US 5,296,703 AGranted

Scanning confocal microscope using fluorescence detection

AssigneeThe Regents of the University of California

InventorRoger Y. Tsien

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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.

Claims18

Dependent claims

A confocal microscope for scanning a sample, comprising:

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.

9 more elements

A confocal microscope for scanning a sample, comprising:

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.

4 more elements

A confocal microscope for scanning a sample, comprising:

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.

12 more elements

Dependencies are inferred from the claim text.