Scanning confocal microscope using fluorescence detection
First Claim
1. A confocal microscope for scanning a sample, comprising:
- a laser for generating a collimated incident beam having a predetermined wavelength;
optical path means includinga primary dichroic mirror, anda first scanning mirror, disposed between the dichroic mirror and the sample and rotatable about a first axis, for scanning the incident beam repeatedly across the sample, whereupon the sample fluoresces and emits radiation having a first predetermined wavelength, different from the wavelength of the incident beam, a portion of the emitted radiation forming a fluorescence beam that retraces the path of the incident beam back to the primary dichroic mirror,wherein the primary dichroic mirror separates the fluorescence beam from the incident beam;
a fluorescence beam detector for detecting the intensity of the fluorescence beam and for generating a corresponding fluorescence scan signal; and
reference beam means includingmeans for directing a reference beam toward the first scanning mirror, where it impinges on the first scanning mirror at a location coincident with the incident beam, to produce a scanning reference beam,a grating having a series of alternating transparent and opaque regions and positioned to receive the scanning reference beam and produce a pulsed reference beam indicative of the scan rate, anda reference beam detector for detecting the intensity of the pulsed reference beam and for generating a corresponding clock signal representative of the scan rate of the scanning reference beam and thereby the incident beam;
wherein the clock signal generated by the reference beam detector can be used to sample the fluorescence scan signal generated by the fluorescence beam detector.
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Accused Products
Abstract
A scanning confocal microscopes scans a sample with an incident beam of ultraviolet radiation, in a raster scan pattern, causing the sample to fluoresce and emit visible radiation, a portion of which retraces a 5 portion of the path optical of incident beam, to a dichroic mirror that separates it from the incident beam for detection by a photomultiplier tube. A clock signal for clocking the output of the photomultiplier tube is provided by a reference beam system that directs a reference beam onto the same scanning mirror as is the incident beam, at a coincident location on that mirror, and from there through a Ronchi grating having a uniform series of alternating transparent and opaque regions. The resulting pulsed reference beam is detected by a second photomultiplier tube, to produce a clock signal that is an accurate representation of the instantaneous scan rate of the reference beam and, thereby, the scanning incident beam.
82 Citations
18 Claims
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1. A confocal microscope for scanning a sample, comprising:
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a laser for generating a collimated incident beam having a predetermined wavelength; optical path means including a primary dichroic mirror, and a first scanning mirror, disposed between the dichroic mirror and the sample and rotatable about a first axis, for scanning the incident beam repeatedly across the sample, whereupon the sample fluoresces and emits radiation having a first predetermined wavelength, different from the wavelength of the incident beam, a portion of the emitted radiation forming a fluorescence beam that retraces the path of the incident beam back to the primary dichroic mirror, wherein the primary dichroic mirror separates the fluorescence beam from the incident beam; a fluorescence beam detector for detecting the intensity of the fluorescence beam and for generating a corresponding fluorescence scan signal; and reference beam means including means for directing a reference beam toward the first scanning mirror, where it impinges on the first scanning mirror at a location coincident with the incident beam, to produce a scanning reference beam, a grating having a series of alternating transparent and opaque regions and positioned to receive the scanning reference beam and produce a pulsed reference beam indicative of the scan rate, and a reference beam detector for detecting the intensity of the pulsed reference beam and for generating a corresponding clock signal representative of the scan rate of the scanning reference beam and thereby the incident beam; wherein the clock signal generated by the reference beam detector can be used to sample the fluorescence scan signal generated by the fluorescence beam detector. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A confocal microscope for scanning a sample, comprising:
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a microscope objective; a laser for supplying a collimated incident beam having a first wavelength that is directed through said microscope objective to a sample to be scanned; beam deflection means for periodically deflecting said incident beam so that said beam scans the sample, whereupon the sample radiates light having a second wavelength, a portion of the radiated light passing back through said microscope objective and said beam deflection means; a dichroic mirror, disposed between said laser and said beam deflection means, for separating the portion of the light radiated by the sample from said incident beam; a detector for receiving the portion of the light radiated by the sample and passed back through said microscope objective and said beam deflection means, and for generating a signal representing the intensity of that radiated light; and aperture means, disposed between said laser and said dichroic mirror, at a position where said incident beam is collimated, for limiting said incident beam from entering a peripheral edge of said microscope objective. - View Dependent Claims (11, 12, 16, 17, 18)
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13. A confocal microscope for scanning a sample, comprising:
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a microscope objective; means for positioning said microscope objective adjacent to the sample to be scanned; a laser for supplying a collimated incident beam having a predetermined wavelength that is directed through said microscope objective to a sample to be scanned; beam deflection means for periodically deflecting said incident beam so that said beam scans the sample, whereupon the sample fluoresces and radiates light having first and second predetermined wavelengths, a portion of the radiated light forming a fluorescence beam that passes back through said microscope objective; aperture means positioned to limit said incident beam from entering a peripheral edge of said microscope objective, but not to receive any portion of the fluorescence beam; a fluorescence beam detector including a dichroic mirror for separating the fluorescence beam into a first beam that includes radiation having the first predetermined wavelength and a second beam that includes radiation having the second predetermined wavelength, a first detector for detecting the intensity of the first beam and for generating a first signal, a second detector for detecting the intensity of the second beam and for generating a second signal, means for sampling the first and second signals in accordance with a clock signal, frame memory means for storing the successive samples of the first and second signals, and means for computing a real-time ratio of the successive samples of the first and second signals; a video display; and means for selecting data from said frame memory means for display on said video display. - View Dependent Claims (14, 15)
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Specification