Radiant energy – Luminophor irradiation
Utility Patent
1999-01-25
2001-01-02
Hannaher, Constantine (Department: 2878)
Radiant energy
Luminophor irradiation
C250S459100, C250S461100
Utility Patent
active
06169289
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains generally to the field of microscopy 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 the reflected light 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.
Where the scanning fluorescence microscope operates by using moving mirrors or other deflectors to deflect the light beam to scan across the specimen, the light emitted from the specimen is typically passed back through the scanning system (descanned) before being separated from the source light beam by a dichroic mirror and directed to the detector. In a confocal scanning fluorescence microscope, the incoming beam is typically passed through an aperture before entering the scanning system, and the emitted light beam, after being descanned, is focussed through a confocal pinhole aperture before being incident upon the detector. The confocal aperture blocks light from portions of the specimen outside of the focal plane so that substantially only light from the focal plane is incident on the detector, thereby greatly improving the depth resolution. Thus, the image data received from the photodetector for storage and/or display comprises image information from substantially only the focal plane. By focussing the incident light on a specimen at different focal planes, a three-dimensional image of a semi-transparent specimen, such as a living cell, can be built up.
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, 6 April 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 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 descan the light or directly, without descanning, 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. The fluorophore excitation is typically restricted to the minimum required to form an optical section fluorescence image thereby minimizing photobleaching and phototoxicity in thick tissues due to fluorophore excitation. The quality of the images obtained in such fluorescence microscope, particularly for live cell imaging, is dependent on the collection of as much of the fluorescence signal as possible. A limitation of scanning fluorescence microscopes generally, including those using two photon fluorescence excitation, is that the amount of fluorescent light collected from the specimen by the microscope objective may be relatively low.
SUMMARY OF THE INVENTION
In accordance with the invention, multi-photon excitation fluorescence microscopy is carried out with significantly increased fluorescent photon collection efficiency and improved image intensity of the available fluorescent signal. Such increased collection efficiency is obtained without requiring an increase in the intensity of the incident excitation light beam. Optionally, the intensity of the incident excitation beam can be reduced while still obtaining comparable image information to thereby reduce photolysis and photobleaching caused by the incident light beam on specimens such as living cells. Such increased fluorescent signal collection efficiency is obtained without requiring the use of an additional photodetector or a major modification of existing laser scanning fluorescent microscope designs, and without inhibiting or affecting the functionality of such microscopes in their epi-illumination transmitted light imaging modes.
A scanning multi-photon fluorescent microscope in accordance with the invention receives light from a laser source that includes a chosen long wavelength, typically a pulsed laser providing short pulses of light in red or near infrared wavelengths, and directs the beam of excitation light from the source on an optical path to the back aperture of an objective lens of a microscope, which focusses the incident beam to a narrow point or waist inside a specimen to be examined. Fluorophores in the specimen absorb two (or more) photons at the wavelength of the incident light and emit—in a random direction—a fluorescent photon at a lower wavelength (higher energy) than the incident light. The fluorescent photons that are emitted from the specimen toward the objective lens are collected by the objective lens and directed back in a beam to a first dichroic mirror which is formed to pass (or, alternatively, reflect) the shorter wavelength fluorescent light to direct such light to a detector such as a photomultiplier tube. The dichroic mirror is further formed to substantially reflect (or, alternatively, pass) wavelengths of light above a selected wavelength including light at the incident beam wavelength. The detector is positioned with respect to the dichroic mirror so that the detector only receives the fluorescent light em
White John G.
Wokosin David L.
Foley & Lardner
Hannaher Constantine
Israel Andrew
Wisconsin Alumni Research Foundation
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