Optics: measuring and testing – By dispersed light spectroscopy – Utilizing a spectrometer
Reexamination Certificate
2000-06-26
2004-02-03
Adams, Russell (Department: 2851)
Optics: measuring and testing
By dispersed light spectroscopy
Utilizing a spectrometer
C356S300000
Reexamination Certificate
active
06687000
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains generally to the field of microscopy and spectroscopy, and particularly to laser scanning fluorescence microscopy.
BACKGROUND OF THE INVENTION
Scanning optical microscopes, such as laser scanning confocal microscopes, are of increasing importance in microscopy, particularly for imaging of dynamic biological structures such as living cells. In a scanning microscope, the light beam from the source, usually a laser, is focussed to a point within the specimen by the microscope objective and the specimen and beam are moved relative to one another in a raster fashion, either by moving the stage on which the specimen is mounted or, more commonly, by deflecting the light beam so that it scans across a stationary specimen. The light from the specimen is collected by the objective and passed back through the microscope to a detector, such as a photomultiplier tube. In addition to detection of light reflected from a specimen (or transmitted through the specimen), scanning microscopes can also be constructed to detect fluorescence induced by the illuminating light beam. Typically, the fluorophores in the specimen absorb the illumination light, which is at a chosen wavelength (usually shorter wavelength visible light), and fluorescently emit photons at a longer wavelength which are received by the objective of the microscope and passed back through the scanning optics to a dichroic mirror which separates the fluorescent light from light at the illuminating light wavelengths and directs the fluorescent light to a separate photodetector. In this manner, particular structures within the specimen, such as parts of cells, can be labeled with fluorescent markers and distinctively imaged by the scanning microscope.
Most fluorophores can also absorb two (or more) photons of longer wavelengths simultaneously when sufficiently intense illumination light is applied thereto and will emit a fluorescent photon at a shorter wavelength than the incident light. This phenomenon is exploited in multi-photon laser scanning microscopes in which an incident beam of relatively long wavelength light in short pulses from a laser source is narrowly focussed onto a specimen so that the light reaches an intensity at the focal point sufficient to excite detectable two (or more) photon fluorescence. The emitted fluorescent photons collected by the objective lens of the microscope are passed back through the optical system, either through the scanning optics to a dichroic mirror which reflects light at longer wavelengths while passing the shorter wavelength fluorescent light to a separate detector, or by bypassing the scanning system, and directing the light from the microscope objective lens to a dichroic mirror which passes the shorter wavelength fluorescent light directly to a detector while reflecting the longer wavelength excitation light. See, Winfried Denk, et al., “Two Photon Laser Scanning Fluorescence Microscopy,” Science, Vol. 248, Apr. 6, 1990, pp. 73-76; Winfried Denk, et al., “Two-Photon Molecular Excitation in Laser-Scanning Microscopy,” Chapter 28, Handbook of Biological Confocal Microscopy, Plenum Press, New York, 1995, pp. 445-458; and U.S. Pat. No. 5,034,613 entitled Two-Photon Laser Microscopy. By focussing the incident light from the objective lens to a relatively narrow spot or beam waist such that the intensity of the incident light is sufficient to excite multi-photon excitation only at the waist within the specimen, multi-photon fluorescence excitation will occur generally only in the focal plane. The shorter wavelength fluorescent light emitted by the specimen can then be passed back, either through the scanning system to de-scan the light or directly, without de-scanning, to a fluorescent light detector to obtain an image corresponding to the focal plane. Therefore, the excitation light alone produces the desired depth resolution (i.e., an optically sectioned fluorescence image), so that there is no need for the use of a confocal aperture.
Fluorescent signal photons can be characterized by their wavelength, the lifetime of the excited state giving rise to the photon, and polarization. These parameters can be used to identify a fluorophore or to provide information on the microenvironment of the fluorophore. Fluorescence microscopy of living specimens generally yields very weak signals. Therefore, any multi-dimensional spectral imaging system must be very photon efficient to be practical for in vivo imaging. In addition, such systems must have the lowest possible values of intrinsic (i.e., system generated) noise. Living cell studies, such as four dimensional imaging or ion imaging, generally require faster imaging speeds than are currently available from commercial multi-photon laser scanning microscopes (MPLSM). Several fast scanning MPLSM systems are currently in use in research laboratories, but these instruments either do not preserve the deep section contrast advantages of multi-photon over confocal microscopy or do not allow use of electronic magnification of the scanned area.
Most biological tissue is autofluorescent. Molecules such as NAD(P)(H), elastin, and chlorophyll act as endogenous fluorophores. These endogenous fluorophores can often be identified by their characteristic spectra. A spectral imaging system would thus be of considerable use in identifying endogenous fluorophores and specifying spectral windows that would either maximally accept or reject these signals, depending on the application.
The use of engineered fluorescent probes as physiological indicators has become a well established technique. Some probes indicate the presence of a bound ligand by changes in fluorescence intensity (e.g., calcium Green 1) while others use spectral shifts (e.g., Indo 1). The latter are favored because ratio imaging at two different wavelengths may be used to provide measurements that are independent of the concentration of the indicator molecule, requiring quantitative measurements. However, each of these probes now requires the use of a custom two-channel filter set.
Fluorescence resonant energy transfer (FRET) is a powerful technique for measuring intermolecular distances in vivo. This technique also now requires custom filter sets that are matched to the donor and the acceptor molecule's emission spectra. Ratiometric measurements are used to measure the extent of resonance transfer. This technique is proving to be valuable for the in vivo visualization of the docking of a receptor with its ligand, and is the basis of operation of a GFP based calcium indicator.
Fluorescence in situ hybridization is another significant area where multiple fluorophores and ratiometric techniques are used. Often, the main requirement in this application is to spectrally resolve as many separate fluorescent probes as possible.
A major consideration in the detection of fluorescence from scanning microscopes is the ability to collect the desired signal in the presence of significant noise (detection noise, system noise, fluorescent background, etc.). Background fluorescence from endogenous fluorophores or from another interfering exogenous fluorophore can severely reduce detection, or interpretation, of the image signal. In samples labeled with multiple fluorophores, the signal from one fluorophore is often much stronger than another and can spill over to an adjacent channel. In such instances, it is often necessary to move the spectral detection windows as far apart as possible to aid discrimination between the two fluorophores being studied rather than choosing spectral windows to give the maximum signal in each channel. The use of multiple fluorescent labels has been commonplace in the study of fixed specimens, and is now being established for use in in vivo studies. There are now many fluorophores that are available, each with its own unique spectral characteristics. The large number of available fluorophores has carried with it the problem that many different filter sets are now required for double or triple labeled samples. Filter sets use expensive
Esplin D. Ben
Foley & Lardner
Wisconsin Alumni Research Foundation
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