Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2000-01-28
2003-08-19
Cherry, Euncha (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C356S479000
Reexamination Certificate
active
06608717
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an optical coherence microscope (OCM) for study of problems in developmental biology and biotechnology. More particularly, the invention is used for imaging cells located up to four millimeters or more below the surface of living tissue.
2. Description of the Related Art
Optical coherence microscopy (OCM) is a technique developed recently to image objects embedded in an opaque medium (e.g., flesh) up to a depth of 1 to 2 mm. It has been applied successfully on a prototype basis in ophthalmology (Swanson et al., 1993) and dermatology (Schmitt et al., 1995) to image tissue structures and interfaces. Moreover, OCM has been used to measure the optical properties of tissue and thereby provide information on the physiological state of tissue. OCM has recently become a subject of interest for the study of developmental biology.
Understanding of developmental mechanisms has come from studies of gene expression patterns, tissue geometry, and/or cell morphology, all performed on fixed tissue. From these “snap-shot” views, researchers must infer the dynamics of the underlying cellular and molecular events. Recently, biological imaging technologies have been introduced that permit the non-destructive analysis of cell migration, differentiation, and neuronal interconnection during embryonic development. For example, fluorescent or absorbing compounds can be used to label cells which are then followed with a conventional light microscope equipped with a video camera or with a confocal microscope. The confocal microscope adds significant depth resolution, offering the possibility of obtaining a three-dimensional image by combining optical sections through the depth of an embryo. The image formation rate of the confocal microscope is sufficiently fast to follow the dynamic behaviors of cells as they migrate or of retinal cell axons as they extend, actively sense, and retract projections toward cells in the tectum (O'Rourke et al., 1994). However, light scattering in embryonic tissue reduces the signal-to-noise ratio of a confocal microscope, limiting the depth of the specimen that can be explored to about 200 &mgr;m (Schmitt et al., 1994b). A second imaging technology is magnetic resonance imaging (MRI), recently extended to the microscopic domain so that it can now resolve a 12 &mgr;m cube in living embryos (Jacobs and Fraser, 1994). Although an MRI microscope is indifferent to optical opacity, it is both expensive and slow, requiring nearly an hour to generate a high-resolution image.
It is worth noting that other recently developed imaging techniques also experience image degradation with depth into tissue. For example, green fluorescence protein (GFP) has been modified and expressed in the plant
Arabidopsis thaliana
, yielding beautiful images of developing roots. However, the images are obtained with a confocal microscope and are limited to depths less than 100 &mgr;m in this preparation. Development of the primary meristem in the seed embryo occurs several hundred micrometers into the tissue, too deep for confocal microscopy. Similar limitations apply to 2-photon microscopy (Potter et al., 1996) and fluorescence resonance energy transfer (Helm and Tsien, 1996).
Optical Coherence Microscopy
An optical coherence microscope uses the principles of confocal microscopy, with an additional coherence gate that excludes back-scattered light from out-of-focus planes, resulting in a signal-to-noise ratio that is enhanced by 6 orders of magnitude (lzatt et al., 1994a,b). A resolution of 10 &mgr;m has been achieved in both the lateral and depth directions (Huang et al., 1991b). Optical fiber and solid state sources/detectors are typically used, so the instrument is inherently rugged. OCM overcomes the depth limitation of confocal microscopy and is currently faster than MRI. And at an estimated cost of under $10,000 the instrument is two orders of magnitude less expensive than the MRI microscope.
The coherence gate in OCM is achieved by superposing a Michelson interferometer on the confocal microscope. Back-scattered light from the specimen interferes coherently with light returning from an added reference arm only when the two optical paths are equal. The amplitude of interference fringes (their “visibility”) becomes the signal; this signal is appreciable only for light back-scattered from a narrow range of depths in the specimen. The depth range over which interference occurs is related to the coherence length of the source. For example, the depth range, which is also the depth resolution, is roughly 10 &mgr;m when the spectral width of the source is 30 nm (&lgr;=830 nm, Swanson et al., 1992). At a particular depth, a lateral image (optical section) can be formed by translating the beam; the spot size of the focused beam (easily less than 10 &mgr;m) determines the lateral resolution.
History of Reflectometry
When optical fibers were introduced into the communications industry in the 1970s, the need immediately arose for a method of testing and locating flaws in fiber cables. The first reflectometers (Barnoski and Jensen, 1976), which operated in the time domain, simply measured the round trip time of flight to a reflecting fiber flaw. Typical pulse widths were a few nanoseconds, so spatial resolution was about one meter.
In the 1980s there appeared low-coherence reflectometers which operate in the frequency domain (Danielson and Whittenburg, 1987; Takada et al., 1987; Youngquist et al., 1987). In this technique a spectrally broad (30 nm) light source operating in the near infrared (800 to 1300 nm) is employed in a Michelson interferometer, one leg of which is the fiber under test. The light source has a coherence time of 70 femtoseconds, a considerable improvement over the timedomain pulse widths. As the reference path length is varied, the interferometer output is monitored for interference fringes that occur when light is reflected or back-scattered from a point a distance along the tested fiber equal to the reference path length. The spatial resolution along the tested fiber is one-half the coherence length because the fiber is traversed twice in that leg of the interferometer. (Actually the geometrical spatial resolution is even smaller by a factor of n, where n is the refractive index of the fiber.) For a spectral width of 30 nm, the geometrical spatial resolution along a fiber is 7 &mgr;m.
Shortly thereafter ophthalmologists adapted this low-coherence reflectometer to measure the length of the eye (Fercher et al., 1988; Hitzenberger, 1991). Finally lateral scans were added, and both lateral and depth data were interpreted in terms of images of the sample, usually 2-D images with one lateral and one depth dimension (Huang et al., 1991 a,b). The image presumably represents the spatial variation of the optical properties of the sample, primarily the scattering coefficient.
Polarization Effects
Interference occurs at the output of the OCM only between the same polarization components of the electric fields returning from the reference mirror and the sample, respectively. Birefringence effects in the optical fibers or in the sample may alter the relative magnitude and phase of the two polarization components emitted by the source and hence reduce the amplitude of the interference fringes at the photodetector. To eliminate problems in the fibers, some workers have used polarization-preserving fibers and linearly polarized light to eliminate polarization-dispersion effects that lead to different optical path lengths for different polarization states (Clivaz et al., 1992). Kobayashi et al. (1991) constructed a polarization-insensitive reflectometer by separating the two polarization states at the output of the interferometer and measuring their interference fringes with two independent detectors. The sum of the detector outputs is independent of birefringence effects in the fibers or the sample. On the other hand, Wang et al. (1994) devised a simple, inexpensive means of circumventing birefringence ef
Haskell Richard C.
Hoeling Barbara M.
Medford June I.
Petersen Daniel C.
Wang Ruye
Cherry Euncha
Colorado State University Research Foundation
Knobbe Martens Olson & Bear LLP
LandOfFree
Optical coherence microscope and methods of use for rapid in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Optical coherence microscope and methods of use for rapid in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Optical coherence microscope and methods of use for rapid in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3075240