DECONVOLVING FAR-FIELD IMAGES USING SCANNED PROBE DATA
I. FIELD OF THE INVENTION The field of the invention is the combination of scanned probe microscopic
data with far field optical and other images in order to deconvolve these images
beyond the diffraction limit.
II. BACKGROUND OF THE INVENTION
Lens based far-field imaging is limited in the resolution that it can achieve by the characteristics of the lens. In general, there are problems of diffraction of the lens, problems with aberration of the lens and problems of out-of-focus radiation.
The latter, out-of-focus radiation problem is generally partially improved by the use of confocal imaging methodologies; in optics, non-linear imaging techniques are also
useful The solution of the former diffraction and aberration problems is partially
addressed by measuring the point spread function of the lens and then using computer deconvolution to remove these effects from the image. Even the latter out-
of-focus problem can be addressed without confocal or non-linear imaging by considering both the in-focus and the out-of-focus point spread function and using deconvolution routines to try and eliminate these effects. Numerous algorithms have
been devised to address these problems of computer deconvolution of far-field
imaging data, but none are completely successful and none of them have the ability to
carry the far-field image to the realm beyond the diffraction limit as defined, for example, by the Rayleigh criterion, which is approximately Vi of the wavelength of
the radiation that is being used. For visible 500 nm light this is 250 nm.
In terms of deconvolution algorithms, a powerful mathematical approach is
based on the use of constraints. For example, in deconvolving a far-field image a
good constraint would be to define with high precision the cell membrane of a cell
that is stained with a dye and is being imaged by a lens. By precisely defining the position of a cell membrane or a portion of the cell membrane, it is possible precisely
to define where the staining in the image is confined and beyond which point or
points there is no staining and its associated optical phenomenon. Such a constraint would give many deconvolution algorithms a powerful advantage. Nonetheless, even
though the idea is mathematically a powerful concept [Carrington et al, Science 268,
1483 (1995)], it is seriously limited in far-field optics by the inability to obtain a constraint that is better than the optical resolution.
III. STATE OF PRIOR ART
No one has previously attempted to incorporate near-field optical data and other scanned probe microscope data such as that which is obtained from atomic
force microscopy to the problem of providing constraints in the deconvolution of far- field optical and other far-field imaging techniques.
IV. SUMMARY OF THE INVENTION
In accordance with the present invention, a method for deconvolving far-field
optical images beyond the diffraction limit includes the use of near-field optical and other scanned probe imaging data to provide powerful and new constraints for the
deconvolution of far- field data sets. Near- field data, such as that which can be
obtained from atomic force microscopy on a region of the far-field data set in an
integrated and inter- digitate way, is used to produce resolutions beyond the
diffraction limit of the lens that is being used. In the case of non-linear optical
imaging or other microscopies, resolutions beyond that which is achievable with these microscopies can be obtained.
V. BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing, and additional objects, features and advantages of the present
invention will become apparent to those of skill in the art from the following detailed
description of a preferred embodiment thereof, taken with the accompanying drawings, in which:
Fig. 1A illustrates a first image of a model object, a second image of the object imaged by a lens having a known point spread function, a third, or sampled image recorded by a CCD device, and a fourth image of a deconvolution of the sampled
image without constraints;
Fig. IB is a graphical illustration of the deconvolution of Fig. 1 A;
Fig. 2A illustrates the same images as Fig. 1A, wherein the deconvolution is
performed with constraints obtained from near-field imaging data; and Fig. 2B is a graphical illustration of the deconvolution of Fig. 2A.
VI. DESCRIPTION OF THE INVENTION
The present invention incorporates data that has never been incorporated previously to resolve issues and problems in far-field imaging. This data comes from
near-field optical microscopy and its scanned probe cousins, such as atomic force imaging (AFM), and presents the far- field microscopist with constraints that will
dramatically improve the far-field imaging of all forms of far-field microscopy. These
improvements are available for both linear and non-linear optical microscopy and
even those microscopies that use particles rather than electromagnetic radiation. The
invention also allows for using one form of scanned probe microscopy to deconvolve
another, to thereby improve the resolution of a scanned probe microscope before this data is used in the present invention, as described below.
In accordance with the invention, it is essential to obtain near-field optical or other scanned probe imaging data in a way that is fully integrated with a far-field
data set that is to be improved. In one embodiment of this invention in which far-
field optical microscopic data is to be deconvolved, one useful approach to achieving the required full integration of the data sets is to use a charge coupled device (CCD)
to record the far-field data that corresponds to an associated scanned probe pixel.
Furthermore, it is additionally useful if the scanned probe data sets that are to be used in the deconvolution are obtained in simultaneous channels. This can be done,
for example, with a tip that is multifunctional such as a tip that is both a
subwavelength light source and an AFM sensor [K. Lieberman, et al., Rev. Sci. Instr.
67, 3567 (1996)] that can be used in contact or near-contact with a surface of a
specimen that is being imaged.
However, even before the far and near-field imaging process is begun, lens
images of a subwavelength light source of known dimension are recorded on a CCD.
These images, both in-focus and out-of-focus, are used to obtain a measure of the lens
point spread function (PSF), even with perturbation of the object, or sample, being
imaged. This PSF is the lens function that convolutes with the functional
representation of a sample to give the blurred image that is the far-field image with
its associated diffraction and other problems mentioned in the Background section,
above. Alternately, the convolution effect of the lens can also be determined if a known high resolution sample is imaged and the error between the real and the ideal
image is represented as a blurring function introduced by the lens. Obviously, if the
imaging task is fluorescence then the high resolution test object will have to be similarly fluorescent. Thus, the first task in this method is to determine the PSF of the lens that is to be used to image the desired object, or sample.
The next step is to record with the CCD the far-field image of the object.
Subsequently, super-resolution optical data is recorded for specified points on the object surface. An example of such data can include defining an exact point at which
the optical contrast in an object terminates; i.e., defining the edge of an object to
much better than the optical resolution if the far-field image is an optical image or defining the x, y and z point, or voxel, at which there is a contrast change, and relating this point to another point of contrast change in the sample. The two points
of contrast change in the sample could, for example, be at different planes as defined by the lens in the far-field, and the near-field scanned probe data, obtained through a
multifunctional AFM sensor tip and simultaneously recorded, could provide not only
the x-y separation of the two points but also the Z separation at a resolution that is
better than any optical approach such as confocal microscopy.
The exemplary constraints listed above, or for that matter any constraints from
scanned probe technology, have never been used in this cross-fertilization mode with
far-field optical microscopy or, for that matter, any far-field microscopic technique. In addition, such cross-fertilization has not been used between scanned probe techniques, i.e. to use near-field optical data as a constraint at deconvolving near-field
atomic force microscopy data, or the reverse. With regard to this latter mode of deconvolution there is quite a bit of synergism between, for example, the near-field
optical and the near- field AFM since the functional dependence of the decay of the
effect, as a function of probe sample separation, is near-exponential for the near-field optical and occurs over a much shorter distance for the simultaneously recorded AFM
technique. In essence, then, the scanned probe microscopy synergism can first be used to improve the near-field scanned probe microscopy data and then that data can
be applied as a constraint to the far-field microscopy deconvolution in question. At this point, it is also important to note that the order of the procedures listed in this
section is not a critical part of the invention; any combination of the order of the
steps or partial combination of steps constitutes this invention. For example, if the
near-field optical data is not used to deconvolve to higher resolution the atomic force
microscopy data, it, and the atomic force microscopy data could still be used to
deconvolve the far-field data.
The CCD mentioned above is a most useful method to obtain the digitized
image of the far-field, but this can also be accomplished with confocal microscopy. In
the case of far-field optical microscopy it should be noted that there could be
innovative ways to record the confocal data without any confocal aperture. For
example, a film of material that produces a non-linear optical signal known as second
harmonic generation (SHG) can be used as part of this invention to record a confocal
image. In this case the light from the plane of focus is focused by the lens onto the
film, such as a plastic film of purple membrane that produces SHG [Z. Chen et al.,
Applied Optics 30, 5188 (1991)], with an intensity that is higher than from any other
plane in the sample that is being focused by the lens. A film that would produce a second harmonic signal only where there is a point of light from the plane of focus in
the sample could be used to replace the confocal pinhole. Such a film would act as a parallel filter for light from the plane of focus in the sample. This could be used
together with an appropriate filter after the film to remove the fundamental wavelength that was illuminating the sample and to pass only the SHG to the detector which could be a CCD rather than the single channel detector that is
normally part of a confocal set-up. This could be done with SHG or other non-linear optically active films.
VII. ADVANTAGES OVER PRIOR ART
Scanned probe microscopy data has not been used as a constraint in
mathematical constraint algorithms to deconvolve far-field optical images. In
addition, the use from multi functional scanned probe microscopy of one parameter,
such as near-field optical data, to deconvolve another parameter such as atomic force microscopy data has also not been applied. The advantage over prior art arises from
the increase in spatial resolution that this approach achieves.
VIII. APPLICATIONS
Methodologies for increased spatial resolution always open new doors in
science and technology, as evidenced by the revolution that was caused by the
introduction of the electron microscope.
To test the essence of this invention a calculation has been performed on a model far-field optical data set, as illustrated in Figs. 1A, IB, 2A and 2B, to which
reference is now made. In Figure 1A there are four images, one in each of four
horizontal rows. At the right-hand end of each row is an intensity legend for
comparison. Starting from the top of Fig. 1A, the first row of the figure represents a model object which has a dimension that cannot be resolved optically; for example,
an object having a dimension of about 0.2 micron. As may be seen in the first row,
the object has a first sharp light-to-dark transition point at its left-hand edge, a
second, dark-to light transition near the center, a third, light-to-dark transition to the right of the second transition, and a fourth, dark-to-light transition at the left-hand
edge of the model object. When the object is imaged by a lens with a known point spread function, each of the transition points on the object is blurred because the
dimensions are too small to be resolved, and the blurred image, which is the second
row from the top in Figure 1, results. When this image is recorded by a CCD there is
further blurring due to the pixel character of the CCD, as illustrated in the third row.
The last image, row 4 in this figure, is produced by processing the image of row 3
with a standard deconvolution algorithm without the imposition of the type of
constraints that are central to this invention, in which a new approach to provide constraints is described. Figure IB is a chart illustrating the intensity variations
produced by the model object and by the restored (deconvoluted) object.
In Figure 2A the same object, lens, and CCD are used, and rows 1-4 illustrate the same object and images as described above with respect to Fig, 1A, except that in
the deconvolution algorithm four points are given the resolution of near-field optics.
These points are, in going from left to right in the model object illustrated in the top row of Fig. 2A, the first, second, third and fourth alterations in contrast, as described
above with respect to Fig. 1A. The results of using such constraints is seen in the vastly improved quality of the deconvolved image in the bottom row of that Figure.
Although the invention has been described in terms of preferred embodiments, it will be understood that numerous variations and modifications may be made
without departing from the true spirit and scope thereof as set forth in the following claims.