WO2000047978A1 - Deconvolving far-field images using scanned probe data - Google Patents

Abstract

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.

Description

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.

Claims

What is claimed is:

1. A method for deconvolving far-field optical microscopic images to a level of resolution that has never been achieved previously by introducing scanned probe

microscopy data in an interdigitated, integrated fashion with confocal or charge

couple device imaging fashion such that every pixel or voxel from the super-resolution scanned probe microscopy data set is directly related to an image pixel or voxel in the

far-field data set and then using the scanned probe data as a mathematical constraint

to deconvolve the far-field image.

2. A method that uses multifunctional scanned probe microscopy data so that one scanned probe microscopy parameter can be deconvolved by integrating data from

another such as, but not excluding other combinations, deconvolving an atomic force

microscopy image with a near-field optical microscopy image or vice versa.

3. The method as in claim 1 in which the procedure in claim 2 is used in combination

with the method in claim 1.

4. A method to obtain the point spread function, by using a known high resolution

sample either fluorescent or non-fluorescent, which is imaged and the error between the real and the ideal image is represented as a blurring function introduced by the

lens and is representative of an accurate point spread function.

5. A method to obtain a digitized image of the optical far-field in addition to simple confocal or charge coupled device imaging that can be used for integrated near-field and far-field imaging that uses a film of material that can produce a non-linear optical

signal known as second harmonic generation in which light from the plane of focus of

the lens would be focused by the far-field lens onto a film, such as plastic purple membrane films, with an intensity that is higher than from any other plane in the

sample that is being focused by the lens and thus such a film could be used to replace

the confocal pinhole in confocal imaging by using a film that would light up only

where there is a point of light from the plane of focus in the sample and thus such a

film would act as a parallel filter for light from the plane of focus in the sample with

this light being imaged through a filter for the fundamental illuminating frequency onto a charge coupled device.

6. A device that collects in an integrated, correlatable fashion that as a result allows

for deconvolving far-field optical microscopic images to a level of resolution that has never been achieved previously by introducing scanned probe microscopy data in an interdigitated, integrated fashion with confocal or charge couple device imaging such

that every pixel or voxel from the super-resolution scanned probe microscopy data set

is directly related to an image pixel or voxel in the far-field data set and then using

the scanned probe data as a mathematical constraint to deconvolve the far-field image.

7. A device that collects in an appropriate integrated fashion multifunctional scanned probe microscopy data so that one scanned probe microscopy parameter can be deconvolved by integrating data from another such as, but not excluding other

combinations, deconvolving an atomic force microscopy image with a near-field optical microscopy image or vice versa.

8. A device as in claim 5 that also has the capabilities of claim 6.

9. A device to obtain the point spread function, by using a known high resolution

sample either fluorescent or non-fluorescent, which is imaged and the error between

the real and the ideal image is represented as a blurring function introduced by the lens and is representative of an accurate point spread function.

10. A device to obtain a digitized image of the optical far-field in addition to simple confocal or charge coupled device imaging that can be used for integrated near-field and far-field imaging that uses a film of material that can produce a non-linear optical

signal known as second harmonic generation in which light from the plane of focus of

the lens would be focused by the far-field lens onto a film such as plastic purple

membrane films, with an intensity that is higher than from any other plane in the

sample that is being focused by the lens and thus such a film could be used to replace the confocal pinhole in confocal imaging by using a film that would light up only

where there is a point of light from the plane of focus in the sample and thus such a

film would act as a parallel filter for light from the plane of focus in the sample with

this light being imaged through a filter for the fundamental illuminating frequency onto a charge coupled device.