Optics: measuring and testing – By light interference – Having polarization
Reexamination Certificate
2000-03-16
2002-11-12
Turner, Samuel A. (Department: 2877)
Optics: measuring and testing
By light interference
Having polarization
C356S497000
Reexamination Certificate
active
06480285
ABSTRACT:
FIELD OF THE INVENTION
This invention is related to optical and acoustical imaging, including utilizing such images to perform optical data storage and retrieval, precision measurements on biological samples, wafers, integrated circuits, optical disks, and other samples, and to perform optical biopsies.
BACKGROUND OF THE INVENTION
The invention relates to techniques for rapidly, accurately producing an in-focus image of an object, or a cross-section thereof, wherein the effect of light signals from out-of-focus foreground and/or background light sources are mostly eliminated with regard to both statistical and systematic errors. Confocal and confocal interference microscopy are finding many applications in, for example, the life sciences, the study of biological samples, industrial inspection, and semiconductor metrology. This is because of the unique three-dimensional imaging capability of these instruments.
Perhaps the most difficult multi-dimensional imaging is encountered when the background from out-of-focus images is significantly larger than the signal from the in-focus images. Such circumstances arise frequently in the study of thick samples, particularly when working in the reflection mode in contrast to the transmission mode of confocal systems.
There are two general approaches for determining the volume properties of three-dimensional microscopic specimens. Such approaches are based on conventional microscopy and confocal microscopy. Generally, the conventional microscopy approach requires less time to acquire the data but more time to process the data for a three-dimensional image, compared to the confocal microscopy approach.
In a conventional imaging system, when a part of the object to be imaged is axially displaced from its best focus location, the image contrast decreases but the brightness remains constant so that displaced, unfocused parts of the image interfere with the view of focused parts of object.
If the system's point-spread function is known and images are obtained for each independent section of the object, known computer algorithms can be applied to such images to effectively remove the signal contributed by the out-of-focus light and produce images that contain only in-focus data. Such algorithms are of several distinct types, are referred to as “computer deconvolutions,” and generally require expensive computer equipment and considerable computing time and considerable amounts of data to obtain the desired statistical accuracy.
The wide field method (WFM) (D. A. Agard and J. W. Sedat, “Three-Dimensional Analysis of Biological Specimens Utilizing Image Processing Techniques,”
Proc. Soc. PhotoOpt. Instrum. Eng., SPIE,
264, 110-117, 1980; D. A. Agard, R. A. Steinberg, and R. M. Stroud, “Quantitative Analysis of Electrophoretograms: A Mathematical Approach to Super-Resolution,”
Anal. Biochem.
111, 257-268, 1981; D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, “Fluorescence Microscopy in Three Dimensions,”
Methods Cell Biol.
30, 353-377, 1989; D. A. Agard, “Optical Sectioning Microscopy: Cellular Architecture in Three Dimensions,”
Annu. Rev. Biophys. Bioeng.
13, 191-219, 1984; Y. Hiraoka, J. W. Sedat, and D. A. Agard, “The Use of a Charge-Coupled Device for Quantitative Optical Microscopy of Biological Structures,”
Sci.
238, 36-41, 1987; W. Denk, J. H. Strickler, and W. W. Webb, “Two-Photon Laser Scanning Fluorescence Microscopy,”
Sci.
248, 73-76, 1990) uses a conventional microscope to sequentially acquire a set of images of adjacent focus planes throughout the volume of interest. Each image is recorded using a cooled charge-coupled device (CCD) image sensor (J. Kristian and M. Blouke, “Charge-coupled Devices in Astronomy,”
Sci. Am.
247, 67-74, 1982) and contains data from both in-focus and out-of-focus image planes.
The technique of laser computed tomography is implemented using a conventional microscope. The system discussed by S. Kawata, O. Nakamura, T. Noda, H. Ooki, K Ogino, Y. Kuroiwa, and S. Minami, “Laser Computed-Tomography Microscope,”
Appl. Opt.
29, 3805-3809 (1990) is based on a principal that is closely related to the technique of X-ray computed tomography, but uses three-dimensional volume reconstruction rather than two-dimensional slice reconstruction. Projected images of a thick three-dimensional sample are collected with a conventional transmission microscope modified with oblique illumination optics, and the three-dimensional structure of the interior of the sample is reconstructed by a computer. Here, the data is acquired in a time short compared to that required to process data for a three-dimensional image. In one experiment by Kawata et al., ibid., the 80×80×36-voxel reconstruction required several minutes to collect all projections and send them to a minicomputer. Approximately thirty minutes then were required for digital reconstruction of the image, in spite of utilizing a vector processor at a speed of 20 million floating point operations per second (MFLOPS).
In a conventional point or pinhole-confocal microscope, light from a point source is focused within a very small space, known as a spot. The microscope focuses light reflected from, scattered by, or transmitted through the spot onto a point detector. In a reflecting point-confocal microscope the incident light is reflected or back-scattered by that portion of the sample in the spot. Any light which is reflected or back-scattered by the sample outside of the spot is not well focused onto the detector, thus it is spread out so the point detector receives only a small portion of such reflected or back-scattered light. In a transmitting point-confocal microscope, incident light is transmitted unless it is scattered or absorbed by that portion of the sample in the spot. Generally, the point source and point detector are approximated by placing masks containing a pinhole in front of a conventional light source and a conventional detector, respectively.
Similarly, in a conventional slit-confocal microscope system, light from a line source is focused into a very narrow elongated space, which is also known as a spot. The slit-confocal microscope focuses light reflected from, scattered by or transmitted through the spot onto a line detector. The line source and line detector can be approximated using a mask with a slit in front of a conventional light source and row of conventional detectors, respectively. Alternately, a line source can be approximated by sweeping a focused laser beam across the object to be imaged or inspected.
Since only a small portion of the object is imaged by the confocal microscope, either the object to be imaged must be moved, or the source and detector must be moved, in order to obtain sufficient image data to produce a complete two-dimensional or three-dimensional view of the object. Previous slit-confocal systems have moved the object linearly in a direction perpendicular to the slit to obtain successive lines of two-dimensional image data. On the other hand, point-confocal systems having only one pinhole have to be moved in a two-dimensional manner in order to acquire two-dimensional image data and in a three-dimensional manner in order to acquire a three-dimensional set of image data. The raw image data are typically stored and later processed to form a two-dimensional cross-section or a three-dimensional image of the object that was inspected or imaged. The reduced sensitivity to out-of-focus images relative to conventional microscopy leads to improved statistical accuracy for a given amount of data and the processing operation is considerably simpler in comparison to that required when processing data obtained in conventional microscopy approach.
In a system known as the Tandem Scanning Optical Microscope (TSOM), a spiral pattern of illumination and detector pinholes are etched into a Nipkow disk so, as the disk rotates, the entire stationary object is scanned in two dimensions [cf. M. Pétran and M. Hadravsky, “Tandem-Scanning Reflected-Light Microscope,”
J. Opt. Soc. A.
58(5), 661-664 (1968); G. Q. Xiao,
Cahill, Sutton & Thomas PLLC
Turner Samuel A.
Zetetic Institute
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