Optical waveguides – With optical coupler – Input/output coupler
Reexamination Certificate
2001-04-03
2003-05-20
Lee, John D. (Department: 2874)
Optical waveguides
With optical coupler
Input/output coupler
C385S048000, C385S902000, C359S368000
Reexamination Certificate
active
06567585
ABSTRACT:
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 &mgr;m while the Z resolution is approximately 500 &mgr;m. 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 R
Larson & Taylor PLC
Lee John D.
Optiscan PTY LTD
Song Sarah U
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