CA2215975A1

CA2215975A1 - Optical fibre confocal imager with variable near-confocal control

Info

Publication number: CA2215975A1
Application number: CA002215975A
Authority: CA
Inventors: Martin Russell Harris; Peter Delany
Original assignee: Individual
Current assignee: Optiscan Pty Ltd
Priority date: 1995-03-24
Filing date: 1996-03-22
Publication date: 1996-10-03
Also published as: JPH11502943A; US5926592A; JP2007128095A; DE19681304T1; JP3945820B2; WO1996030796A1

Abstract

A confocal imaging system using optical fibres is provided which has a flexible near confocal optical transmission means having a light collection end (71) adjacent to a light collection end (70) of the confocal optical transmission means and adapted to transmit only near confocal light (S', T') emerging from points in the object (S, T) located within a range of distances above and below the focal plane, in such a manner that a selected portion of the near confocal light emerging from greater than any selected distance within said range is substantially separable from the remainder. The system also has variable selection means (73, 76) to exclude from detection said selected portion.

Description

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CA 022l~97~ l997-09-22 W096l30796 PCT/AU96/00159 OPTICAL FIBR~ COl.r-G~AL TMA~T~ WITH ~ART~RT-T~ NEaR-~GN~ocAL
CONTROL

Field of the Invention ThiC invention relates to confocal imaging cystems which use a flexible optical transmission means such as optical fibres as a substitute for the return pinhole, ana more particulariy but not limited to confocal microscopes constructed using optical fibres.

Description of the Prior Art Confocal microscopy can be considered to have originated with the work of Marvin M;nc~y. Hic ~.S. Patent, No.
3,013,467 aescribes a system in which light passes through a ~inhole, traverses a beamsplitter ana is focusea by an objective to a ~pot on or within a specimen. In an epi-illumination embo~; ~ t, light retl~r~;n~ from the spot region i~ converged by the same objective lenc, reflected by its xecon~ encounter with the beamsplitter and passes through a cecond pinhole to a photo aetector. The geometry of the arran~ ~nt is such that the focucea spot (Gaussian wai~t volume) is the only volume within the specimen from which the general set of ray paths ret~n;ng through the lens will retrace their outgoing paths to pass through the cecond pinhole to the photo detector. Light reflectea from above or below this $ocu~ which pa~ses through the objective lens will be largely blocked off by the opa~ue sheet material forming the pinhole surro~n~;n~ area.

The electrical signal from the photo aetector will give a value for the light reflected from the spot. If the specimen is moved the changes in electrical output from the photo detector inaicate changes in the level o~ light return from the material of the specimen along the path of CA 022l~97~ 1997-09-22 the spot. If the specimen is moved in a two ~; ~ional raster then a two ~; ~n~ional rastered map of the return light intensity can be build up based on the raster synchronous moaulation of the electrical output ~rom the photo detector. This can be displayea on a cathode ray screen or by other means giving an image Wh; ch i~ a sharp optical slice, substantially el;m;n~ting the con~ribution of light from above or below the focal plane. Such light no -1 ly reduces the contrast a~d blurs the image in conventional microscopy, particularly from translucent biological specimens, and renders high power microscopic observation of thick tissue sections impossible. The use of an optical fibre in the place of one or more of the pinholes is disclosed in ~.S. Patent 5,120,953. In such a fibre confocal microscope, the core of the optic fibre e~fectively acts a the pinhole, and, when the fibre is single moded, the light leaves the fibre as a single set of concentric eYrAn~;ng wavefronts and the system becomes diffraction limited and -Y; resolution is obtA;n~.

The chief advantage Of using a fibre to replace the pinhole i~ that the two sides of the pinhole are effective optically conn~cted by the core of the fibre, but are physically in~e~n~nt and can be indep~n~ntly and separately positionea. The major advantages conferre~ by this are (a) the ~everal large and hea~y component~ of the micro~cope can be located in any convenient po~ition and do not need to be rigialy located with respect to the specimen;
(b) the fibre tip it~elf can be ~~chAn;cally scAnne~ to give the raster reguired to build-up the image data set;
(c) an "in fibre" evanescent wave beam~plitter can be employed.

A disadvantage of existing fibre confocal microscopes i~

CA 0221~97~ 1997-09-22 that for these sy~tems there is no direct functional equivalent of opening u~ the pinhole. Most bulk optic laser scA~;~g confocal microscopes include a function in which the secona pinhole can be progressively enlargea.
While for the purpo~es of the highest resolution image, the smallest sizea secona pinhole i~ desirable consistent with a reasonable optical signal strength, in practice it is aesirable to enlarge ana contract the secona pinhole as the microscopist r-~ ;~S the object, by the ~ of operating a continuously variable ~;~rh~agm. The ~;~rh~agm opens the a~erture and collects an increasing fraction of light from the double cone volume on either siae of the Gaussian waist region. This increases the strength of the electrical signal but at the expense of optical resolution. This procedure is usea (a) auring a "search moae" in the early stages of observation where quick single scan images are being usea to locate the aesirea structure;
(b) where a rapidly moving structure is to observed which is not repetitive ana thus cannot be scan synchronisea;
(c) where an increasea aepth of fiela is aesirea ~or large aepth three ~; - ~ional reconstructions;
(a) where the fluorescence or the reflection signal is very weak;
(e) where the fluorophore is fugitive (ie.
easily photobl~Ached or spo~tA~eously ~c~- _osing).

Summary of the Invention It is an object of the current invention to be able to con~truct a fibre optic confocal imaging system which retains the aavantages of the use of the optical fibre but in addition has an equivalent function of opening up and closing down a pinhole without the necessity of providing a physical pinhole or ~;~p~agm adjacent to the specimen optics.

CA 0221~97~ 1997-09-22 W096/30796 PCTI~U96/00159 Therefore in accordance with a broad aspect of the invention there is ~rovide~ a confocal imaging system comprising:
a light ~ource for supply of a light beam;
light focusing means for focusing light from the beam onto a point observational fiela on or within an object and for recei~ing object emanated light emanating from the vicinity of the point observational field;
a detector for detecting the object emanated light;
sC~nn; ng mean~ operable to cause relative ~~v~L_nt between the object and the point obser~ational ~ield such that the point observational fiel~ scans over a focal plane transverse to an optical axis o~ the imaging system; an~
flexible optical transmission means for transmitting the source light beam from the light source to the light focusing means and ~or tran~mitting the object emanate~ light to the detector, and ha~ing light separator mean~ to separate the object emanatea light ~rom the light beam for passing to the detector and con~ocal optical tr~n~m;~xion means to transmit the object emanated light emerging only from the point obse~vational field;
wherein the optical transmission means further comprises (i) flexible near con~ocal optical tr~n~mi~sion means ha~ing a light collection end adjacent to a light collection end of the con~ocal optical trAn~;ssion means and adapted to transmit only near confocal light emerging from points in the object locatea within a range of distances above and below the focal plane in guch a ~ nn~
that a selected portion of the near confocal light emerging from greater than a correspon~;n~ selected distance within said range is substantially separable from the r~m~;n~n;
and (ii) an exit region for exit of at least a portion of said near confocal light from the flexible near CA 0221~97~ 1997-09-22 confocal optical transmi~sion means;
ana wherein there i~ further proviaea variable selection means to define ~aid ~electea portion ana exclude it from the aetector.

By proviaing ~eparable transmission through f lexible ana selectable means, a variable pinhole effect can be providea which may be locatea remotely of the ~pecimen.

In one class of embo~; ~nt, the near confocal optical tr~nl ;~sion means comprises a plurality of opticall~
isolatea ch~nn~l~ having aajacent ends at said light collection end to proviae said substantially ~eparable transmis~ion. The plurality of ch~nnel~ may be proviaea by a bundle of optical fibres, or a large aiameter optical fibre with multiple cores. Alternatively, the plurality of ch~nn~l~ may be a plurality of COA~; ~ 1 concentric waveguiaes, mutually separated by optically insulating material.

In this first class of embo~; - ts, an exit region of the near confocal optical trAn~ ;~sion means may be proviaed by a plurality of etchea ~ections of fibre exposing aifferent ones or ~ubset~ of aia plurality of ~hAnn~15 ana cont~;n;~g optical c~ ~nt to aivert light travelling in the correspon~;ng one or subset of ch~nn~lc to correspon~;ng photo aetectors. In this case, the variable selection means comprises switching circuitry or t~e like to select output from aifferent photo aetectors. ~lternatively, the exit region may be providea by opposite enas of the plurality of isolate~ ch~nnels forming an emission end of the fibre or fibre bundle, ana the variable selection means may comprise ~ 30 focusing means to project an image of the emission ena onto a region contA;n;ng a variable ~;~ph~agm to progressively excluae from detection saia selectea portion, the detector being aisposea behina the ~;~agm.

CA 0221~97~ 1997-09-22 W096~0796 PCT/AU96/00159 In a second class of :- ~o~; ~ s, the near confocal optical trA~ ;ffsion means com~rises a wide diameter fibre or the Cl ~;ng of a single mode optical fibre. In this class of ~ ~o~; - ~, the subst~t;~ separability of said selected portions may be att~;~ if the focussing means causes rays entering the light collection end of optical fibre to be transmitted through the fibre at an angle which i~creases with the distance of a point o~ entry of the ray into the collection end from the optical axis of the fibre. The variable selection means may include a ~ariable ~;~h~agm disposed adjacent the exit region of the o~tical fibre to exclude light emerging at greater than a selected angle.

In : ho~; - t~ where the exit region is ~rovided by an e_ission end of the fibre or fibre bundle, the variable selection means may also include near confocal focussing means to focus an image of the emission end of the fibre onto a secona variable ~;~ph~agm.

In other ~m~o~; ~ ts of the second class the exit region is provide~ by an expose~ side of the fibre such as an area of the fibre ~trippe~ of its outer jacket and contacting a material with refractive in~ex suitably matched to the fibre so as to extract the near confocal light. The near confocal light may be extracted from a single such region and the variable selection performed by variable ~;~ph~agm means. Alternatively, the near confocal light may be extracted from a ~lurality of regions along the length of the fibre co~t~cting materials having progressively greater refractive index to progressively extract rays of lower angle, the variable selection means compri~ing optical or electronic switching means.

In a thir~ class o~ embo~; - ts, the near confocal optical transmission means comprises a gradient index fibre. In CA 0221~97~ 1997-09-22 this class, the exit region may be provided by ~uccessively dee~n;ng etched areas in the fibre side with correspon~;n~
photo aetectors. Alternatively, the exit region may be provided by an emis~ion en~ of the fibre. In quch ca~es, a first variable ~;Arh~a~m may be ~rovi~ea to admit only low angle light through near confocal focussing means to project an image of the fibre tip onto a ~econd variable ~;Arh~agm in front of the detector.

Description of the Preferred Rmbo~; - ts In order that the invention may be more clearly ascertA;ne~, preferred embodiment~ will now be described with reference to the accompanying drawings, in which Figure l is a diagram of ray paths emerging f rom the vicinity of the point observational fiela of a confocal micro~cope an~ being focused onto the collection en~ of an optical trAn~ ;~sion means;
Figures 2a, 2b, 2c and 2~ are examples of the plurality of isolatea chAnn~l~ of the fir~t embo~ of the invention;
Figure 3 is a diagram showing a schematic optical arrany. - t of a variation of the first embo~; ~nt using "four leave~ clover~ fibres (see 26) as the near confocal optical tr~n~ ;~sion means, an~ etche~ region~ of fibre to provi~e the progressive selection;
Figure 4 is another variation of the first embo~; ~ t showing the use of a conc~nt ~iC wave guide structure;
Figure 5 is another variation of the first embo~; ~nt showing the progressive selection means providea by the projection of an image of an emission en~ of the f ibre;
Figure 6 is diagram showins the principle of trAn~r;~sion of light rays through the cl A~; n~ of a single mode optical fibre;
Figure 7 shows one embo~iment of the second class CA 0221~97~ 1997-09-22 W096/30796 PCT/AU96/OOlS9 of e_bs~;-? ts where the near confocal optical tr~n~m;~sion means is providea by the cl~;~g of ~ingle mode optical fibre;
Fi~ure 8 shows a variation of the secona class of : ~o~; ts using clA~;~g modes of a single moae fibre where the near confocal light is extractea from the side of the fibre;
Figure 9 shows another variation of the e_bo~; - t of Figure 8;
Figure 9A shows a variation of the second class with the near confocal light extracted progressively;
Figure lO shows detail of another e_bo~;m~t of the secona class using a ball lens to angularly code the light before it enters the collection ena of the fibre;
Figure lOA shows a variation of the emboaiment of Figure lO.
Figure 11 shows an example of the thir~ class embo~ using a gradient inaex fibre an~ etchea sections of fibre for the selection means;
Figure llA shows the gradient inaex profile of the fibre of Figure 11;
Figure 12 is a aiagram showing an emission ena of a graaient inaex fibre ana selection means in emboaiments of the third class which make use of the projected image of a fibre tip;

Referring now to Figure 1, there is shown a schematic diagram o~ ray paths from points in an object above ana below the focal plane. Specifically, a single moae optical fibre in a fibre confocal microscope typically may use the core lO of a single mode optical fibre to transmit laser source light from a laser (not shown). At an end 11 of the core lO of the optical fibre, the laser light projects outward in a cone of divergence angle approximately 8 to lO~ for a typical fibre (exaggeratea in the figure) through a focusing lens 12, focusing the light to a point observational fiela P within an object to be observed CA 0221~97~ 1997-09-22 g (object not shown). Since the light ret~n; n~ through the focusing lens 12 ana back into the en~ 11 of the core of single mo~e fibre mu~t pass through the point P, it is pr~A~ ;n~ntly light ; nAting from point P which re-enters the core, proviaing the desirea isolation of the light from the focal plane F ana enabling this "confocal" light to be collectea by the use of separations means such as a h~- ~litter or optical fibre coupler. Light from points Q
and R in the ~icinity of P but a ~istance D above ana below respectively the focal plane F is not focusea into the end of the core 11, but has a focal point either ; -~;Ately in front of or behin~ the en~ of the core 11. As a resu:t, at the front face 14 of the optical fibre, light from p~ints Q
and R aiffusively impinges on the clAAA;ng material of the fibre. ~or~-lly the c~AAA;n~ of a single mode fibre is surroun~ea by a jacket 13 ha~ing a refractive inaex greater than that of the cl AAA; ng ana therefore inhibiting the propagation of rays called clAAA;~g moaes through the clAAA; ng, calle~ clAAA; ng - ~~ .

Clearly, light emanating from points closer than a aistance D falls within a circle of raaius R at the face 14 of the optical fibre, ana as D is increasea, 80 aoes R increase.
Accordingly, the aistance R from the axis at which light impinges on the optical fibre is related to a aistance D
from the focal plane from within which the light has emanate~.
This well-known aperture relationship is what allows the opening an~ closing of the pinhole in a stAnAA~a confocal microscope to proviae increasea aepth of fiela.

Accoraingly, i~ the light can be transmittea in such a way that this aistance relationship is preservea or otherwise encoaed, then the light i~ transmittea in a separable ~ n~ 8uch that when it r~Ache~ an exit region of the fibre, it may by various means be selectea in a progressive m~nne~ to aefine the eguivalent of a variable pinhole.

CA 0221~97~ 1997-09-22 In the first class of pre~erred ~ ~oA; -nt~, which are the simplest to visualise, this ~istance relationshi~ is preserved by providing a plurality of isolate~ ch~nne~, as shown in ~igure 2. For example, as shown in Figure 2a this may be realisea by a coherent fibre bundle, with the laser light delivery and confocal return ~ibre 20 at its centre, ana a plurality of collection fibres surrol~nA; n~ the delivery core. Alternatively, a multi-core fibre may be used such as the "four lea~ clover" design shown in Figure 2b, or a multiple clover design shown in Figure 2c. An alternative variation involves conc~n~ic cylindrical wave guide region~ as shown cut-away in Figure 2d, separated by lower re~ractive index regions 23 (~or example, silica glass) which space the higher re~ractive index cylinders by a aistance su~icient to re~uce coupling between the cylinaers to an acceptable level over the lengths used in the ~ibre optic ~atch cord A jacket 24 ~n~om~asses the ~ibre. It may be desirable for the outer cylindrical wave guide structures to be thicker than inner ones.

Referring now to Figure 3, there is shown a means of tapping the near confocal light in a progressive ~-nne~ to provide the variable selection means of the first class, applicable to the four leavea clover fibre arrangement. An etchea region 30 of the fibre exposes a ch~nnel 31 and i5 filled with optical c~ - t 32 having a refractive index e~ual to or greater than that of silica. Within the optical cement 32 is embedded a photo detector 33 as a part of the detector means. Light travelling along chAnnel 31 encounters the etched region 30 and is divertea into the optical cement, activating the photo detector 33.
Similarly, a second etched region 34 is provided which exposes a section of a second one 35 of the four leaved clover ch~nn~ls, and is again filled with optical cement 36 Co~tA i n; ng a second photo detector 37. The variable 3S selection means is provided by switching means, and hardware or software, to select light from the desired CA 022l~97~ l997-09-22 detector~. Further down the fibre, a ~A~n~h mode stri~per 38 is ~rovided which expo~eq all but the central core and prevents laser li~ht in the light beam from the la~er ~rom travelling down the four leavea clover cores and into the detectors 33 and 37.

At a remote en~ 39 of the fibre, light from the confocal core emerges and returns through light source focussing optics 390 and i8 partially deflected by a light separator in the form of beam splitter 391 into a ~hotomulti~lier tube 392 to proviae detection of the confocal return light, in a similar mAnn~ to known laser scAnn;ng confocal microscopes.

The -- ho~; - t shown in Figure 3 uses only one single mode fibre core for both trAn~ ;~sion of the light beam from the laser to the object ana for collection and trAn~m;~sion to the detector of the confocal light (~ -n~ting from the confocal plane). ScAnn;n~ can be achieved, as i~ disclo~ed in ~.S. Patent 5,120,953, by a number of means, including vibration of the fibre tip, and/or conventional s~Ann;ng mirrors between the fibre tip and the ~pecimen. S; ;
~mho~ can be envisaged, in accordance with the disclosures of ~.S. Patent 5,120,953, which involve a separate fibre for trAn~ ;~sion of the light beam. All the de~criptions given here including the embodiment of Figure 3 correspona to emboA; ~nts in which the light i8 conveyed to the microscope head and specimen by means of a core of a ~ingle mode fibre. In the~e embo~; - ts the alternative chAnn~l~ for conveying the near confocal light back to the photo~etector are within the same ~ibre which conveys the light to the specimen. In accordance with the teachings of ~S Patent 5,120,953, a beamsplitter may be uxea and the fibre conveying the confocal light back to the photodetector may be a second completely separate fibre.
All the methods describea in the current speci~ication can also be appliea to the two fibre system in which the moaal CA 0221~97~ 1997-09-22 selection means are applied to the second return fibre to selectively extena or restrict the depth of field.

Referring now to Figure 4, there is shown a similar variable selection means for the concentric wave guide structure shown in Figure 2d, with etched region~ of progreqsively deeper extent being a~plied along an exit region of the fibre. The first region 40 extracts light only from the outermost core. The next region 41 is slightly deeper ana it extracts light from the ~econd outermo~t core, light from the outermost core already having been extracted. Further and deeper regions may be arrangea in succession. A ~ nch mode gtripper (not shown) is ~rovided at the end of the fibre, as in Figure 3, ana similar switch; ng means are provided.

Referring now to Figure 5, there i~ shown an alternative means of providing variable selection means in the first class of ~ ~o~; - t~. The exit region is provided by an emission end 50, the same end which receive~ the light beam 51 from the laser 52. The laser focusing optics 53 also act~ for the return light a~ a near confocal focussing means to provide an enlarged projected image of an emission ena 50 of the fibre at a remote ~oint 54. An iris A; ~rh~agm 55 is used to progressively exclude the selecte~
portions of the light from entering the photomultiplier tube (not shown). This method is a~plicable to any of the isolated ch~nn~l arrany ,~ ~8 shown in Figure 2. For isolated ch~nnel arran~ - ts other than the con~nt~ic waveguide structure of Figure 2(d), the preservation of XY
information could also be used to advantage if a multiple photo detector is used. For example, if a quadrant photo detector is used in association with the four leaf clover design, a difference between the out~uts of the four ch~nnels can be used in displaying other imaging moaes such as differential interference contrast.

CA 022l~97~ l997-09-22 W096/30796 ~ PCT/AU96/00159 Referring now to Figure 6 there i~ shown near confocal light exemplifiea by ray~ 61 and 62 propagating a~ clAAA;~g mode~ through the clA~A;~ of a ~ingle mo~e fibre with core 63 accepting the confocal light 64. Single moae fibres are compo~ea of a Ge dopea core 63, typically of about 3 ~m aiameter, ~urroundea by silica cl A~; ng 65 of lower refracti~e inaex thAn the core, the A; r ~ ~er of the clAAA;ng typically being about 125 ~m. Surro~nA;n~ the clAAA;n~ is a jacket. In such an arrany~- - L, the clAAA;ng moaes are accepted ana propagate by total internal reflection if they are ;nc;Ae~t on the collection ena 41 at an angle of less than about 30~. If it i5 desirea to allow the clAAA;n~ modes to propagate, the jacket should be constructed from a material of lower refractea index than the clAAAing. Transparent silicone rubber is a suitable material. Normally, the jacket is conctructea from acrylic material which inhibits the propagation since clAAA;~g modes are normally unaesirable.

In a microscope of co--ve,-~ional A; - ~ions ana u~ing 125 micron fibre, the clAAA;n~ 65 cannot be used on it~ own to transmit the near confocal light in a se~arable mAnn~ such that a ~electea portion of the near confocal light emerging from one or more selectea distances within a range of ai~tance~ above ana below the focal plane may be separated from the -; nA~ of the near confocal light. This is firgtly becau~e the light rays are ;YeA a~ they propagate through the cl AAA; n~ and emerge at the other ena of the fibre in a disoraerea fashion, such that the oraered relationship between distance from the optical axis of entry and di~tance range from the focal plane is lo~t.
Seconaly, the angle of inciaence of the near confocal light will not vary sufficiently when collectea by 125 micron fibre in a -nne~ dep~nA~nt on depth of field.

Howe~er, the fact that the angle of exit of the rays is always equal to the angle of entry can be u~ed to aavantage CA 022l~97~ l997-09-22 by the addition of o~tical element on or near the fibre tip. Referring now to Figure 7, there is ~hown ~art of the o~tics of one of the secon~ class of em~o~; ~nts which uses the clA~;n~ modes by co~;n~ the distance from the axis into incidence angle of propagation within the c~ ;n~.
Rays emerging from the confocal point P are shown entering the 8 in~le mode core at 70 (exaggerated angle). The collection ena 71 of the fibre is fashioned into a curved sha~e to provide a lensing effect which bends ra~ to a greater extent the more aistant they enter from the optical axis. The confocal light effectively enters the core end 70 without refraction, ~ince the curved ~ha~e is behind the core end 70. This shape may be m~ufactured by heating the end of the fibre 80 that soften;ng and surface tension produces a curved shape. Light emerging from points S and T progressively farther from the focal plane enter~ along rays S' and T' progressively farther from the o~tical axis, and therefore corresponds to rays S'' and T'' of increasing angle of propagation. Jacket 7la is composed of a suitable low RI material such as silicone plastic. At an emission end 72 of the fibre, the distance from the axi~ at which the rays S'' and T'' emerge is not oraered in accordance with the angle of propagation, but the angle of emergence is 80 ordered. This can be use~ to a~vantage by ~rovision of far-fiel~ iris ~;Arh~agm 73 in front of the near confocal focusing means 74, which also may act a~ the laser focussing optics. In a fully opened position the iris ~;~rh~agm admits substantially all of the light e~erging from the emission end 72, which is then focused onto an image of the fibre tip at detection focal plane 75. A
further near-field iris ~;~rh~agm 76 in front of the detection focal plane 75 will not operate in a progressive m~nn~n gimilar to irig ~;~rh~agm 73 since the spatial variation of intensity in a projected image of the fibre end i~ not correlatea in this embodiment with distance from the axis of entering light, but may be used to exclude the near confocal light from entering the photo detector when CA 022l~97~ l997-09-22 W096/30796 PCTtAU96/OOlS9 operating at maximum resolution to detect only confocal light. When the far-field iris ~;~rh-agm 73 is partially o~ened, the near-field iris ~i~h~a~m 76 will vary the proportion of near confocal light being admitted.
-It may be advantageou~ to provide a section near the tip ofthe fibre having re~c~ overall diameter (not shown) by hydrofluoric acid etching or other techniques so that the radius of curvature of the tip can be decreased to give a reduced ~ath length ~or the required separation of the near confocal light. This section may be reduced in diameter in a single step or gradually as an ~ h~tic taper.

Referring now to Figure 8, there is shown an alternative means of extracting the near confocal light from one of the secon~ class of embo~; - ts which uses cl~;ng mode propagation. A glass block 80 is optically connected by optical glue 81 to an ex~osed part of the cl~;n~ of the fibre. The refractive index of the glass block must be higher than or equal to the refractive index of the optical glue which must be higher or equal to the refractive index of the fibre clA~;~g. ~ens 83 focuses the light emerging through variable iris 84 onto photomultiplier tube 85. A
mirror 86 reflects light emerging from the other side of the fibre to follow substantially the same path. A
cl~;~g mode stri~per 87 prevents laser light from the laser travelling along the cl~;n~. The confocal return light travelling along the core of the ~ibre, which passe~
through the centre of lens 83 is extracted at the fibre end in a st~n~d -~e~ and passed via beamsplitter 89 to photo detector 88. In fluorescence imaging a~lications, where the wavelength of the object emanate~ light differs from the wavelength of the laser ~ource light, a laser exclusion filter 890 can be u~ed to exclude any stray laser source light which is reflected from the ti~ back into the fibre as claading mo~es. Anti-reflection coatings or other tip treatments could be employed without filter 890 if the CA 022l~97~ l997-09-22 W096~0796 PCT/AU96/OOlS9 a~paratus i~ to be u~ed in reflection mode confocal microscopy.

An alternative similar arrangement i~ shown in Figure 8A, where a second glass fibre 8Al , preferably of larger diameter ana the same or higher refractive index as the c~;~ of the first ~ibre 8A2 (corre~ponding to the ~ibre of Figure 8), i8 fused to the first fibre 8A2 over a length of some millimetres. The light travelling down the ClA~;~
of first fibre 8A2 i~ chA~l led into the larger Recond fibre 8Al in proportion to the cross-sectional areas o~ the two fibres, and the angular ordering of the light rays i~
maintA;~e~. If a 500 micrometer fibre is u~ed for the second fibre 8Al, and a 125 micrometer fibre for first fibre 8A2, then approximately 94 percent of the cl A~
modes will be chAnn~lled into the second fibre 8Al. ~ens 8A3 and iris A;Arh~agm 8A4 may then be positioned remote from fibre 8A2, ~Gving the need for encompassing the lens around fibre 8A2.

Figure 8B show~ another alternative mean~ o~ providing the variable ~election meang, where a material 8Bl of variable refractive index is co~t~cted with an exposed part 8B2 of the clA~;ng of the fibre (correspo~;~ to the fibre of Figure 8). A variable amount of higher order mode~ can then be extracted through surface 8B2 and discardea. The ~ ;~;~g complementary fraction passes to photomultiplier tube 8B3. The material of variable refractive index may be a collection of different liquias selectively being made to contact the surface 8B2, or a series of soft polymer blocks .

A further alternative similar arrany~ ?nt is shown in Fiyure 9 where a perspex box 90 surrounds the ~ibre, including a region contA;~;~g expo~ed cl~;~ at 92.
Clear polyester resin 93 is poured into the box 90 and sets .

CA 022l~97~ l997-09-22 Referring now to Figure 9A, there is chown an alternative exit re~ion for embo~; nt~ of the second class. Rather than have a single exit region as in the em~oA; - tq of Figures 8 ana 9, whereby the selection means is provided by lenses and irises, it is ~ossible to use succe~sive regions of the fibre with the jacket 9A1 removed and drops of optical glue with successively increasing refractive index to cause ray~ of successively lower angle of internal propagation to be extractea. If the cl~AA; n~ 9A2 typically has a refractive index of 1.45 and the jacket 9A1 typically has a refractive index of 1.40, in a first stripped region a blob 9A4 of optical glue may have a refractive index of, for example, 1.41 to extract a first portion of high angle propagation ray~ into the glue in which i~ placea a photo detector 9A3. At a second region optical glue 9A5 having refractive index of 1.42 and detector 9A6 extracts further light greater than a lower angle, and so on. As in the emboA; ~nts shown in Figures 3 ana 4, l~nch moae strippers are ais~osed at one end and switching mean~ ~rovide the variable selection means (not shown). The blobs 9A4 and 9A5 are not to scale and are typically 3 to 4mm or more in size, sufficiently long in an axial direction of the fibre to extend at least as far as the internally reflecting ray path "pitch".

A conventional objective lens may be used in place of the curved fibre ti~ if the light is allowed to ~ro~agate on an extended path to allow sufficient lateral divergence of the near confocal light from the confocal light cone to thereby produce the required coAi~ of lateral separation into angular separation. The advantage of the curved fibre end is that it allows for a much shorter di~tance between fibre tip and specimen. Any arrany - t where the confocal ch~nnel is disposed such that the confocal light is not adversely refracte~ by the lens may be ~uitable to allow short distance ~eparation between tip and specimen.

CA 0221~97~ 1997-09-22 The integral f OCU8 ing providea by the curvea en~ 71 in Fi~ure 7 may be pro~idea by se~arate ~mall lens ~luea onto the fibre such as a ball lens 100 ~hown in Figure 10, ty~ical ray ~aths 101 for which are shown. A di~aavantage of this ~m~oA; - t is that the confocal light an~ the la~er emission light is also focussea by the ball lens, again requiring a larger aistance between fibre tip an~ sp~c;m~n.
This ~m~o~; - t also doe~ not correct for chromatic aberration. However, one advantage is that relative ,v- ~nt between fibre tip and lens is maae possible. In order to match the projectea laser beam to a variety of lense~ in the micro~cope turret, each having a aifferent back focal ~iameter it is aesirable to have an aajustment mechAn;~m by mean~ of which the fibre tip entering the heaa can be mo~ea towaras or away from the lens adjacent the fibre tip. Figure 10A shows lens 10A1 attAch~ by a flexible optical glue 10A3 to fibre tip 10A2 housea within a piezoelectric cylin~er 10A4. The cylinder 10A4 i~
contractible in length, which shifts the fibre tip longit~; n~1 ly by a few microns. This motion increase~ the wiath of the light beam with negligible alteration in the beam angle, ana after passing through the transfer optic~
this adjustment can be usea to match the aperture of the objective lens being used, -~;~;~ing the optical efficiency and resolution ~or each len~.

One of a thira class of embo~ 8 is shown in Figure 11 where insteaa of a lens on or in front of the face o~ the fibre, a gradient index " - -~y profile" fibre i8 u8ea in place of a ~ingle moae fibre. In this fibre, the optical material surro~n~;ng the ~ingle moae core ha~ gra~ation~ of refractive inaex, provi~ing a curvea ray path~ 110 for the moae~ propagating in the fibre out~iae the core. Figure 11A
show~ a refractive inaex profile for the '~m~y profile"
fibre. Regions llA1 corre~pond to the polymer jacket, regions llA2 to the gla~ clA~;ng, region llA3 to the multimode-supporting region of gradation~ of inaex an~ llA4 CA 022l~97~ l997-09-22 W096/30796 PCT/AU96/OOlS9 to the single mode core region. The ~Y; angle of propagation of ray paths is related to the distance from the optical axis at which the ray path enters as a ; n;
distance of approach of the ray to the outer face 111.
Thus, rays entering at a greater distance $rom the o~tical axis have greater -~; angle of propa~ation as they cross the optical axis and go closer to the outer face 111.

Thus progressive selection of the near confocal light by means of a series of progressively ~eeper etchea regions with optical glue at 112a, 112b and 112c is possible. The diagram in Figure 11 of ray paths i8 schematic only. The rays in fact do not enter the fibre in a -nn~ which would cause the maintenance of node regions more than a short distance along the fibre. Each etched region is in fact not placed strategically with respect to a node, but is elongate along the fibre axis for about 3 to 4mm, being several times the pitch length of the oscillatory ray path, whereby a single etched region on one side of the fibre to a depth A will absorb effectively all light rays which come within ~ of the sur~ace at their ~-~; of oscillation.

~eferring now to Figure 12, there is shown the emission end 120 of a gradient index fibre according to another variation of the third class. A double iris diaphragm arrany- - t similar to that shown in Figure 7 is employed here, although the principle of operation is somewhat different. The light emerging from the remote end of the fibre is either far from the axis and has a low angle of incidence such as rays 121 and 122, or is near to the axis and has a high angle o~ incidence such as rays 123 and 124.
The light which proceeds to iris ~;~ph~agm 126 is light which is emerging from the fibre at a low angle, aperture 125 block;~g out high angle light. This light is approximately ordered in distance from the core in a correspo~; ng -nn~ to the light entering the fibre, in CA 022l~97~ l997-09-22 turn corres~onA;~g to aistance from the focal ~lane in the s~ecimen. For example, ray 121 is shown emerging slightly further from the core than ray 122, ana is excluaed from detection by the variable iris ~;Arh~a~m 126, while ray 122 i~ acceptea. The situation is more complex than this in practice, h~cA~e the iaealisation of noaes depictea in Figure 11 is not realised. This results in there being also rays orderea in angle rather than aistance form the core, and these may be progressively selectea by operation of iris ~;Arh~agm 125. In practice, there is a conti~uous range of int~ ?d;Ate cases also. The entire range can however be progressively selected to an acceptable degree by operating iris ~;Arh~agm 125 and 126 simultaneously.
The projected image of the fibre tip at iris ~;Arl~agm 126 i~ then displAc~ - t-codea from the axis in the desirea -~n~, and its operation of the iris A; Aph~agm 126 is such as to produce a variable pinhole effect for the near ~ield modal pattern rays. Operatea in conjunction with variable occlu~ion of the for field modal pattern rays by ris ~;Aph~agm 125 this will provide operation which i~
functionally equivalent to a conventional variable physical pinhole in a confocal microscope system.

Still another arrang~ -~t in shown in Figure 13 where a first fibre 131 is shown ca~t into a polymer block 132, the surface 133 having been polished away to expose the Cl A~;~g almost to the core 134. A variable amount of the higher oraer moaes can then be extracted through surface 133 and discaraed by sliding a second polymer block 135 progressively over the surface 133, as is known analogously in variable ratio fibre coupler technology.

In order to achieve appropriate separation of confocal and near confocal light, it may be necessary in many embodiments of the invention to use beam extenders in order to provide an ade~uate distance between the fibre tip and the objective while maintA;~;~g manageable product , CA 0221~97~ 1997-09-22 ion~. Thi~ can be de3igned in a c~ _-5t r~c~e using qt~ ~a o~posing mirror tech~;~ue~ as is well known in the art.

The principle of coupling out modes from a fibre by means of a su v~ 7;~ medium, the refractive inaex of which can be changed, might also be applied to the light form the la~er on the way to the microscope head. If a ~ew --~A
c ;cation~ fibre wa~ u~ed a~ the optical transmi~sion meanC and the cl~;~g glas~ was etched away from a ~ection of this fibre and replaced with a controllable variable RI
material then the modes passin~ into the microscope ~ould be controlled at the same time as the mode~ c~~;~g back to the detector~. Thi~ would have certain advantages in giving extra ~ignal strength for low fluorophore concentrations where there is fluorescence ~aturation and where non linear bl~ Ch; ~ may be a problem.

Modifications may be made to the invention a~ would be a~parent to a person skilled in the art of confocal optical design. For instance, the invention i~ not restricted to application~ re~uiring a diffraction-limited confocal spot and imaging systems other than microccopes which can make u e of the same optical principle~ are within its ~cope.
Further ~till, the near-field iri~ rh~agms which are disposea adjacent a projected image of the fibre ena ana it~ a~sociated photo detector may be replaced by CCD array~
if desirea and the selective exclusion of light per$ormed in software. CCD arrays may similarly be u~ed with far-f ield pattern decoaing.

Also, a number of embo~;me~t~ have been ~hown which variously use exit regions in either a mia region of the near confocal return ~ibre or an ena; ~election mean~ which may be classifiea as ~near field" or "far field", being composea of lenses an~ irise~ or switching mean~; ana "coding~ ~ystems in three classes using isolated c , W096t30796 PCT/AU96/00159 or angular co~;~. Other combinations of these basic ideas may be en~isagea ana are also within the scope of the inve~t;~.

Further, as explA;~ above the single-fibre e_bo~;me~t shown here can be replacea by dual fibre systems, with ~ource fibre and return fibre~ being separate or al~o with the confocal return being provided by a separate fibre to the near-confocal return. These and other moaifications may be maae without aeparting from the ambit of the invention, the nature of which i~ to be a~certA;~ from the foregoing description ana the drawings.

Claims

CLAIMS:

1. A confocal imaging system comprising:
a light source for supply of a light beam;
light focusing means for focusing light from the beam onto a point observational field on or within an object and for receiving object emanated light emanating from the vicinity of the point observational field;
a detector for detecting the object emanated light;
scanning means operable to cause relative movement between the object and the point observational field such that the point observational field scans over a focal plane transverse to an optical axis of the imaging system; and flexible optical transmission means for transmitting the source light beam from the light source to the light focusing means and for transmitting the object emanated light to the detector, and having light separator means to separate the object emanated light from the light beam for passing to the detector and confocal optical transmission means to transmit the object emanated light emerging only from the point observational field;
wherein the optical transmission means further comprises (i) flexible near confocal optical transmission means having a light collection end adjacent to a light collection end of the confocal optical transmission means and adapted to transmit only near confocal light emerging from points in the object located within a range of distances above and below the focal plane in such a manner that a selected portion of the near confocal light emerging from greater than a corresponding selected distance within said range is substantially separable from the remainder;
(ii) an exit region for exit of at least a portion of said near confocal light from the flexible near confocal optical transmission means;
and wherein there is further provided variable selection means to define said selected portion and exclude it from the detector.

2. A confocal imaging system as claimed in claim 1 wherein the near confocal optical transmission means comprises a wide diameter fibre or the cladding a single mode optical fibre.

3. A confocal imaging system as claimed in claim 2 wherein the focussing means causes rays entering the light collection end of the optical fibre to be transmitted through the fibre at an angle which increases with the distance of a point of entry of the ray into the collection end from the optical axis of the fibre such that the substantial separability of said selected portions is thereby attained.

4. A confocal imaging system as claimed in claim 3 wherein the focussing means comprises a ball lens glued onto the collection end of the optical fibre.

5. A confocal imaging system as claimed in claim 3 wherein the focussing means comprises the collection end of the fibre fashioned into a curved shape to provide a lensing effect which bends rays to a greater extent the more distant they enter from the optical axis.

6. A confocal imaging system as claimed in claim 5 wherein there is provided a narrow section near the tip of the fibre having reduced overall diameter such that the radius of curvature of the tip is decreased to give a reduced path length for the required separation of the near confocal light.

7. A confocal imaging system as claimed in claim 6 wherein the narrow section is reducing in diameter in a single step.

8. A confocal imaging system as claimed in claim 6 wherein the narrow section is reduced in diameter adiabatically.

9. A confocal imaging system as claimed in claim 3 wherein the variable selection means includes a variable diaphragm disposed adjacent the exit region to exclude light emerging at greater than a selected angle.

10. A confocal imaging systems as claimed in any one of claims 2 to 9 wherein the exit region is provided by an emission end of the fibre.

11. A confocal imaging system as claimed in claim 10 wherein the variable selection means includes near confocal focussing means to focus an image of the emission end of the fibre onto a second variable diaphragm.

12. A confocal imaging system as claimed in claim 3 wherein the exit region is provided by one or more regions where the side of the fibre is exposed and contacts an extracting material with refractive index suitably matched to the fibre so as to extract some or all of the near confocal light.

13. A confocal imaging system as claimed in claim 12 wherein the exit region is provided by a single such exposed region.

14. A confocal imaging system as claimed in claim 13 wherein the extracting material is a glass block optically connected to the exposed region.

15. A confocal imaging system as claimed in claim 13 wherein a clear box surrounds the fibre, including the exposed region and the extracting material is a clear resin set inside the box to optically connect with the exposed region.

16. A confocal imaging system as claimed in claim 13 wherein the variable selection means includes a variable diaphragm disposed adjacent the exit region of the optical fibre to exclude light emerging at greater than a selected angle.

17. A confocal imaging system as claimed in claim 12 wherein the exit region is provided by a plurality of said exposed regions arranged along the fibre contacting materials having progressively greater refractive index to progressively extract rays of lower angle, the variable selection means comprising optical or electronic switching means.

18. A confocal imaging system as claimed in claim 2 wherein the near confocal optical transmission means comprises a gradient index fibre.

19. A confocal imaging system as claimed in claim 18 wherein the exit region is provided by successively deepening etched areas in the fibre side with corresponding photo detectors.

20. A confocal imaging system as claimed in claim 18 wherein the exit region is provided by an emission end of the fibre.

21. A confocal imaging system as claimed in claim 20 wherein a first variable diaphragm is provided to admit only low angle light through near confocal focussing means to project an image of the fibre tip onto a second variable diaphragm in front of the detector.

22. A confocal imaging system as claimed in claim 1 wherein the near confocal optical transmission means comprises a plurality of optically isolated channels having adjacent ends at said light collection end to provide said substantially separable transmission.

23. A confocal imaging system as claimed in claim 22 wherein the plurality of channels is provided by a bundle of optical fibres.

24. A confocal imaging system as claimed in claim 22 wherein the plurality of channels is provided by a large diameter optical fibres with a plurality of cores.

25. A confocal imaging system as claimed in claim 24 wherein the plurality of channels is a plurality of coaxial concentric waveguides, mutually separated by optically insulating material.

26. A confocal imaging system as claimed in any one of claims 22 to 25 wherein the exit region of the near confocal optical transmission means is provided by a plurality of etched sections of fibre exposing different ones or subsets of said plurality of channels and containing optical cement to divert light travelling in the corresponding one or subset of channels to corresponding photodetectors.

27. A confocal imaging system as claimed in claim 26 wherein the variable selection means comprises switching means to select output from different ones or subsets of said photodetectors.

28. A confocal imaging system as claimed in any one of claims 22 to 25 wherein the exit region is provided by opposite ends of the plurality of isolated channels forming an emission end of the fibre or fibre bundle.

29. A confocal imaging system as claimed in claim 28 wherein the variable selection means comprises focusing means to project an image of the emission end onto a region containing a variable diaphragm to progressively exclude from detection said selected portion, the detector being disposed behind the diaphragm.

30. A confocal imaging system as claimed in any one of claims 1 to 29 wherein the confocal optical transmission means is integral with the near confocal transmission means.

31. A confocal imaging system as claimed in claim 30 wherein the confocal optical transmission means comprises a single mode core disposed inside the near confocal optical transmission means.

32. A confocal imaging system as claimed in any one of claims 1 to 30 wherein the confocal optical transmission means is separate from the near confocal optical transmission means.