Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2001-12-14
2004-10-12
Evans, F. L. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
C250S458100, C359S305000, C356S326000
Reexamination Certificate
active
06804000
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to optical instrumentation and relates more particularly to the beam-steering of light using acousto-optical deflectors.
Optical instruments have long played an important role in the study of physical and biological phenomena. Light microscopes, in particular, have been used for more than one hundred years to gain insight into the structure of biological media. As can readily be appreciated, achieving high spatial resolution remains one of the foremost objectives of a light microscope. This objective, however, is often hampered by the fact that biological media, by their very nature, are typically highly scattering with respect to light. Consequently, objects located beneath the surface of a biological medium are often difficult to observe with high resolution using light microscopy. As a result, a number of different approaches have been undertaken in an effort to counteract the light scattering effects of biological media.
One such approach is the confocal microscope, an example of which is disclosed in U.S. Pat. No. 4,863,226, inventors Houpt et al., which issued Sep. 5, 1989, and which is incorporated herein by reference. In a confocal microscope, light is brought to focus on or within a sample, and the light emitted from the illuminated sample is then brought to focus on a pinhole positioned in front of a detector, the pinhole being used to prevent light scattered by the sample from reaching the detector. Often in a confocal microscope, the illuminating light is laser light, and a galvanometer or the like is placed along the optical path of the illuminating laser light to create a scanning beam of illuminating laser light. Laser scanning confocal microscopes are often used to create fluorescence images of a sample, with the illuminating laser light being used to excite native and/or extrinsic fluorophores present within the sample, and the non-scattered component of the fluorescent light emitted from said fluorophores being passed through the pinhole and detected by the detector.
One of the problems associated with the use of laser scanning confocal microscopes in fluorescence imaging is that the detected light signal is typically weak. This is because, of all the fluorescence photons generated by the sample, only the non-scattered (i.e., ballistic) photons generated at the illuminating focus (i.e., on-focus) are permitted to pass through the pinhole and are detected by the detector. In other words, not only are the undesirable fluorescence photons generated at loci other than the illuminating focus (i.e., off-focus) excluded from detection but so are the desirable scattered on-focus fluorescence photons, said scattered on-focus fluorescence photons representing a significant portion of the on-focus fluorescence photons.
Another problem associated with the use of laser scanning confocal microscopes in fluorescence imaging is that the intensity of the illuminating light necessary to generate an appreciable detected signal often has the undesirable consequence of adversely affecting the fluorophore (i.e., photobleaching) or adversely affecting the sample through a fluorophore-mediated event (i.e., photodamage). Moreover, because the illuminating light must travel through the sample to the illuminating focus, the above-mentioned effects of photobleaching and photodamage are not confined to the illuminating focus.
One form of laser scanning microscopy that has been devised to address the types of shortcomings discussed above in connection with laser scanning confocal fluorescence microscopy is multi-photon excited fluorescence laser scanning microscopy. In multi-photon excited fluorescence laser scanning microscopy, excitation of a fluorophore is achieved by the simultaneous absorption by the fluorophore of two or more photons of low energy that combine their energies to provide the requisite energy for transition of the fluorophore to its excited state. For example, two photons of lower energy red or infrared light may be used to excite a fluorophore typically excitable by one photon of higher energy ultraviolet light. Because multi-photon absorption requires two or more photons for each excitation, its rate depends on the square of the instantaneous intensity and is, therefore, almost completely confined spatially to the high-intensity region at the focal point of the strongly focused excitation laser.
Consequently, because the requisite energy for excitation is spatially confined to the focal point of the illuminating laser, multi-photon excited fluorescence laser scanning microscopy does not result in off-focus photobleaching or photodamage and does not require the placement of a pinhole in front of the detector, as in confocal fluorescence microscopy, to reject off-focus fluorescence photons. Because such a pinhole is unnecessary in multi-photon excited fluorescence laser scanning microscopy, both scattered and ballistic on-focus fluorescence photons are detected, thereby yielding a stronger signal than if only ballistic on-focus fluorescence photons were detected. Furthermore, because longer wavelength photons typically scatter less in biological media than do shorter wavelength photons, one can achieve improved depth penetration of the media using multi-photon excited fluorescence laser scanning microscopy than using laser scanning confocal fluorescence microscopy.
Additional information relating to multi-photon excited fluorescence laser scanning microscopy is provided in the following published documents, all of which are incorporated herein by reference: U.S. Pat. No. 5,034,613, inventors Denk et al., which issued Jul. 23, 1991; Denk et al., “Two-Photon Laser Scanning Fluorescence Microscopy,”
Science,
248:73-6 (1990); Denk et al., “Photon Upmanship: Why Multiphoton Imaging Is More than a Gimmick,”
Neuron,
18:351-7 (1997); Denk et al., “Two-Photon Molecular Excitation in Laser-Scanning Microscopy,”
Handbook of Biological Confocal Microscopy
, pages 445-57, edited by James B. Pawley, Plenum Press, New York (1995); Fan et al., “Video-Rate Scanning Two-Photon Excitation Fluorescence Microscopy and Ratio Imaging with Cameleons,”
Biophysical Journal,
76:2412-20 (1999); Koester et al., “Ca
2+
Fluorescence Imaging with Pico- and Femtosecond Two-Photon Excitation: Signal and Photodamage,”
Biophysical Journal,
77:2226-36 (1999); Mainen et al., “Two-Photon Imaging in Living Brain Slices,”
METHODS: A Companion to Methods in Enzymology,
18:231-9 (1999); and Parthenopoulos et al., “Three-Dimensional Optical Storage Memory,”
Science,
245:843-5 (1989).
Another form of laser scanning microscopy that has been devised to address the types of shortcomings discussed above in connection with laser scanning confocal fluorescence microscopy is multi-harmonic generation laser scanning microscopy. In one type of multi-harmonic generation laser scanning microscopy, namely, second-harmonic generation laser scanning microscopy, the combined coherent electric fields of two incident photons interact with a dipolar molecule. The incident field is scattered and, in the process, a single photon of exactly half the incident photon wavelength and twice the incident photon energy is formed instantly. This photon is then detected.
As a second-order reaction in the concentration of incident photons, second-harmonic generation laser scanning microscopy possesses the same intrinsic resolving power as two-photon excited fluorescence laser scanning microscopy. In addition, second-harmonic generation laser scanning microscopy, like multi-photon excited fluorescence laser scanning microscopy and unlike laser scanning confocal fluorescence microscopy, does not require the placement of apinhole in front of the detector. However, unlike multi-photon excited fluorescence laser scanning microscopy, multi-harmonic generation laser scanning microscopy does not require that the object being imaged possess a fluorescent molecule. Instead, multi-harmonic generation laser scanning microscopy merely requires that the object possess th
Miesenböck Gero
Roorda Robert Dixon
Evans F. L.
Geisel Kara
Kriegsman & Kriegsman
Sloan-Kettering Institute for Cancer Research
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