WO1986007148A1

WO1986007148A1 - Differential imaging device

Info

Publication number: WO1986007148A1
Application number: PCT/US1986/001096
Authority: WO
Inventors: Marcos Francisco Maestre; Ignacio Tinoco, Jr.
Original assignee: The Regents Of The University Of California
Priority date: 1985-05-20
Filing date: 1986-05-19
Publication date: 1986-12-04
Also published as: EP0227739A4; JPS62503055A; EP0227739A1

Abstract

An apparatus and method for forming an image based on the interactions of polarized electromagnetic radiation with the specimen being imaged. The image formed by the apparatus is the difference of two images of the specimen (18, 40, 86) each made with electromagnetic radiation having a different polarization. The choice of wave length and polarization will depend on the structure of compounds to be accentuated in the image. The present invention may be operated in any of four modes selected by choosing the geometric relationship between the image forming means (28, 46, 88) and the polarized electromagnetic radiation source (30, 44, 82), the wavelength of polarized electromagnetic radiation produced by the polarized electromagnetic radiation source (30, 44, 82) relative to the wavelength detected by the image forming means (28, 46, 88), and the timing of the detection of the electromagnetic radiation relative to the timing of the emission of the polarized electromagnetic radiation by the polarized electromagnetic radiation source (30, 44, 82).

Description

PIFFERENTIAL IMAGING DEVICE BACKGROUND OF THE INVENTION The present invention relates generally to the field of imaging_r and more specifically, to imaging techniques for displaying details of a structure which result from the orientation of the specific structure or the components which make-up that structure.

The Government has rights in this invention pursuant to Contracts No. DE-ACO3-76SF00098 and DE-ATO3-82-OR60090 awarded by the ϋ. S. Department of Energy as well as pursuant to grants Al 08427 and GM. 10840 awarded by the National Institutes of Health.

An image may be defined to be a mapping of the spatial distribution of some property of the specimen being. imaged into another spatial distribution of that property or of a different property. For example, a typical black and white photographic negative of an object is a mapping of the spatial distribution of the intensity of light leaving each point on the object. The mapping process consists of setting the density of silver grains at each point in the negative in proportion to the intensity of light leaving the corresponding point on the specimen.

Imaging systems based on the mapping of a number of different physical properties of a specimen are well known to the art. For example, an x-ray image is a mapping of the ability of each point in the imaged part of the specimen to absorb electromagnetic radiation in the x-ray frequency range. A simple N R image is a mapping of the hydrogen nucleus density at each point in a plane intersecting the patient,'s body.

Significant progress has been made in recent years in biological imaging systems. For example, specific stains have been developed for forming images based on the concentration of specific proteins in the specimen being imaged. A stain is constructed using a monoclonal antibody to which a molecule which has ^"a specific absorption band is attached. An image is then formed by measuring the absorption of light in the band in question. Although such staining systems provide greatly improved images in those cases in which they are applicable, they typically only provide information about the concentration of the particular protein to which the antibody binds. No information is provided about the orientation of the protein in question or its organization into larger structures unless the organization alters the density of protein molecules significantly. Such staining systems also may produce artifacts resulting from the disruption of the specimen needed to introduce the staining agent. Finally, a specific stain must be made for each imaging problem which involves considerable expense and time.

There are many cases in which the concentration of a particular protein is the same in two specimens but the organization of the protein molecules into larger structures is different. Proteins are often organized into larger structures. Some of these structures are "bundles" of protein molecules in which the individual molecules are aligned with respect to each other. Different specimens may contain different organizations. Such organizational details are difficult to detect using even the highly specific staining systems based on monoclonal antibodies.

In a large number of imaging problems, one does not know in advance the best parameter to use to construct an image. In these cases, one typically has two specimens, one normal and one abnormal, and one wishes to construct an image which distinguishes the normal specimen from the abnormal one. If the two specimens differed in the concentration of some known compound, an imaging system based on a stain might be possible. However, in general, no such compound is known in advance and, hence one is left to try a number of different imaging modalities in an attempt to find one that distinguishes the two specimens. This will be especially difficult if the two specimens differ mainly in the organization of some compound rather than in its concentration.

Imaging systems which display organizational differences based on polarized light are known to the prior art. For example. Smart, (U. S. Patent No. 3,525,803) teaches an imaging system in which the specimen is illuminated with plane polarized light and the image is viewed with and without a polarization filter. The polarized light is composed of two components. The polarization filter selects one of these components for viewing. A structure in the image which is composed of molecules which are bundled with a specific orientation, will absorb one component of the polarized light in preference to the other of said components. Hence in principle, the image produced without the polarization filter will be different from that produced with the polarization filter if such structures are present. Unfortunately, in practice this difference is often masked by other processes.

The plane polarized light used in the apparatus of Smart is composed of two components, a component with polarization in one direction, V, and a component, H, with polarization in a second direction at right angles to the first component. The image measured without the polarization filter is the sum of the absorbances of the V and H components which are transmitted by the specimen. The image measure with the polarization filter set to select the V component is the the V component which was transmitted by the specimen plus the V component of the light which resulted from the scattering of the H component. Since the scattering of the H component gives rise to light having a mixture of both the V and H polarizations, if this scattering is large, it may mask the differences in absorption of the V component which resulted from the oriented structure. This problem arises from attempting to measure the difference in absorbance for the two different polarization components by illuminating with a mixture of the two components and then sorting the light transmitted by the specimen using a filter.

A further problem with the type of apparatus exemplified by the Smart patent is that the apparatus is limited to polarized electromagnetic radiation in the frequence range for which an appropriate polarization filter can be constructed. For polarized electromagnetic radiation of very high energies, i.e., gamma-rays, this is not practical.

F.inally, the type of apparatus exemplified by Smart is not useful for viewing chiral structures which do not occur in some form of ordered larger structure. A chiral structure is one whose mirror image is different from the structure. For example, helical molecules are either left or right handed depending on the twist of the helix. These molecules are chiral, since the mirror image of a right handed helix is a left handed helix, not a right handed one. Such molecules preferentially absorb circularly polarized electromagnetic radiation, but do not preferentially absorb linearly polarized electromagnetic radiation unless they are organized into some larger structure which preferentially absorbs linearly polarized electromagnetic radiation as described above.

Accordingly, it is an object of the present invention to provide a means for producing an image of a specimen which accentuates ordered structures in said specimen. It is a further object of the present invention to provide a means of imaging a specimen based on the presence of chiral molecules.

It is a still further object of the present invention to provide a means of imaging a specimen which does not require that the specimen be treated with a staining agent.

These and other objects of the invention will become obvious from the following detailed description of the invention and the accompanying drawings.

SUMMARY OF THE INVENTION The present invention comprises an apparatus and method for forming an image based on the interactions of polarized electromagnetic radiation with the specimen being imaged. The image formed by the apparatus of the present invention is the difference of two images of the specimen each made with electromagnetic radiation having a different polarization. The apparatus of the present invention consists of a source of polarized electromagnetic radiation which produces electromagnetic radiation of either one of two preselected polarizations and a means for forming a difference image. The intensity of each point in the difference image is equal to the difference of the intensity of electromagnetic radiation that would be measured if two images were formed using each polarization separately and the intensities of electromagnetic radiation at each point in each of these two images were subtracted from one another to produce the intensity of electromagnetic radiation represented by the corresponding point in said difference image. The polarized electromagnetic radiation source may produce electromagnetic radiation of any wave length from the low frequency radio waves to high energy gamma rays, as well as electromagnetic radiation in the visible range. The choice of wave length and polarization will depend on the structure or compounds to be accentuated in the image. The images may be constructed in either of two scanning modes. First, the specimen may be scanned sequentially at each point to be included in the image with a beam of polarized electromagnetic radiation and the resulting electromagnetic radiation which reaches a detector measured. Here, a data processing system connected to the detector records the intensity of electromagnetic radiation measured by the detector when each point on the specimen is illuminated with polarized electromagnetic radiation of one polarization and then the other polarization. Second, the difference image may be constructed by first illuminating the specimen with the polarized electromagnetic radiation of one polarization and forming an image in a plane with an imaging lens. This image is then be inputed to a data processing system together with an image similarly produced using the other polarization.

For a given scanning mode, the apparatus of the present invention may be operated in any of four modes described in detail below. These operational modes are selected by choosing the geometric relationship between the image forming means and the polarized electromagnetic radiation source, the wavelength of polarized electromagnetic radiation produced by the polarized electromagnetic radiation source relative to the wavelength detected by the image forming means, and the timing of the detection of the electromagnetic radiation relative to the timing of the emission of the polarized electromagnetic radiation by the polarized electromagnetic radiation source. Each of these modes produces a difference image in which different features of the specimen under investigation are accentuated.

The choice of which form of polarized electromagnetic radiation to be used is based on the type of structure which is to be accentuated. Ordered structures are imaged using linearly polarized electromagnetic radiation. Chiral structures are imaged using circular polarized electromagnetic radiation. DETAILED DESCRIPTION OF THE DRAWINGS FIGURE 1 is a block diagram of an apparatus according to the present invention.

FIGURE 2 is a block diagram of the preferred embodiment of the present invention.

FIGURE 3 is a block diagram of a second apparatus according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

The apparatus of the present invention constructs an image of a specimen by measuring the difference in the intensity of electromagnetic radiation leaving each point on the specimen when the specimen is illuminated with electromagnetic radiation of different polarization. The detected electromagnetic radiation may be the result of incident electromagnetic radiation which passed through the specimen without being absorbed or which was scattered by the specimen. It may also be the result of incident electromagnetic radiation which was absorbed by the specimen and then re-emitted either immediately (referred to as fluorescence) or after some time delay (referred to as phosphorescence) . The re-emitted electromagnetic radiation will, in general, be at an energy less than that of the incident electromagnetic radiation. Electromagnetic radiation is said to be polarized if the vibrations of the electric, or magnetic, field vector representing the radiation at a fixed point in its path exhibits a preference to direction or sense of vibration. Thus the electric field vector representing linearly polarized radiation vibrates parallel to a fixed direction. Electromagnetic radiation is said to be circularly polarized if the tip of the electric field vector, at a fixed point in the path of the radiation, generates a circular pattern in time. A circularly polarized wave is mathematically equivalent to two orthogonal linearly polarized waves of equal magnitude but differing in phase by a quarter wave. The sense of circular polarization, right or left, is determined by the sense of the circular pattern, clockwise or counterclockwise.

In optically active media, the interaction of polarized electromagnetic radiation depends on the direction or sense of the polarization. For example, if a structure consisting of a bundle of hemoglobin molecules, similar to a bundle of straws, is illuminated with linear polarized electromagnetic radiation in which the direction of polarization is parallel to the axis of the bundle, the electromagnetic radiation will be less strongly absorbed than it would be if the direction of polarization was orthogonal to the axis of the bundle. Similarly, if a solution of chiral molecules is illuminated with circularly polarized electromagnetic radiation, the amount of circularly polarized electromagnetic radiation absorbed by the solution will be different for left handed circularly polarized electromagnetic radiation than for right handed circularly polarized electromagnetic radiation.

Although this differential absorption has been used to characterize the structure of molecules, it has not been used for imaging. For example, if it is found that a solution of the molecule in question absorbs left handed circularly polarized electromagnetic radiation in preference to right handed circularly polarized electromagnetic radiation, or vice versa, then one can presume that the molecule in question is chiral, i.e., it differs from its mirror image. The present invention is based on the observation that a portion of a specimen which contains chiral molecules such as DNA will absorb circularly polarized electromagnetic radiation of one handedness in preference to the circularly polarized electromagnetic radiation of the other handedness. Hence an image constructed by subtracting an image of the specimen taken with right handed circularly polarized electromagnetic radiation from an image taken of the specimen using left handed circularly polarized electromagnetic radiation will be nonzero only in those regions of the specimen which contain chiral molecules. This is equivalent to a "staining" system in which chiral molecules are stained. By choosing different wavelengths, different chiral molecules can be accentuated in the image. Similarly, by subtracting images taken with two different polarizations using linearly polarized electromagnetic radiation, areas of the specimen containing linearly ordered structures may be "stained". An apparatus according to the present invention is shown at 10 in FIGURE 1. Broadly it consists of a means 30 for illuminating a specimen 18 with monochromatic polarized electromagnetic radiation having either one of two polarizations, a detector 28 for measuring the intensity of electromagnetic radiation leaving each of the points on the specimen which is to be included in the image, and a means for forming the difference 26 of the intensities so measured when the specimen is first illuminated with electromagnetic radiation of one of said polarizations and then illuminated with electromagnetic radiation of the other of said polarizations.

In the frequency range from the infrared to the ultraviolet, the means for illuminating the specimen consists of a monochromator 12 which provides monochromatic unpolarized electromagnetic radiation which is polarized by a polarizer 14 and a lens 16 which focuses the electromagnetic radiation leaving the polarizer onto the specimen 18. The monochromator is of conventional design. In the preferred embodiment, it is a high intensity filament based light source and an appropriate filter to remove light of undesired wave lengths. Monochromators based on lasers will be apparent to those skilled in the art.

The polarizer 14 selects the appropriate components of the electromagnetic radiation produced by the monochromator to convert the output of the monochromator to a polarized source. The output of the polarizer 14 is either linearly polarized electromagnetic radiation or circularly polarized electromagnetic radiation. If linearly polarized electromagnetic radiation is choosen, the two polarizations correspond to the electric field vector being aligned along two orthogonal directions indicated by the arrows shown at 31 and 32 respectively. If circularly polarized electromagnetic radiation is chosen, the polarized electromagnetic radiation leaving the polarizer 24 will either be left or right handed circularly polarized electromagnetic radiation.

Over the frequency range from the infrared to the ultraviolet, the polarizer 14 may be constructed using the Pockels Effect which is known to those skilled in the art. Here, a suitable crystal, for example, potassium dideuterium phosphate (KD2PO4) is subjected to an applied electric field. By choosing the angle of incidence of the light on the crystal and the applied voltage, electromagnetic radiation which is either circularly polarized or linearly polarized may be obtained. This effect is discussed in detail in "Circular Dichroism-Theory and Instrumentation", Analytic Chemistry. Vol. 38, P. 29A, June 1966, which is incorporated by reference.

Circularly polarized electromagnetic radiation at wave lengths in the radio frequency range may be produced using a helical antenna. Linearly polarized electromagnetic radiation in the radio frequency range may be generated by use of a phased array of dipole antennae. Both helical antennae and phased arrays of dipole antennae are conventional in the art. In the x-ray and gamma-ray region, both linearly polarized electromagnetic radiation and circularly polarized electromagnetic radiation may be produced from synchrotron radiation using the method of Kim which is described in detail in an article by Kwang Kim entitled "Synchrotron Radiation Source with Arbitrarily Adjustable Elliptical Polarization", Nuclear Instruments and Methods. Vol. 219, P. 425, 1984 which is hereby incorporated by reference.

The choice of wave length of polarized electromagnetic radiation to be used for illuminating the specimen will depend on the type of compound or object which is to be emphasized in the image. If chiral objects are to be emphasized, the maximum difference in absorbance and difference in scattering of the polarized electromagnetic radiation will occur at a wave length which is the same order of magnitude as the diameter of the chiral object in question. However,, satisfactory signals can be obtained with wavelengths which are a thousand to ten thousand times larger or smaller than this diameter in many cases. If one wished to scan the surface of the earth for large objects which were chiral in nature using polarized electromagnetic radiation, wave lengths in the radio frequency band would be appropriate. Images of biological specimens based on clusters of protein molecules can be produced by using wave lengths in the visible regions and linear and circular polarization.

If it is known that a particular molecular species is present in the specimen and the distribution of that species is to be accentuated, a wave length which coincides with a strong absorption band of the species in question would be appropriate. For example, an image could be constructed to accentuate chiral molecules which contain a known chemical group which absorbs electromagnetic radiation strongly at a particular wave length by setting the wave length of the source of polarized electromagnetic radiation to that wave length and using circular polarization.

If the apparatus is to be used to distinguish a "normal" sample from an "abnormal" sample, the wave length may be varied until a wave length which maximizes the difference between the two samples is found. The choice of detector 28 will depend on the frequency region of the polarized electromagnetic radiation being used. For electromagnetic radiation with frequencies between infrared and ultraviolet, this detector may be constructed from a lens 20 and a two dimensional detector 24 which measures the intensity of electromagnetic radiation at each point in a plane associated with the detector. The lens 20 focuses the electromagnetic radiation leaving the specimen onto this plane. Image detectors in which this image is first focused on a surface other than a plane will be obvious to those skilled in the art.

For electromagnetic radiation in the visible region, a conventional television camera may be used for the detector 24. Vidicon cameras with sensitivities in the infrared are also conventional in the art. For' electromagnetic radiation with frequencies above that of visible light, i.e., ultra-violet, x-rays, or gamma-rays, detectors constructed from a screen which emits light in the visible region when electromagnetic radiation in the frequency region above the visible region strikes it and a television camera are conventional in the art. Also detectors constructed from a material which emits electrons when struck by electromagnetic radiation in or above the visible region followed by an electron multiplier and an electron detector are also conventional in the art. In those energy regions for which a suitable two dimensional imaging detector 24 cannot be constructed, the apparatus may operated in the scanning mode described below. The detector 28 is coupled to an image processing system 26 which provides a means for forming the difference of the two intensities measured at each point by the detector 28. In the preferred embodiment, the image processing system 26 is a digital computer of conventional design. The image processing system 26 is coupled to the source of electromagnetic radiation 30. For each of the two polarizations, the imaging processing system records the intensities measured by the detector 28 at each point on the specimen and then forms an image of the specimen in which each point in the image is related to the difference of the measured intensities taken with the two alternative directions of polarization. In the preferred embodiment, each point in the image has an intensity proportional to the difference in intensities measured with the different directions of polarization at the corresponding point on the specimen. Other methods of representing the difference in the measured intensities as an image will be apparent to those skilled in the art. The apparatus of the present invention may be used to form images in one of 4 modes. The particular selected mode is determined by the geometric relationship of the illuminating source 30 and the detector 28, by the relationship between the frequency of electromagnetic radiation emitted by the illuminating source and the frequency of electromagnetic radiation detected by the detector, and by the temporal relationship of the emission of the electromagnetic radiation relative to the detection of electromagnetic radiation by the detector.

The embodiment shown in FIGURE 1 is intended for operation in the first of these modes. Here, the optical axis of the illuminating source 30 is the same as the optical axis of the detector 28. The detector 28 is adjusted to measure electromagnetic radiation of the same wavelength as that emitted by the illuminating source 30. The electromagnetic radiation measured by the detector 28 is essentially the electromagnetic radiation which was not absorbed or scattered by the specimen. To generate images in the other modes of operation, the optical axis of the illuminating source must be moveable relative to the optical axis of the detector, the wavelength of the electromagnetic radiation measured by the detector must be variable over a range of wavelengths, and the illuminating source must be capable of emitting electromagnetic radiation in short pulses.

A schematic diagram of an apparatus according to the present invention which is capable of making measurements in all four modes is shown at 50 in FIGURE 2. It comprises an illuminating source 44 which illuminates a specimen in a sample plane 40 with monochromatic electromagnetic radiation which consists of primarily either left or right handed circular polarized electromagnetic radiation or of primarily linearly polarized electromagnetic radiation with the direction of the polarization being parallel to one of two orthogonal directions indicated by the arrows at 54 and 55, a means such as a lens 48 for focusing the electromagnetic radiation leaving the sample plane 40 onto an image plane 47, and a detector 46 for measuring the intensity of electromagnetic radiation having a frequency in a specified frequency interval, at each point in the plane 47. The angle of illumination 42 at which the illuminating electromagnetic radiation strikes the plane 40 containing the specimen is specified by the angle of the normal to said plane indicated by the arrow 43 and the direction of emission of the electromagnetic radiation indicated by the arrow 45. The output of the detector is used as input to an image processing circuit 52 which is also coupled to the illuminating source 44. The image processing circuit 52 forms an image of the specimen by taking the difference of the intensities measured at each point in the plane 47 when electromagnetic radiation of different polarizations is used to illuminate the specimen. A digital computer is preferred for the image processing circuit 52.

For electromagnetic radiation in the energy range above the infrared, a detector 46 consisting of a vidicon camera is preferred. For electromagnetic radiation energies between the infrared and the near ultraviolet, the image plane 47 is contained in the vidicon camera, i.e., the photocathode of the camera. At energies higher than the near ultraviolet (i.e., ultraviolet, x-rays, and gamma-rays) , the image plane 47 may be constructed from a material which emits light when it absorbs electromagnetic radiation. Such image planes are commonly used in fluoroscopic imaging. Screen based on zinc sulfide will also be apparent to those skilled in the fluoriscopic arts.

In the first mode of operation, referred to as the absorption mode, the angle 42 is 180 degrees and the frequency of electromagnetic radiation detected by the detector 46 is the same as the frequency of electromagnetic radiation emitted by the illuminating source 44. Here, each point in the image will reflect the difference of the absorption of electromagnetic radiation measured with the two different polarizations. Since different chemicals absorb electromagnetic radiation at different energies, by varying the wavelength of the illuminating source and the frequency sensitivity of the detector, different images based on different chemicals may be produced. -16-

In the second mode of operation, referred to as the dark field mode, the angle 42 is less than 180 degrees. This angle is set so that electromagnetic radiation from the illuminating source 44 does not reach the image plane 49 if no sample is present in the sample plane 40. The frequency of electromagnetic radiation detected by the detector 46 is again adjusted to be the same as the frequency of electromagnetic radiation emitted by the illuminating source 44. Here, the electromagnetic radiation from the illuminating source 44 is scattered by the specimen in the sample plane 40. Some of the scattered electromagnetic radiation is imaged onto the image plane by the imaging means 48. If the angle 42 is small, the detector will measure "back-scattered" electromagnetic radiation. Such backscattering emphasizes objects which are large compared to the wave length of the polarized electromagnetic radiation emitted by the illuminating source 44. This mode is also useful for forming an image of the surface of an opaque object in which chiral or ordered structures are to be accentuated.

In the third mode of operation, referred to as the fluorescent mode, the frequency of electromagnetic radiation detected by the detector 46 is adjusted to be less than the frequency of electromagnetic radiation emitted by the illuminating source 44. The angle of illumination 42 may be less than or equal to 180 degrees. In the preferred embodiment, this angle is set such that no electromagnetic radiation reaches the detector 46 when no specimen is present in the sample plane 40. In this mode, the detector 46 detects electromagnetic radiation which is emitted as the result of the absorption of the polarized electromagnetic radiation used to illuminate the specimen followed by the immediate re-emission of this electromagnetic radiation at lower frequencies. The frequencies at which the electromagnetic radiation is absorbed and the frequencies at which it is re-emitted will depend on the chemical composition of the specimen. By adjusting the frequencies at which the detector 46 measures electromagnetic radiation, images based on different chemicals may be made. Similarly, the frequencies at which electromagnetic radiation is absorbed will depend on the chemical composition of the specimen. Different chemical groups have different known absorption bands. Hence this mode is useful for accentuating ordered structures or chiral objects which contain a chemical group which may be excited by the incident electromagnetic radiation to fluoresce. In prior art fluorescent imaging, the image is made using unpolarized light and accentuates the chemical group which emits the fluorescent electromagnetic radiation. In the apparatus of the present invention, only those fluorescent groups which are bound to a chiral or a linearly ordered structure will be emphasized. Hence this mode of operation of the apparatus of the present invention provides much greater specificity than the prior art fluorescent based imaging systems.

In the fourth mode of operation, referred to as the phosphorescent or time-resolved fluorescent mode, the frequency of the electromagnetic radiation detected and the angle of illumination are adjusted as in the case of the fluorescent mode; however, only electromagnetic radiation which is emitted from the specimen having a predetermined temporal relationship with respect to the illuminating electromagnetic radiation is measured by the detector 46. In this mode, the illuminating source 44 emits electromagnetic radiation in short pulses and the detector 46 only measures the intensity of electromagnetic radiation in the image plane 49 during the time periods in which said illuminating source 44 is not emitting electromagnetic radiation. Many chemicals of interest absorb electromagnetic radiation and then emit electromagnetic radiation at one or more lower frequencies at a later time. As in the fluorescent mode, which chemicals form the basis of the image may be determined by setting the frequency range over which the detector 46 measures electromagnetic radiation intensities. This mode provides another means of forming images based on linearly ordered or chiral objects which contain a chemical group which has a known emission spectral line, in this case a phosphorescent emission line which is excited by the incident electromagnetic radiation. This mode of operation has the advantage of being less sensitive to effects resulting from chemicals which emit electromagnetic radiation of similar frequencies in the fluorescent mode.

A second embodiment of the present invention is shown at 100 in figure 3. This embodiment is well suited to measurements at frequencies outside the visible range. At frequencies above and below the visible range, it is not always possible to construct a satisfactory detector 28 consisting of a lens and a two-dimensional detector on which an image of the specimen is formed. At short wave lengths, i.e., x-rays and gamma-rays, a pinhole lens can be used to image the electromagnetic radiation on a fluorescent screen which is viewed by a vidicon or other two dimensional detector in the visible range. However, the time required to make the measurements may be too long, because of the small aperture of the pinhole lens. Similarly, at long wave lengths, i.e., radio frequencies, it is difficult to find a satisfactory two-dimensional detector.

This embodiment differs from the one shown in FIGURE 2 in that the specimen is illuminated at only one point at a given time. In the embodiment shown in FIGURE 2, the illumination source 44 illuminates the entire specimen and the lens 48 in conjunction with the image plane 49, separates the electromagnetic radiation which originated at each point on the specimen so that the detector 46 can measure the intensity of electromagnetic radiation that left each of said points. In this second embodiment, only one point on the specimen is illuminated at a given time; hence all of the electromagnetic radiation reaching the detector is known to have originated at the point being illuminated. Thus the lens 48 and image plane 49 are not needed.

Referring to FIGURE 3, this second embodiment of the present invention consists of a source of monochromatic polarized electromagnetic radiation 82 which produces a narrow beam of polarized electromagnetic radiation 80, a means 84 for causing said narrow beam 80 to scan a specimen in a sample plane 86 in a sequential manner, and a detector 88 which measures the intensity of electromagnetic radiation leaving the specimen. The detector 88 is connected to a data processing system 90 of conventional design which controls the state of polarization of the source of polarized electromagnetic radiation 82 and the point (x,y) on the specimen at which the polarized electromagnetic radiation is directed. Since only one point on the specimen is illuminated at a given time, the lens 20 and two-dimensional detector 28 shown in FIGURE 1 may be replaced by a single detector 88 which measures the total electromagnetic radiation coming from the specimen.

The means for causing the beam 80 to scan the specimen may be an x-y drive of conventional design which causes the specimen to move in relation to the source 82. Alternatively, the source could be mounted on a platform which may be caused to move relative to the specimen. This embodiment of the apparatus of the present invention may be used in the four modes described above for the embodiment shown in FIGURE 2.

This embodiment of the apparatus of the present invention would be particularly useful for scanning the surface of the Earth from a satellite using a polarized electromagnetic radiation source in the radio frequency range. The apparatus would be operated in the dark field mode described above. If linearly polarized electromagnetic radiation is used, ordered structures such as rows of buildings on the surface of the Earth could be detected. If circularly polarized electromagnetic radiation is used, chiral structures would be detected. The source of polarized electromagnetic radiation may be constructed from an ordinary transmitter in the relevant radio frequency range driving an appropriate antenna. In the case of circularly polarized- electromagnetic radiation, a helical antenna or. phased array antenna would be used. If linearly polarized electromagnetic radiation were used, a phased array of dipole antennae would be employed. The source would scan the "specimen" on the surface of the Earth by varying the angle of the antennae by conventional mechanical means.

Narrow beam x-ray sources can be constructed from conventional x-ray sources and a collimating device such as the hole in a lead shield. These sources can be made to scan the specimen by mechanically moving the source relative to the specimen. Scanning x-ray sources which function by electrical means are also known to those skilled in the art of CT-scanning. The choice of the detector 88 will depend on the frequency range of the electromagnetic radiation being detected. In the radio frequency range, the detector would be an ordinary antenna for receiving electromagnetic radiation of the desired frequency. In the infrared to ultraviolet range, numerous detectors are known to those skilled in the art. A photomultiplier tube or solid state light detector are preferred. At frequencies above the ultraviolet, a detector consisting of scintillation counter of conventional design is preferred.

While various embodiments of the invention have been discussed herein, it will be appreciated that various changes and modifications may be made without departing from the present invention as claimed.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for forming an image of a specimen comprising: means for illuminating said specimen with polarized monochromatic electromagnetic radiation having two different preselected states of polarization; means for detecting the difference in the intensity of electromagnetic radiation leaving preselected points on said specimen when said specimen is first illuminated with electromagnetic radiation having one of said preselected states of polarization and then illuminated with electromagnetic radiation having the other of said preselected states of polarization; and means for displaying said difference of intensities as a two dimensional distribution in which each point in said two dimensional distribution corresponds to one of said preselected points.

2. The apparatus of Claim 1 wherein said illuminating means comprises a source of unpolarized monochromatic electromagnetic radiation; and polarizing means for converting unpolarized electromagnetic radiation into polarized electromagnetic radiation having one of the two possible circular polarizations.

3. The apparatus of Claim 2 wherein said source of monochromatic electromagnetic radiation includes a polychromatic light source and filtering means for removing light not within a specified range of wave lengths.

4. The apparatus of Claim 2 wherein said polarizing means includes a KD2PO4 crystal and a means for applying an electrostatic potential to said KD2PO4 crystal.

5. The apparatus of Claim 1 wherein said illumination means comprises: a source of monochromatic electromagnetic radiation; and linear polarizing means for converting unpolarized electromagnetic radiation to linearly polarized electromagnetic radiation having an electric field vector parallel to one of two preselected orthogonal directions.

6. The apparatus of Claim 5 wherein said source of monochromatic electromagnetic radiation includes a polychromatic light source and filtering means for removing light outside a specified range of wave lengths.

7. The apparatus of Claim 5 wherein said linear polarizing means includes a KD2PO4 crystal and a means for applying an electrostatic potential to said

KD2PO4 crystal.

8. The apparatus of Claim 1 wherein said detecting means comprise a lens means for imaging the electromagnetic radiation leaving said specimen onto a surface and means for detecting the intensity of electromagnetic radiation at each point on said surface.

9. The apparatus of Claim 1 wherein said illumination means are oriented such that no electromagnetic radiation from said illuminating means is detected by said detection means unless said electromagnetic radiation is first scattered by said specimen.

10. The apparatus of Claim 1 wherein said detection means detects electromagnetic radiation in a specified frequency range and the maximum frequency detected is less than the frequency of the electromagnetic radiation emitted by said illumination means.

11. The apparatus of Claim 10 wherein said illumination means further comprises means for causing the electromagnetic radiation emitted by said illumination means to be interrupted and said detection means further comprises means for detecting electromagnetic radiation only when said illumination means is not illuminating said specimen.

12. The apparatus of Claim 1 wherein said illumination means comprises: means for generating a narrow beam of monochromatic polarized electromagnetic radiation having two preselected states of polarization; and means coupled to said detecting means for causing said narrow beam to sequentially scan said specimen.

13. The apparatus of Claim 12 wherein said detecting means and said illumination means comprises antennae.

14. The apparatus of Claim 12 wherein said ^• detecting means comprises a scintillation counter.

15. The apparatus of Claim 12 wherein said detecting means comprises a means for detecting electromagnetic radiation in the visible frequency range.

16. The apparatus of Claim 12 wherein said sequential scanning means comprises means for moving said specimen relative to said narrow beam.

17. The apparatus of Claim 12 wherein said sequential scanning means comprises means for directing said narrow beam at preselected points on said specimen.

18. A method for forming an image of a specimen comprising the steps of (a) illuminating said specimen with monochromatic polarized electromagnetic radiation of a preselected frequency having a first preselected state of polarization; (b) detecting the electromagnetic radiation having a frequency in a preselected range leaving preselected points on said specimen when said specimen is so illuminated; (c) illuminating said specimen with monochromatic polarized electromagnetic radiation having a second preselected state of polarization; (d) detecting the electromagnetic radiation having a frequency in a preselected range leaving said preselected points on said specimen when said specimen is so illuminated; and (e) for each of said preselected points, subtracting the intensity of electromagnetic radiation detected in step (b) from that detected in step (d) and displaying the result of said subtraction as a two dimensional distribution.

19. The method of Claim 18 wherein said preselected range of frequencies includes the frequency of said monochromatic electromagnetic radiation.

20. The method of Claim 18 wherein said preselected range of frequencies includes only frequencies which are less than the frequency of said monochromatic electromagnetic radiation.

21. The method of Claim 18 wherein said preselected frequency is chosen to correspond to a known absorption band of a chemical group contained in said specimen.

22. A method for forming an image of a specimen comprising the steps of: (a) illuminating said specimen with monochromatic electromagnetic radiation of a preselected frequency having a first preselected state of polarization; (b) interrupting said illumination; (c) detecting the electromagnetic radiation leaving preselected points on said specimen having frequencies in a preselected range which does not include said preselected frequency while said illumination is so interrupted; (d) illuminating said specimen with monochromatic electromagnetic radiation of said preselected frequency having a second preselected state of polarization; (e) interrupting said illumination; (f) detecting the electromagnetic radiation leaving said preselected points on said specimen having frequencies in said preselected range while said illumination is so interrupted; and (g) for each of said preselected points, subtracting the intensity of electromagnetic radiation detected in step (c) from the intensity of electromagnetic radiation detected in step (f) and displaying the result of said subtraction as a two dimensional distribution.