Multi-color laser scanning confocal imaging system
First Claim
1. A multi-color confocal microscopy system for use with an optical microscope in viewing a specimen stained with a plurality of stains, which stains fluoresce when excited by light of particular frequencies, comprising:
- laser means for producing multi-line incident laser light having multiple excitation lines from a single laser light source, each of said excitation lines corresponding to an excitation frequency of one of the stains;
means for directing said incident laser light into the microscope and for receiving emitted light from the microscope, said emitted light having multiple fluorescent emissions, each of said fluorescent emissions corresponding to an excitation frequency of one of the stains;
detector means positioned to receive said emitted light for converting said emitted light into electrical signals; and
control means connected to said detector means for accumulating said electrical signals and for producing a plurality of images of the specimen at a precise focal plane, each of said images corresponding to one of said lines of said laser.
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Abstract
A true multi-color laser scanning confocal imaging system for use with a microscope is described in which a single laser having a multi-line output is used to simultaneously or individually excite a plurality of dyes. The images may be simultaneously viewed by a plurality of photomultiplier tubes to reconstruct an image showing distinct features of a specimen strained with different dyes. The images may also be constructed using a single detector in a time-multiplex fashion and using a computer to construct the image.
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Citations
16 Claims
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1. A multi-color confocal microscopy system for use with an optical microscope in viewing a specimen stained with a plurality of stains, which stains fluoresce when excited by light of particular frequencies, comprising:
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laser means for producing multi-line incident laser light having multiple excitation lines from a single laser light source, each of said excitation lines corresponding to an excitation frequency of one of the stains; means for directing said incident laser light into the microscope and for receiving emitted light from the microscope, said emitted light having multiple fluorescent emissions, each of said fluorescent emissions corresponding to an excitation frequency of one of the stains; detector means positioned to receive said emitted light for converting said emitted light into electrical signals; and control means connected to said detector means for accumulating said electrical signals and for producing a plurality of images of the specimen at a precise focal plane, each of said images corresponding to one of said lines of said laser. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. The multi-color confocal microscopy system for use with an optical microscope viewing a specimen stained with a plurality of stains, comprising:
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a multi-line laser producing laser light having a plurality of lines, some of said lines being selectively prevented from exiting said laser, and some other of said lines being selectively allowed to exit said laser as excitation lines, each of said excitation lines selected to correspond to an excitation frequency of only one of the plurality of stains; a plurality of excitation filters each selectively placed in the path of said laser light; a first dichroic mirror placed in the path of said laser light behind said excitation filters, said first dichroic mirror selected to reflect excitation lines and to pass emitted fluorescent light; a scanning unit placed to receive said laser light from said first dichroic filter and reflect said laser light in a raster-scanned form into the optical microscope; said scanning unit further placed to receive said emitted fluorescent light from the optical microscope, to descan said raster-scanned form and to reflect said emitted fluorescent light back through said first dichroic filter; a second dichroic mirror placed in the path of said fluorescent light behind said first dichroic filter, said second dichroic mirror selected to reflect some wavelengths of said fluorescent emission of said emitted light and pass other wavelengths of said emitted fluorescent light; and a first detector placed to receive said reflected fluorescent light passed from said second dichroic mirror and a second detector placed to receive said reflected fluorescent light reflected from said second dichroic mirror.
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13. A method of viewing a specimen stained with a plurality of stains, which stains fluoresce when excited by light of particular frequencies, comprising the steps of:
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producing multi-line incident laser light having multiple excitation lines from a single laser light source, each of said excitation lines corresponding to an excitation frequency of one of the stains; directing said incident laser light into a microscope holding the specimen; receiving emitted light from the microscope, said emitted light having multiple fluorescent emissions, each of said fluorescent emissions corresponding to an excitation frequency of one of the stains; detecting said emitted light and converting said emitted light into electrical signals; and accumulating said electrical signals and producing a plurality of images of the specimen at a precise focal plane, each of said images corresponding to one of said lines of said laser. - View Dependent Claims (14, 15, 16)
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Specification