Optics: measuring and testing – Of light reflection
Reexamination Certificate
2001-04-04
2003-04-22
Lee, Michael G. (Department: 2876)
Optics: measuring and testing
Of light reflection
C356S244000, C356S246000, C356S440000, C356S446000
Reexamination Certificate
active
06552794
ABSTRACT:
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to a method and an article for improving optical detection and sensitivity. More particularly, the present invention relates to a method and an article for improving optical detection and sensitivity in situations in which emission of fluorescence light is monitored.
Optical detection is used intensively in many fields and for a variety of applications. In many cases, the optical signal emitted by or from a viewed or analyzed object is very low, on the border of detection. Vast efforts are therefore directed at increasing the sensitivity of detection of optical and electro-optical systems, or, in other words, at increasing the ability of optical or electro-optical systems to detect light signals of lesser intensity.
Fluorescence microscopy provides an example. Fluorescence microscopy is one of the most powerful techniques for analyzing tissues and cells [J. S. Ploem (1987) Introduction to Fluorescence Microscopy, Oxford Science Publications, New York]. Unlike bright field microscopy where light is transmitted through an analyzed sample, in fluorescence microscopy, a signal appears only with respect to specific entities that emit light, whereas the background is left dark. This fact makes fluorescence microscopy a very sensitive method for detecting both the existence and distribution of materials in a sample and their quantities. Fluorescence microscopy is therefore one of the most important experimental methods used in light microscopy [Lakowicz (1983) Principles of fluorescence spectroscopy, Plenum Press, New York, London].
Thus, in fluorescence microscopy, an analyzed sample is emitting light, a phenomenon known as fluorescence. The fluorescence light can be native to the analyzed sample, or it can be as a result of an interaction between the analyzed sample and a probe. Some probes are chemicals that fluoresce under certain conditions. For example, probes are known that chemifluoresce differently according to a level of a chemical, e.g., an ion, such as hydrogen or calcium ions, present in the sample or portions thereof. Such probes are therefore useful in determining the concentration and/or distribution of a particular ion in the sample. Other probes include a binding portion and a fluorescent tag. The binding portion can be, for example, a first member of a binding pair, capable of binding a second member of a binding pair present in the sample. The members of a binding pair can be, for example, a ligand that binds a receptor and vice versa, an antibody that binds an antigen and vice versa, a nucleic acid that binds it complement, a substrate, product, inhibitor or analog that binds its enzyme and vice versa, etc. The fluorescent tag is typically a fluorochrome covalently linked to the first member of a binding pair and serves to monitor binding to the second member of the binding pair present in the analyzed sample. Many fluorochromes are presently known each is characterized by a unique absorption spectrum and absorption peak and emission spectrum and peak. Examples of fluorochromes include, fluorescent proteins, such as green, yellow, cian and red fluorescent proteins and smaller chemical compounds such as fluorescein-5-iso-thiocyanate (FITC), rodamine, SpectrumOrange™, SpectrumGreen™, Aqua, Texas-Red, 4′,6-diamidino-2-phenylindole (DAPI), Cy3, Cy5.5. Hundreds of other fluorochromes are known. A partial list of commercially available fluorochromes can be found in the catalog of Molecular Probes. For a detailed review of fluorescent probes see, Mason (editor) (1993) Fluorescent and Luminescent Probes for Biological Activity, Biological Techniques Series, edited by Sattelle, Academic Press Limited, London; Waggoner (1986) Applications of fluorescence in the biomedical sciences, Eds. Taylor et al., New York: Alan R. Liss, Inc. pp. 3-28; and Taylor et al. (1992) The New Vision of Light Microscopy, American Scientist, Vol. 80, pp. 322-335.
In the last few years, further advances have been made in both the detection methods and the fluorochromes. Side by side with the development of fluorochromes that are brighter, more stable and easier to attach to different other compounds, other fluorescent materials have been developed commonly called quantum dots or nanocrystals [see, Bruchez et al (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013-2016 and Chan, W. C. et al (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281, 2016-2018]. These structures are in effect nanosized semiconductors that fluoresce. These structures are far more stable than organic-materials based fluorochromes, and in addition, it is possible to design and manufacture the nanocrystals so that they emit light at a desired spectral range and with a narrower bandwidth.
Improvements were also introduced in the detection of fluorescence. Imaging microscopy employing highly sensitive charge coupled devices (CCD) are used intensively and improve many aspects of detection, including, but not limited to, higher sensitivity, larger number of probes that can be co-detected, accurate quantitative analysis and automation. In addition, confocal microscopy which employs laser scanning mechanisms combined with confocal optics that improves the accuracy in the depth of field is also intensively used [see, Wlison, T. (1990) Confocal Microscopy. Academic Press, London]. These detection methods have broadened the use of fluorescence microscopy.
Fluorescent microscopy was improved to allow detection of different probes simultaneously. A remarked improvement in multicolor fluorochromes is the introduction of combinatorial fluorochromes which are various combinations of few basic fluorochromes [see, Ried et al., (1992) Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. USA 89, 1388-1392; and, Ried (Jan. 1994) Fluoreszenz in situ Hybridizierung in der genetischen Diagnostik, Faculty of theoretical medicine, Ruprecht-Karls University Heidelberg].
Spectral imaging combined with fluorescence microscopy provides an even better spectral resolution and is presently developed to allow simultaneous detection of several dozens of different combinatorial fluorochromes.
A spectrometer is an apparatus designed to accept light, to separate (disperse) it into its component wavelengths, and measure the lights spectrum, that is the intensity of the light as a function of its wavelength. A spectral imaging device, also referred to herein as “imaging spectrometer” is a spectrometer which collects incident light from a scene and measures the spectra of each picture element thereof.
Spectroscopy is a well known analytical tool which has been used for decades in science and industry to characterize materials and processes based on the spectral signatures of chemical constituents therein. The physical basis of spectroscopy is the interaction of light with matter. Traditionally, spectroscopy is the measurement of the light intensity emitted, scattered or reflected from or transmitted through a sample, as a function of wavelength, at high spectral resolution, but without any spatial information.
Spectral imaging, on the other hand, is a combination of high resolution spectroscopy and high resolution imaging (i.e., spatial information). Most of the works so far described in spectral imaging concern either obtaining high spatial resolution information from a biological sample, yet providing only limited spectral information, for example, when high spatial resolution imaging is performed with one or several discrete band-pass filters [See, Andersson-Engels et al. (1990) Proceedings of SPIE—Bioimaging and Two-Dimensional Spectroscopy, 1205, pp. 179-189], or alternatively, obtaining high spectral resolution (e.g., a full spectrum), yet limited in spatial resolution to a small number of points of the sample or averaged over the whole sample
Applied Spectral Imaging Ltd.
G.E. Ehrlich Ltd.
Le Uyen-Chau N.
Lee Michael G.
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