with a light source (which will preferably be configured to avoid interference with endoscopic imaging) to assist in directing the sample region to the desired location within the patient's body. It may also be appropriate for a suction mechanism to be provided for drawing the sample into the sample region. Similarly, a heating device may be incorporated within at least those components of the apparatus that will be internal during endoscopic use, to assist with patient comfort and ensure optimal operation of the equipment. It is also preferred for components of the apparatus that will be internalised during endoscopy to be provided with a soft covering, coating or laminate to both protect the equipment from exposure to potentially damaging acids, alkali, enzymes and the like within bodily fluids and to minimise discomfort to the patient and/or damage or disruption to organs and tissues. It is also preferred that the covering, coating or laminate is of a form that can readily be lubricated and sterilised, by conventional means.

The two-photon fluorescence microscope/endoscope of the present invention can be configured to incorporate other imaging modalities. For example, the present invention may be combined with optical coherence tomography and/or sonohysterography equipment, which would offer the capability for real time acquisition of detailed sample information that would ordinarily require two separate processes.

It is also possible to incorporate in conjunction with the scanner head of the invention a micropositioning functionality which will serve to allow controlled location of the scanning head in vivo. The features that will allow this positioning capability are well known in conventional endoscopic apparatus.

The present invention will now be described further, by way of example only, with reference to the following non-limiting examples: EXAMPLE 1 P.oPEtRIKRSfC.ncn..p-AU- il.03 14- Arrangement of two-photon fluorescence microscope The arrangement of a two-photon fluorescence microscope is given in Fig. 1. Ultrashort optical pulses generated from a turn-key Ti:Sapphire laser (Spectra Physics, Mai Tai) of a wavelength range from 730 nm to 870 nm, pulse width 80 fs and repetition rate 80 MHz were used as the illumination. This laser beam was coupled into a port (port 3) of a threeport single-mode fibre coupler via a 0.25 NA objective 01. Rotation of a neutral density filter, ND, placed before the objective 01 allowed variation of the input power. The output beam from port 1 of the coupler was collimated by a 0.25 NA objective 02 and then passed through a variable aperture, AP, to fill the back aperture of the imaging objective 03 (0.85 NA, 40x, /170). The emitted fluorescence from a sample was collected by the objective 03 and returned via the same optical path used for pulse delivery. The signal was delivered via port 2 of the coupler into a photomultiplier tube. A bandpass filter, BF, operating at wavelength 550 nm 20 nm) was placed in the beam path to ensure that only the fluorescence signal was detected. Each port of the fibre coupler was placed in a chuck holder in an X-Y-Z micro-positioner to allow precise positioning of the fibre tip at the focus of an objective.

To obtain the optimum delivery of the pulsed laser beam a fibre coupler (Newport) was used that was designed for operation at wavelength 785 nm with an equal splitting ratio.

The length of each arm of the coupler was 1 m. The measured coupling efficiency from ports 2 and 3 to port 1 varied between 20% 38% in the wavelength range between 770 nm to 870 nm. This property implies that the laser power of up to 80 mW 150 mW in this wavelength can be delivered to a microscope objective. As most commercial objectives have a transmittance of approximately 50% to 60% in the infrared region and the ultrashort pulsed beam is broadened approximately to a few picoseconds after it transmits through the coupler, this coupler is capable of delivering a laser beam of sufficient power for two-photon excitation.

P.OPERWMKR'SPECI.inupp-AU doc-1 3003 The optical fibre used is of a single mode type (at the operating wavelength) and has a core/cladding ratio of approximately 5/125 and a NA of 0.11. The operating wavelength of this coupler is 785nm.

EXAMPLE 2 Assessment of coupling efficiency In the visible range in which two-photon fluorescence falls, the coupling efficiency of this coupler (from the apparatus exemplified in Example 1) may be reduced and an equal splitting ratio between ports 2 and 3 may not necessarily be maintained. To confirm these features, the mode profile and coupling efficiency at ports 2 and 3 were measured while port 1 was illuminated by a continuous wave beam at wavelength 532 nm (Spectra Physics, Millenia). It was found that the field distribution from ports 2 and 3 is a single-mode profile, which is consistent with the estimation based on the core size and numerical aperture of the coupler in the visible range. The coupling efficiency at port 2 was approximately 1% with a splitting ratio of 90:10 between ports 2 and 3. As a result, using port 2 for the signal collection of two-photon fluorescence and port 3 for delivery of the pulsed beam means that the coupler acts as a low-pass filter and that the strength of twophoton fluorescence signal can be maximised.

EXAMPLE 3 Depth discrimination characterisation The depth discrimination of the new imaging system (as demonstrated in Fig. 1) was characterised by measuring the axial response to a thin fluorescent layer. The layer was produced on a cover slide by placing a few fluorescent drops comprising AF-50 dye dissolved in isopropanol until they were dried. The sample was excited by two-photon absorption at a wavelength of 800 nm. The measured fluorescence intensity as a function of the power input to port 3 of the coupler is shown in Fig. 2 in a log-log scale. It can be seen that the gradient of the curve is approximately 2.0 0.1 indicating that the P.'OPERWKVRrkpEClimpp-AU doc- 13)O -16fluorescence signal varies with the square of the input power, as expected for two-photon excitation.

EXAMPLE 4 Assessment of axial response Typical axial responses obtained using the apparatus shown in Fig. I are shown in inset (a) of Fig. 2. It is shown that the full width at half maximum (FWHM) of the axial response (solid curve in Fig. 2a) measured in the new two-photon fluorescence microscope, Az, is approximately 2.1 pm, which is reduced by 30% compared with that measured by a large area detector without a pinhole 6 (solid curve in Fig. 2a). The decrease in the FWHM indicates the enhancement of the optical sectioning effect and therefore the improvement in axial resolution. In this case, the resolution improvement results from the aperture of port 1, which acts as an effective confocal aperture. In inset of Fig. 2, the dependence of the FWHM Az on the incident power to port 3 is depicted, showing that the resolution is improved as the input power is increased. This feature is caused by the fact that the spectrum of the pulsed beam is broadened and blue-shifted due to self-phase modulation and self-steepening, respectively, in a fibre.

EXAMPLE Three-dimensional imaging To demonstrate the three-dimensional imaging capability of the system (as shown in Fig.

Fig. 3 shows a series of image sections taken at I pm depth step into an ensemble of pm fluorescent polymer microspheres. Fig. 4 displays a series of image sections of the autofluorescence signal from sea algae, Griffithsia, demonstrating the applicability of the instrument for biological study. Both image sets exhibit high contrast and the pronounced optical sectioning property of the system.

PVOPERM KR'SECqri -inAU d4 1-%&3 -17- EXAMPLE 6 Inclusion of scanning head in two-photon endomicroscope Fig. 5 shows a schematic representation of a two-photon endomicroscope, in all other respects the same as that shown in Fig. 1, which includes a scanning head (also referred to as a micro-scanning head) in place of the fixed objective/s and variable aperture at the sample end. The scanning head essentially comprises miniature beam focusing and directing optics. The single-mode optical fibre that is port 1 of the fibre coupler is attached to a 1mm diameter Plano-Plano, 0.25 pitch, 0.46 NA GRIN lens (Newport: LG1830-2) designed for operation at a wavelength of 830 nm. The pitch of the lens was chosen to yield the required Gaussian beam parameters. A 0.5 mm BK7 right angle microprism (Edmund Industrial Optics: A45-946) is mounted at the distal end of the GRIN lens to direct the beam perpendicular to the axis of the endoscope. In other experiments a I.0mmBK7 right angle microprism has been used. The microprism, GRIN lens and optical fibre were all attached with ultraviolet curing optical adhesive (Norland) to form a single unit, which has an approximate working distance of 2.0 mm.

EXAMPLE 7 Coupling efficiency of endomicroscope that includes scanning head Using the arrangement shown in Fig. 5 and with the scanning head attached to port 1 of the fibre coupler, the coupling efficiency for illumination of wavelength 800 nm was measured to determine the magnitude of power that can be delivered to a sample. The results are shown in Fig. 6, which depicts the measured output power at the focus as a function of input power to port 3 of the coupler. For illumination of wavelength 800 nm, the coupling efficiency of the system including the micro-head is reduced to approximately 28% compared with approximately 35% obtained with the system using bulk imaging components. This decrease arises for a number of reasons, including the slight mismatch in the refractive indices of the fibre core, GRIN lens, microprism and the optical adhesive.

Furthermore, if one considers the geometry of the arrangement shown in Fig. 5 it can be seen that a certain fraction of the incident beam will be transmitted through the microprism P.OPER MKRSPEC ina pp-AU doc-1.003 18along the principal axis and will therefore be lost. This fraction was measured and found to be approximately 5-8% of the power input to port 3.

EXAMPLE 8 Axial response of endomicroscope that includes scanning head Scanning using the arrangement shown in Fig. 5 may be achieved by using the input fibre port 1 of the coupler in this case) in conjunction with an optical coupling element (fibre ferrule) to couple the incident illumination across a small air gap to a second singlemode fibre with the micro-optic assembly attached. In this way it is possible to scan (rotate) the beam in a circumferential manner to image cross sectionally through a biological structure into which it was inserted. However, to demonstrate the feasibility of the design for use in fibre-optic TPFM, a rotational scan was not performed. Rather, the axial response in two cases was investigated.

First, the response to a plane mirror was measured by continuous scanning through focus in the z-direction. Second, the mirror was replaced with a fluorescent polymer block to measure the axial fluorescence response using the same scanning method. These two axial responses give measures of axial resolution of the endoscope in the case of nonfluorescence and two-photon fluorescence imaging. The measured response for the reflection (ie. mirror) case is shown in Fig. 7. Here, the optical sectioning ability of the instrument with the micro-head assembly attached is clearly demonstrated through the FWHM of the response curve, which is approximately 2.8 pm.

The practicability of the instrument for use in biological studies is reliant on the ability of the micro-head to collect two-photon excited fluorescence from a sample. Figure 8 demonstrates this ability and depicts the axial response of a planar fluorescent polymer sample obtained by coupling 400 mW of 800 nm laser illumination to port 3 of the fibre coupler. The FWHM of the response curve is approximately 2.6 ipm, which indicates a good optical sectioning ability. It should be pointed out that the measured axial resolution of the fibre coupler based system for an input power of 400 mW was approximately P.'OPER'MKRSPECtniN.tpp-AU doc. 1 603 19pm. One reason for the decrease in the optical sectioning performance of the system using the micro-head is due to the lower NA of the GRIN lens compared to the NA of the bulk imaging objective used in earlier experiments.

EXAMPLE 9 Simulation of internal imaging To demonstrate the feasibility of the micro-endoscope head for imaging an internal structure, rotating of the distal optics inside an internal body cavity was simulated by rotating a hollowed cylindrical sample holder around a fixed micro-endoscope head. The sample holder as shown in Fig. 10 was machined from a 25 mm length of Perspex to form a hollowed cylindrical rod of 3 mm inner diameter and mounted to the shaft of a variable speed 12V DC rotary motor. The speed of rotation could be accurately controlled by the potential supplied to the motor. A two-dimensional image was constructed by progressively increasing the insertion depth of the micro-endoscope head into the cylinder with a step size of 0.5 mm and performing a single rotational scan. The sample consisted of a fluorescent paper sheet cut into the shape of a 5.0 mm equilateral triangle. The sheet was prepared by uniformly coating a piece of 75g/m2 paper with a mixture of fluorescent dye and isopropyl alcohol.

4 Double-sided adhesive tape was used to secure the sample inside the cylinder oriented such that the base of the triangle was parallel with the circumference of the cylinder.

Fig. 4 shows five acquisitions into the cylinder. The first series is obtained by rotating the cylinder with the focus of the micro-endoscope positioned 1.0 mm along the fluorescent sample. The trigonometric shape of the top constructed by the five curves reveals that the region of the detected fluorescence intensity as a function of the angular displacement of the cylindrical sample holder is consistent with the actual shape of the fluorescent material.

It is clearly shown that as the micro-endoscope head is inserted further into the cavity, the rotation angle range over which fluorescence is detected decreases accordingly.

P.'OPER.IYR'SPECr iMpp-AU doc-13'0603

REFERENCES

1. W. Denk, J.H. Strickler and W. W. Webb, Science, 248, 7 (1990).

2. T. Dabbs and M. Glass, Appl. Opt., 31, 705 (1992) 3. J. Benschop and G. van Rosmalen, Appl. Opt. 30, 1179 (1991).

4. S. Kimura and T. Wilson, Appl. Opt., 30, 2143 (1991).

M. Gu, C. J. R. Sheppard and X. Gan, J. Opt. Soc. Am. A, 8, 1755 (1991).

6. D. Bird and M. Gu, "Resolution improvement in two-photon fluorescence .microscopy using a single-mode fiber", Appl. Opt. 41, 1852 (2002).

7. M. V. Iravani, Electron. Lett. 22, 103 (1986).

8. Y. Fujii and Y. Yamazaki, J. Microscopy, 158, 145 (1989).

9. H. Zhou, M. Gu and C. J. R. Sheppard, Optik 103, 45 (1996).

G. J. Tearney, S. A. Boppart, B. E. Bouma, M. E. Brezinski, N. J. Weissman, J. F.

Southern and J. G. Fujimoto, "Scanning single-mode fibre optic catheter-endoscope for optical coherence tomography", Opt. Lett., 21, 543-545 (1996).

11. F. Ouellette, "All-fiber filter for efficient dispersion compensation", Opt. Lett., 16, 303 (1991).

12. B. J. Eggleton, P. A. Krug and L. Poladian, "Experimental demonstration of compression of dispersed optical pulses by reflection from self-chirped optical fiber Bragg gratings", Opt. Lett., 19, 877 (1994).

P.'OPR f KR'SPECI4miina AU doc-IPOo03 -21 13. M. E. Fermann, K. Sugden and I. Bennion, "Environmentally stable high-power soliton fiber lasers that use chirped fiber Bragg gratings", Opt. Lett., 20, 1625 (1995).

14. D. Bird and M. Gu, Opt. Lett., 27, 1031 (2002).

Claims (18)

1. A two-photon fluorescence endoscope comprising: an ultrashort pulsed laser source of coherent light suitable to initiate two- photon fluorescence in a sample of interest located at a sample region; a first single mode fibre optic light conductor positioned to receive at its terminal light from said light source and direct said light to a three-port single mode fibre coupler; a second single mode fibre optic light conductor having at its sample end a scanning head comprising a gradient index lens located to receive light from said sample end, and an optical prism which directs light from said gradient index lens transverse to axis of said gradient index lens, the second conductor located to receive light from said first conductor and direct said light to said sample via the sample end, said sample end also located to receive fluoresced light from said sample that is collected by said optical prism, passes through said gradient index lens and is thereby directed to said sample end; a third single mode fibre optic light conductor located to direct fluoresced light from said coupler to a light detection device via a terminal of said third light conductor.

2. The endoscope according to claim 1 wherein a neutral density filter is provided, rotation of which allows power variation of said coherent light.

3. The endoscope according to either of claims 1 or 2 wherein a lens is provided to direct coherent light from said source to said terminal of said first conductor.

4. The endoscope according to any one of claims 1 to 3 wherein a fibre chuck is located at said terminal of said first light conductor. The endoscope according to claim 4 wherein position of said fibre chuck located at said terminal of said first light conductor is controlled by a positioning device.

P:\OPERUMKR\SPECI\203204717 2spa 366 dc- 101105 -23-

6. The endoscope according to any one of claims 1 to 5 wherein said gradient index lens, said optical prism and said sample end are contained within a protective housing.

7. The endoscope according to any one of claims 1 to 6 wherein a filter is provided between said detection device and said terminal of said third light conductor which substantially prevents passage to said detection device of light other than said fluoresced light.

8. The endoscope according to claim 7 wherein said filter is a bandpass filter.

9. The endoscope according to any one of claims 1 to 8 wherein a fibre chuck is located at said terminal of said third light conductor.

The endoscope according to claim 9 wherein position of said fibre chuck located at said terminal of said third light conductor is controlled by a positioning device.

11. The endoscope according to any one of claims 1 to 10 wherein said detection device is a photomultiplier tube.

12. The endoscope according to any one of claims 1 to 11 wherein the laser is a Ti:Sapphire laser.

13. The endoscope according to any one of claims 1 to 12 wherein said coherent light is at a wavelength of between about 730nm to about 870nm.

14. The endoscope according to any one of claims 1 to 13 provided with a suction mechanism for drawing said sample into said sample region. The endoscope according to any one of claims 1 to 14 provided with a camera and light source to assist in directing said sample region to a desired location.

P: PER\MKR\SPECI0032047I7 2p 366.doc- 1/01/05 24

16. The endoscope according to any one of claims 1 to 15 comprising a heater for heating some or all components of the endoscope located internally during use.

17. A two-photon fluorescence endoscope comprising: an ultrashort pulsed laser suitable to initiate two-photon fluorescence in a sample of interest to be located at a sample region; a first single mode fibre optic light conductor having at its terminal a fibre chuck under positional control of a positioning device, wherein light from said source passes through a neutral density filter and a lens which directs the light to the terminal of the first conductor, which in turn directs said light to a three-port single mode fibre coupler; a second single mode fibre optic light conductor having at its sample end a scanning head comprising a gradient index lens located to receive light from said sample end, and an optical prism which directs light from said gradient index lens transverse to axis of said gradient index lens, the second conductor located to receive light from said first conductor and direct light to said sample via the sample end, said sample end also located to receive fluoresced light from said sample that is collected by said optical prism, passes through said gradient index lens and is thereby directed to said sample end; a third single mode fibre optic light conductor having at its terminal a fibre chuck under positional control of a positioning device, the third conductor located to direct light from said coupler to a photomultiplier tube light detection device via said terminal of said third conductor and a bandwidth filter that substantially prevents passage to said detection device of light other than said fluoresced light.

18. A two-photon fluorescence endoscope according to either claim 1 or claim 17, substantially as hereinbefore described with reference to the figures and/or examples. DATED this 1 1 th day of January 2005. SWINBURNE UNIVERSITY OF TECHNOLOGY by its Patent Attorneys DAVIES COLLISON CAVE