Abstract:
A method and apparatus for image sharpening in confocal microscopy or endoscopy observation, the method comprising: collecting true confocal return light from an observational field of an object; focussing the true confocal return light into a core of a fiber wave-guide; collecting near confocal return light from a volume partially overlapping the observational field; focussing the near confocal return light so as to be transmitted principally in a cladding of the fiber wave-guide; separately detecting the true confocal return light and the near confocal return light to produce a true confocal output signal and a near confocal output signal; and adjusting the true confocal output signal on the basis of the near confocal output signal to substantially eliminate from the true confocal output signal a component due to the near confocal output signal; whereby the effective volume of the observational field is reduced and the resolution is effectively increased.

Description:
FIELD OF THE INVENTION 
     The present invention relates to an apparatus and method for performing image sharpening in confocal microscopy, of—in particular—the Z axis of confocal data sets in real time with a single scan. The present invention is of particular but by no means exclusive application in increasing the density of information storage of optical date storage devices, particularly of three dimensional digital data store devices. 
     BACKGROUND OF THE INVENTION 
     In confocal microscopy it is generally desirable to minimise the thickness of the focal plane section. This is achieved by reducing the return pinhole to the smallest size which will give a reasonable signal. 
     With a 1.4 NA oil immersion objective lens, the XY resolution is approximately 200 μm while the Z resolution is approximately 500 μm. This means that the voxels or boxels making up the image have a long axis that is 2.5 times the two orthogonal voxel dimensions. This is true for all laser scanning confocal microscopes (LSCMs) and affects all 3D reconstructions. 
     This ratio is greater for lower NA lenses and the result has a deleterious effect in 3D reconstructions. Rotations of images show a lack of resolution in the Z direction and perhaps more seriously, an artefact in which give a perception an anisotropy in views of tissues which include a Z dimension. 
     Image processing software can be used to improve the image. For example, in a first existing technique, Z sharpness is increased by concentrating on a voxel and then deconvolving it to a sharper value by subtracting from it a small proportion of the value of the voxel above it and below it. 
     A second existing technique utilises a similar principle in conjunction with XY sharpening algorithms. This is actually marketed as a synthetic aperture confocal system which can deconvolve sharp pictures from successive depth blurred low contrast brightfield images. However, it has been suggested that confocal data sets would also benefit from this approach. More sophisticated correction takes into account the brightness of pixels two levels above and below the focal plane being Z sharpened. 
     These are in effect a digital versions of unsharp masking techniques by means of which a correction is provided for the brightness of each individual voxel, which takes into account the brightness and ‘spillover’ addition of light from voxels above and below. The successful use of the aforementioned methods also depends on the operator having a fairly good understanding of the nature of the sample, the lens characteristics, the pixel sampling interval, the distance between successive image planes and other factors and entering these into the variables and chart of the algorithm. 
     A third existing technique that effectively achieves an identical Z sharpening result involves carrying out two separate scans of each plane, one scan being with the pinhole stopped right down and a second scan with the pinhole opened to about double the XY resolution optimum size. The second scan includes light from fluorescence from objects in the adjacent planes above and below and gives an analog sum of light intensities which can be used to obtain a correction factor equivalent to the digital correction algorithm used in the technique described above (in which one concentrates on a voxel and then deconvolves it to a sharper value by subtracting from it a small proportion of the value of the voxel above it and below it). 
     However, the above methods are time consuming and require a knowledge of the lens characteristics and sampling intervals. They require more than one scan to be made together with post acquisition processing. The software deconvolution (which is effectively digital unsharp masking) requires 3 or 5 scan depths to obtain corrections for 1 and 2 planes above and below the plane to be sharpened and, in some techniques, 2 or 3 scans with 2 or 3 different pinhole sizes. 
     Similarly, many methods have been proposed for high density digital storage using optically addressable elements within the three dimensional structure. Typical of these is the work by Rentzepis and by Min Gu. Previously proposed methods use confocal techniques to address the individual bit storage elements. The resolution in XY and Z of these methods has pretty thoroughly been established by Sheppard, Gu and others. 
     FIG. 1 illustrates the formation of a Gaussian Waist  10  when a TEM 00  beam  12  comprising a set of plane parallel wavefronts  13  from a laser  14  passes through a beamsplitter  16  (in which the first reflection is omitted for clarity) and objective lens  18 . The lens  18  produces a convergent concentric wave front  20 . If the Gaussian Waist  10  is focussed in a uniform fluorescent medium (not shown) then the points of re-emission of light which will return more than a given percentage of the excitation light energy through the return pinhole  22 , after reflection and re-direction by beamsplitter  16  and focussing by lens  23 , will constitute a volume  24  which is roughly football or elliptically shaped, symmetrically located in the waist  10 . This elliptical volume  24  could be termed an isofluorescence boundary for confocal pinhole return. In fact for a perfect lens the ‘football’ has two haloes above and below it (not shown). These do not affect the discussion and have been omitted for clarity. The 1/e 2  Gaussian profile is also indicated in this figure, as is the region  28  shown in subsequent figures and encompassing the Gaussian Waist  10  and environs. 
     Clearly the principle of unsharp masking involves the subtraction of return light from just above and just below the pixel to be sharpened in which the ‘overlap’ return light is taken away from the central pixel. 
     Two such prior art techniques (such as those employed in the first and second existing techniques discussed above respectively) are illustrated in FIGS. 2A and 2B, in which all the boxels are the same size. The pinhole is not altered but the ‘overlap’ required for the unsharp masking is obtained from the pixels in the scans on either side. FIG. 2A illustrates a prior art digital image sharpening technique using three scans at three separate levels within a specimen. In FIG. 2A, the plane to be sharpened is indicated at  30 , and cross sections of the Gaussian Waist and confocal volume (or isofluorescence intensity voxel perimeter) for each of three scans are shown at  32 ,  34  and  36 ; the Gaussian Waist and confocal volume are respectively on, above and below the desired focal plane. In the sharpening procedure (see schematic representation at  38 ), a portion of both dotted volumes  40  and  42  (corresponding to the confocal volumes of the second and third scans  34  and  36 ) are removed from the central volume  44  (corresponding to the confocal volume of the first scan  32 ), leaving a sharpened voxel  46 . 
     In the prior art technique illustrated in FIG. 2B, the central voxel  50  is sharpened by removing a portion of a 3×3 voxel matrix  52  from above and another 3×3 voxel matrix  54  from below the desired focal plane. The schematic image of FIG. 2B is shown undersampled from the Nyquist point of view to increase clarity. 
     FIG. 3 illustrates the traditional unsharp masking of the third existing technique discussed above, in which—after a first scan  60  is made with the pinhole stopped down—a second scan  62  is made with the pinhole opened but at the same focal plane. Next the pixel values for the image produced in the second scan  62  are subtracted—where an overlap exists—from the image produced in the first scan  60  (with the pinhole stopped down); the resulting difference signal contains the ‘overlap’ information  64  and is used to correct each of the pixels to be sharpened to produce the sharpened voxel  66 . 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention, therefore, to provide a method that avoids the necessity for multiple scanning and post acquisition processing. 
     It is another object of the present invention to provide a method and apparatus for reducing the Bit Error Rate (BER) of reading and of increasing the storage capacity (typically measured in gigabits per cubic millimeter) of a data storage material. 
     In a first broad aspect, therefore, the present invention provides a method of image sharpening in a confocal microscopy or endoscopy observation, comprising: 
     collecting true confocal return light emanating from an observational field of an object; 
     focussing said true confocal return light into a core of a fiber wave-guide; 
     collecting near confocal return light from a volume partially overlapping said observational field and thereby defining an overlap volume; 
     focussing said near confocal return light into said fiber wave-guide so as to be transmitted principally in a cladding of said fiber wave-guide; 
     separately detecting said true confocal return light and said near confocal return light to produce a true confocal output signal and a near confocal output signal; and 
     adjusting said true confocal output signal on the basis of said near confocal output signal to substantially eliminate from said true confocal output signal a component due to said near confocal output signal; 
     whereby the effective volume of said observational field is reduced and the resolution of said observation is effectively increased. 
     Preferably said overlap volume is in the Z axis of said observational field. 
     Preferably said true confocal return light and said near confocal return light are collected and focussed by means of a light condenser. More preferably said light condenser comprises a lens or a compound lens. 
     In one embodiment, adjusting said true confocal output signal comprises subtracting said near confocal output signal from said true confocal output signal. 
     This may constitute an over-correction, but the component of the near confocal signal due to light from other than the overlap volume will be small compared to the component of the near confocal signal due to light from the overlap volume, so the adjustment of the true confocal output signal will nevertheless improve, overall, the resolution of the observation. 
     More preferably said method includes absorbing or otherwise excluding higher angle rays from said near confocal return light, whereby said near confocal return light comprises principally light from said overlap volume. 
     Preferably said method includes excluding higher angle rays from said near confocal return light by transmitting said near confocal return light through a region of said fiber provided with an outer cladding with a refractive index such that said higher angle rays are transmitted into said outer cladding while lower angle rays of said near confocal light are internally reflected and thereby retained in a glass inner cladding of said fiber. 
     Preferably said method includes absorbing light transmitted within said outer cladding. 
     In one embodiment, said fiber is a single moded optic fiber with a glass inner cladding and an outer cladding having a low refractive index such that modes of said near confocal return light in said glass cladding are normally internally reflected by said outer cladding, wherein said method includes cooling said outer cladding within a region of said fiber so that within said region said higher angle rays are transmitted into said outer cladding. More preferably said outer cladding comprises silicone rubber. 
     Preferably said outer cladding is surrounded at least partially within said region with an optically absorbing medium. 
     Preferably said cooling is by means of a Peltier effect cooler. 
     In one embodiment, said object is a data storage medium. 
     In a second broad aspect, the present invention provides an image sharpening apparatus for use in making a confocal microscopy or endoscopy observation, comprising: 
     a light condenser for collecting true confocal return light emanating from an observational field of an object, for focussing said true confocal return light into a core of a fiber wave-guide, for collecting near confocal return light from a volume partially overlapping said observational field and thereby defining an overlap volume, and for focussing said near confocal return light into said fiber wave-guide so as to be transmitted principally in a cladding of said fiber wave-guide; 
     detection means for detecting said true confocal return light and said near confocal return light, and to produce respectively a true confocal output signal and a near confocal output signal; and 
     signal processing means for adjusting said true confocal output signal on the basis of said near confocal output signal to substantially eliminate from said true confocal output signal a component due to said near confocal output signal; 
     whereby the effective volume of said observational field is reduced and the resolution of said observation is effectively increased. 
     Preferably said overlap volume is in the Z axis of said observational field. 
     Preferably said light condenser comprises a first light condenser and a second light condenser, wherein said first light condenser is arranged to collect and focus said true confocal return light and a second light condenser is arranged to collect and focus said near confocal return light. 
     More preferably said light condenser comprises a lens or a compound lens. 
     Preferably said detection means comprises a first detector and a second detector, wherein said first detector is arranged to detect said true confocal return light and said second detector is arranged to detect said near confocal return light. 
     In one embodiment, said signal processing means is operable to adjust said true confocal output signal by subtracting said near confocal output signal from said true confocal output signal. 
     Preferably said apparatus includes absorption means for extracting and absorbing higher angle rays from said near confocal return light, whereby said near confocal return light comprises principally light from said overlap volume. 
     Preferably said fiber has an glass inner cladding and a region provided with an outer cladding with a refractive index such that within said region higher angle rays of said near confocal return light are transmitted into said outer cladding while lower angle rays of said near confocal light are internally reflected and thereby retained in said glass cladding. 
     In one embodiment, said fiber is a single moded optic fiber with a glass inner cladding and an outer cladding having a low refractive index such that modes of said near confocal return light in said glass cladding are normally internally reflected by said outer cladding, wherein said apparatus includes means for increasing said refractive index of said outer cladding within a region of said fiber so that within said region said higher angle rays are transmitted into said outer cladding. More preferably said outer cladding comprises silicone rubber. 
     Preferably said outer cladding is surrounded at least partially within said region with an optically absorbing medium. 
     Preferably said means for increasing said refractive index of said outer cladding within a region comprises a cooling means, and more preferably a Peltier effect cooler. 
     Preferably said apparatus includes optical path varying means for varying the optical path of said true and near confocal return light to compensate for variations in said optical path due to changes in the depth of said observational field within said object, said optical path varying means having regions of greater and lesser optical path, whereby said optical path varying means can be located with a region of lesser optical path in said optical path when said observational field is deep within said object and with a region of greater optical path in said optical path when said observational field is less deep within said object. 
     Preferably said optical path varying means comprises an optical wedge. 
     In a third broad aspect, the present invention provides a data reading apparatus for reading data from a data storage medium, including the image sharpening apparatus described above. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     In order that the present invention may be more clearly ascertained, a preferred embodiment will now be described, by way of example, with reference to the accompanying drawing, in which: 
     FIG. 1 illustrates the formation of a Gaussian Waist; 
     FIG. 2A is a schematic view of a prior art Z sharpening technique in which each voxel is sharpened by subtracting from it a small proportion of the value of the voxel above it and below it; 
     FIG. 2B is a schematic view of a prior art Z sharpening technique similar to that shown in FIG. 2B, but in conjunction with XY sharpening algorithms; 
     FIG. 3 is a schematic view of a prior art Z sharpening technique that employs a traditional unsharp masking technique; 
     FIG. 4 is a schematic view illustrating a preferred embodiment of the present invention; 
     FIG. 5 illustrates the relative locations of the Gaussian Waist, the core and near confocal fluorescences, and the sharpened voxel according to a preferred embodiment of the present invention;. 
     FIG. 6 is a schematic view of a Z sharpening confocal microscope according to a preferred embodiment of the present invention; 
     FIG. 7 is a schematic view of a Z sharpening confocal microscope according to a further preferred embodiment of the present invention; 
     FIG. 8 is a schematic view of a confocal microscope without Z sharpening, modified from the apparatus of FIGS. 6 or  7 ; 
     FIG. 9 is a schematic representation of a data reading apparatus according to another preferred embodiment of the present invention; 
     FIG. 10 is a schematic representation of another data reading apparatus according to a further preferred embodiment of the present invention; and 
     FIG. 11 is a schematic view of an apparatus for use with the apparatus of FIG. 9 or FIG. 10, for compensating for spherical aberration on focussing into an optical data storage medium. 
    
    
     DETAILED DESCRIPTION 
     Referring to FIG. 4, in a Z sharpening method according to a preferred embodiment of the present invention, the near confocal fluorescence or reflection is conveyed back simultaneously via separate coded channels with the one fiber to photodetectors and the intensity converted to an electric signal. The voltage produced from these other channels can be convolved with the confocal return channel voltage and an on-line single scan sharpened image can be generated. FIG. 4 illustrates the volumes around the Gaussian Waist from which confocal and near confocal fluorescence originates and how the overlap can be used for voxel sharpening. In this figure are shown the Gaussian Waist  70 , the fluorescence  72  that goes back into the core of the fiber (not shown), the fluorescence  74   a,b  that goes into the near confocal channel, and the sharpened voxel  76  (i.e. where there is no overlap between the ‘true’ confocal fluorescence  72  and near confocal fluorescence  74   a,b ). 
     FIG. 5 is a schematic view of an apparatus according to an embodiment of the present invention (though for simplicity using a long path variable pinhole). In this embodiment the light  80  from laser  82  passes through beamsplitter  84  and is focussed by lens  86  into the core  98  at the tip  88  of a silicone rubber clad single moded optic fiber  90 . The cladding modes in the glass cladding  92  are internally reflected by the low refractive index silicone rubber  94  but are absorbed by cladding mode stripper  96 . The light is transmitted along the core  98  of the fiber  90  to the end  100  of the fiber  90 , from which it emerges as a beam  102  of low NA which is reflected by lens  104  to a beam  106  which is intercepted by lens  108  and focussed through a coverslip  110  to a spot (or Gaussian Waist)  112  within a specimen in the form of a cell  114  on a slide  116 . The light re-emanating from “focal volume” of the Gaussian Waist  112  returning through lens  108  retraverses the same optical path as the excitation light  106 . It is reconverged by lens  104  to enter back into the core  98  of the optic fiber  90  at the end  100  and retraverses the core  98  to the other end  88  at which point it remerges and is collimated by lens  86 . 
     Passing to the beamsplitter  84  the beam is reflected by the dichroic or semireflective layer  118  to emerge as a beam  120  which impinges on photomultiplier  122 . The electrical signal from this photomultiplier tube  122  provides the ‘true’ confocal data set bitmap information. Fluorescence which is generated above  124  or below the focal plane arrives back at lens  104  with a degree of lateral displacement  126  which is proportional to the distance of the light returning body from the focal plane (Gaussian Waist  112 ). Because of this lateral displacement it is converged to the core  98  at the tip  100  of the fiber  90  at angles which are unable to be carried as bound modes within the core  98 . The light is therefore carried as cladding mode rays  128  and  130 . 
     Ray  128  came from a plane which was closer to the Gaussian Waist  112  than ray  130 . Ray  128  is therefore carried as a cladding mode of lower angle than ray  130 . A Peltier effect cooler  132  reduces the temperature of an optically absorbing medium  134  surrounding the fiber  90  and also cools the silicone rubber and the glass in that vicinity  136 . The silicone rubber has its refractive index increased by the cooling (relative to the silica) and the critical angle at the interface  138  will no longer guide the higher angle ray. Ray  130  therefore passes through the silicone rubber and is absorbed at  140 . Ray  128  which is carried by the fiber  90  at a shallower glancing angle passes through area  136  and proceeds to area  142  where the fiber  90  is cooled by a second Peltier effect device  144 , operated at a temperature that is lower than that of the first Peltier device  132 . 
     This then allows the light to escape from the glass into the silicone rubber at  146  where it can proceed into a second fiber  148 . The light then proceeds along this fiber to the tip  150  from which it emerges and impinges on the photosensitive surface of a photomultiplier tube  152 . It is desirable that all cladding mode light is extracted and directed to this photomultiplier tube  152 . The electrical output from photomultiplier tube  152  quantifies the intensity of this light. The two temperatures of the Peltier effect devices  132  and  144  can be varied to control the mode fraction which passes to photomultiplier tube  152 . 
     FIG. 6 illustrates a variation of the embodiment of FIG. 5, in which two photomultipliers  160  and  162  are used in conjunction with three Peltier Coolers  164 ,  166  and  168 . The temperature of the Peltier coolers is T(P 164 )&lt;T(P 166 )&lt;T(P 168 ). The principle of operation is similar to the embodiment of FIG. 5 except that light from two separate successive planes above and below the focal plane is sampled by the two photomultiplier tubes  160  and  162 , as follows. 
     FIG. 7 indicates the areas (representing volumes) within the Gaussian Waist  170  from which the sets of modes going to the photomultiplier tubes  160  and  162  of the embodiment of FIG. 6 are derived. Photomultiplier tube  160  derives light from the areas  174   a,b  corresponding to the lowest order modes, while photomultiplier tube  162  derives light from the areas  174   a,b  corresponding to the next set of modes. In carrying out the analog computation a portion of the output from photomultiplier tube  160  is subtracted from the ‘true’ confocal signal from area  176  (from photomultiplier tube  122 ), which effectively removes the signal from the overlap areas  178   a,b  and thereby reduces the depth of field of the image obtained. A smaller fraction of the output of photomultiplier tube  162  is added to the ‘true’ confocal signal from area  176 , to sharpen the overlap areas  180   a,b . The temperatures of the three Peltier effect coolers  164 ,  166  and  168  are optimized to give a best sharpening effect. 
     FIG. 8 illustrates the change which would be made to convert the system into a conventional variable pinhole LSCM. The cladding mode coupler fiber  182  is relocated from photomultiplier tube  160  and its output is allowed to fall on photomultiplier tube  122 . Electrical output addition of the two photomultiplier signals is also possible. 
     The above approach can also be employed to provide a method and apparatus by again utilising the return light from the regions which are slightly out of focus of a reading lens. According to the method of this embodiment, a part of the signal from these slightly out of focus regions is convolved or subtracted from the signal which has been generated by the confocal return light. The separation of the “near confocal” return light from the “true” confocal return is achieved using modes in an optical fiber. 
     FIG. 9 is a schematic representation of a data reading apparatus according to this embodiment of the invention, in which laser  210  generates laser beam  212 , which is focussed by lens  214  into single mode optic fiber  216 . The light  212  exits fiber  216 , is collimated by lens  218  and directed into beam splitter  220 . One portion of the light  212  is focussed by lens  222  onto digital data storage medium  224 ; the interrogated spot lies at focal point  226 . 
     Return light from a digital data storage medium  224  is directed by beam splitter  220  to mirror  228 , which directs the light through beam compressor  230  (including lenses  232   a  and  232   a ). Both true confocal rays  234  and near confocal rays  236  emerging from the beam compressor  230  are focussed by lens  238  into silica multimode fiber  242 , encased in low refractive index silicone cladding  240 . Towards the end of fiber  242 , higher (relative to cladding  240 ) refractive index cladding  244  strips out higher order modes; the remainder of the light (confocal return light  246 ) is detected by photodetector  248 , which the higher order modes (near confocal return light  250 ) is detected by photodetector  252 . 
     Note that this method can operate in reflected light or in fluorescence mode, and in single photon (linear) mode or in non linear (multiphoton molecular fluorescence) mode. The method can be used with the one fiber delivering the laser light and returning the signal or with a separate fiber returning the signal (see FIG.  9 ). It can be made to operated in reflection (the preferred mode, as depicted in FIG. 9) or in transmission. 
     FIG. 10 depicts an alternative embodiment, which can be used to obtain “unsharp masking” data density improvement. In this figure, laser light from blue laser diode  260  is collimated by lens  262  into beam splitter  264 . A portion of the light is focussed by lens  266  onto data storage medium  268  (as above). Return light is directed by beam splitter  264  through beam compressor  270 ; then, both true confocal light  272  and near confocal light  274  are focussed by lens  276  into silica fiber  278 . Again, confocal return light exits fiber  278  and is detected by photodetector  282 , while higher modes are stripped out by higher refractive index cladding  280  and detected by photodetector  284 . In this way, “unsharp masking” data density improvement can be obtained by subtracting a part of the output of photodetector  284  from the output of photodetector  282 . 
     FIG. 11 depicts an apparatus for use with the apparatuses of FIGS. 9 and 10, for compensating for spherical aberration on focussing into the optical data storage medium or material. In this figure, light from optical data storage medium  290  passes through glass optical wedge  292 ; the optical data is then “read” by lens  294 . As the focussed “reading” spot  296  moves deeper into the medium  290 , the wedge  292  is moved to the left (in this view) so that a thinner portion of wedge  292  is in the beam path, thereby keeping the optical path effectively constant. 
     Modifications within the spirit and scope of the invention may readily be effected by persons skilled in the art, so it is to be understood that this invention is not limited to the particular embodiments described by way of example hereinabove. For example, it should particularly be noted that any reference to microscopy or to endoscopy is intended also to refer to endomicroscopy.