Laser excited confocal microscope fluorescence scanner and method

• US 5,091,652 A
• Filed: 06/01/1990
• Issued: 02/25/1992
• Est. Priority Date: 01/12/1990
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Mathies, Richard A., Peck, Konan
• Primary Examiner(s): DELACROIX MUIRHEI, CYBILLE
• Application Number:
  US07/531,900
• Time in Patent Office: 634 Days
• Field of Search: 250/234, 250/461.2, 250/461.1, 250/459.1, 250/458.1
• US Class Current: 250/458.1
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6476 Front end, i.e. backscatter, geometry
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 30/74 Optical detectors measurement of intensity, velocity, spectral content, polarisation, or phase of infrared, visible or ultraviolet light G01J
• CPC Class Codes: G01N 30/95 Detectors specially adapted therefor; Signal analysis
• CPC Class Codes: G02B 21/0028 specially adapted for specific applications, e.g. for endoscopes, ophthalmoscopes, attachments to conventional microscopes
• CPC Class Codes: G02B 21/004 fixed arrays, e.g. switchable aperture arrays
• CPC Class Codes: G02B 21/0076 arrangements using fluorescence or luminescence

Abstract

A fluorescent scanner for scanning the fluorescence from a fluorescence labeled separated sample on a sample carrier including a confocal microscope for illuminating a predetermined volume of the sample carrier and/or receiving and processing fluorescence emissions from said volume to provide a display of the separated sample.

Claims

1. An improved gel scanner comprising

means for supporting a gel to be scanned,

means for forming a light beam of predetermined wavelength;

a dichroic beam splitter for receiving and directing said beam toward a supported gel to be scanned;

an objective lens for receiving said beam and focusing the beam in a selected volume of gel to cause fluorescence emission of light at a different wavelength and collecting the emitted light from samples in said selected volume and directing the emitted light to said dichroic beam splitter which passes said emitted light of different wavelengths and reflects light at said predetermined wavelength;

a spatial filter for receiving and passing the emitted light from said volume while rejecting background and scattered light;

means for providing relative movement between said focused beam and the gel being scanned,

means for detecting said passed, emitted light and providing output signals, and

means for processing said signals to provide an image of the fluorescence from the gel.

2. The method of detecting fluorescence from DNA fragments on a gel which comprises

exciting a predetermined volume of said gel with light energy of predetermined wavelength focused therein by an objective lens to cause said fragments to fluoresce at a different wavelength;

collecting the fluorescently emitted light from said predetermined volume with said objective lens;

spectrally and spatially filtering said fluorescently emitted light energy of different wavelength to reject light at said predetermined and other wavelengths; and

applying the filtered light energy to a detector to generate an output signal representative of the fluorescence from said fragments.

3. The method of claim 2 in which the excited volume is scanned across the gel to obtain emission from adjacent volumes to provide an output signal for each of said volumes, and processing the output signals to provide an image of the fluorescence from said fragments.

4. A scanner for scanning a gel to detect fluorescence from fluorescence labeled separated sample components comprising

means for supporting a gel to be scanned,

means for applying excitation light at a predetermined wavelength to a volume in the gel to cause labeled, separated sample components to fluoresce at a different wavelength, and for collecting the fluorescence emitted light from said sample at said different wavelength from said volume and reject the first wavelength;

means for moving said excitation light and gel relative to one another to scan the gel;

at least one spectral filter for receiving the collected emitted light and passing light at said different wavelength while rejecting light at said predetermined wavelength;

a spatial filter for receiving the emitted light and passing light from said sample volume while rejecting background and scattered light;

detecting means for receiving said collective fluorescently emitted light passed by said spectral and spatial filters and providing output signals, and

means for receiving and processing said signals to provide an image of the labeled separated sample components.

5. A scanner as in claim 4 including a laser for supplying said excitation light.

6. An improved scanner as in claim 5 including a spatial filter for receiving said light beam and transmitting light to said dichroic beam splitter.

7. An improved scanner for detecting fluorescence from a fluorescence labeled sample material carried by a sample carrier comprising

means for forming a light beam having a predetermined wavelength;

a dichroic beam splitter for receiving said light beam and directing it towards an objective lens which receives said beam and focuses the beam at a spot on the sample carrier whereby to cause the sample material to fluoresce at a selected volume and for collecting the fluorescently emitted light from said selected volume and directing the emitted light to said dichroic beam splitter, said fluorescent light being at a second wavelength and said dichroic beam splitter serving to pass the second wavelength;

a spatial filter for receiving light at said second wavelength and passing light emitted from said selected volume;

a spectral filter for further filtering said light and passing light at said second wavelength;

means for moving the light beam and sample carrier relative to one another to scan the sample carrier,

means for detecting the passed, emitted light of said second wavelength and providing output signals representative of the intensity of said detected light, and

means for receiving and processing said signals to provide an image of the fluorescence of said samples.

8. An improved fluorescence scanner as in claim 7 in which the carrier is a gel and the sample is a nucleic acid or derivative thereof.

9. An improved gel scanner comprising

means for supporting a gel to be scanned,

means for forming a light beam of predetermined wavelength;

a dichroic beam splitter for receiving and transmitting said beam toward a supported gel to be scanned;

an objective lens for receiving said beam and focusing the beam in a selected volume of gel to cause fluorescence emission of light at a different wavelength and collecting the emitted light from samples in said selected volume and directing the emitted light to said dichroic beam splitter which reflects said emitted light of different wavelengths and rejects light at said predetermined wavelength;

a spatial filter for receiving and passing the emitted light from said volume while rejecting background and scattered light;

means for providing relative movement between said focused beam and the gel to be scanned,

means for detecting said passed, emitted light and providing output signals, and

means for processing said signals to provide an image of the fluorescence from the gel.