Compact multiwavelength phase fluorometer
First Claim
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1. An apparatus for changing the frequency of excitation light incident upon a sample, said apparatus comprising:
- a plurality of excitation light sources, each said excitation light source having an optical axis and emitting a different frequency of excitation light when energized, said plurality of excitation light sources being radially equidistant from an axis;
two parallel reflecting surfaces rotatable about said axis to redirect excitation light from the optical axis of a selected one of said excitation light sources along said axis, whereby the excitation light from the selected excitation light source is incident upon said sample.
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Abstract
The present invention is a compact instrument capable of operating as a steady-state fluorimeter or as a phase fluorometer, and thus it is able to measure steady-state fluorescence intensity and fluorescence lifetime from a fluorescent sample. The light source provides four user-selectable excitation wavelengths generated by light-emitting diodes (LEDs), and has an autocalibration feature and a means to compensate for phase measurement errors due to phase-amplitude crosstalk in the detection electronics.
34 Citations
16 Claims
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1. An apparatus for changing the frequency of excitation light incident upon a sample, said apparatus comprising:
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a plurality of excitation light sources, each said excitation light source having an optical axis and emitting a different frequency of excitation light when energized, said plurality of excitation light sources being radially equidistant from an axis;
two parallel reflecting surfaces rotatable about said axis to redirect excitation light from the optical axis of a selected one of said excitation light sources along said axis, whereby the excitation light from the selected excitation light source is incident upon said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for selecting the frequency of excitation light incident upon a fluorometer sample comprising:
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arranging a plurality of excitation light sources at a fixed radial distance from a central axis, each of said excitation light sources producing a different frequency of excitation light along an optical axis;
rotating first and second reflecting surfaces around said central axis, said first reflecting surface being intersected by said central axis and said second reflecting surface being in fixed parallel relationship to said first reflecting surface, said second reflecting surface traversing an arc that intersects each of the optical axes of the plurality of excitation light sources; and
stopping said rotation with said second reflecting surface substantially centered on the optical axis of a selected one of said plurality of excitation light sources, whereby light is offset from the optical axis of a selected one of said excitation light sources to a path along said central axis and incident upon said sample. - View Dependent Claims (9, 10)
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11. A method for reducing phase-amplitude crosstalk in a phase fluorometer comprising a photomultiplier tube which produces an output whose amplitude varies with the intensity of detected fluorescence, said amplitude also varying according to the level of high voltage applied to a dynode of the photomultiplier tube:
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applying the amplitude to a feedback circuit to vary the high voltage applied to the dynode of the photomultiplier tube, whereby the output amplitude is maintained at a selected set point. - View Dependent Claims (12, 13)
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14. An apparatus for self calibration in a fluorometer comprising:
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a fiber optic reference channel having an input end arranged to receive a portion of excitation light produced by an excitation light source and an output end coupled to a fluorescence detector, said reference channel having a known optical delay; and
a calibration channel shutter arranged to prevent excitation light from traversing the reference channel during fluorescence measurement and to prevent a fluorescence produced by a sample exposed to the excitation light from reaching the detector during calibration, wherein excitation light traversing the calibration channel has a phase delay including the known optical delay of the reference channel and a variable delay due to propagation of optical and electrical signals through the fluorometer, the phase delay of excitation light through the calibration channel being compared to a previous value of phase delay through the excitation channel to determine a calculated time drift attributable to changes in the propagation of optical and electrical signals through the fluorometer.
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15. A method for continuously calibrating a fluorometer comprising:
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diverting a portion of excitation light directed at a sample into a calibration channel having an output coupled to a fluorescence detector;
activating a shutter to prevent fluorescence from the sample from reaching the detector, said shutter permitting excitation light traversing the calibration channel to reach the detector;
measuring a second phase delay between the signal used to modulate a source of the excitation light and an output of the detector;
comparing the measured second phase delay to a previous first phase delay measurement through the calibration channel to determine a drift in the propagation of optical and electrical signals through the fluorometer;
activating the shutter to permit fluorescence from the sample to reach the detector, said shutter preventing excitation light from reaching the detector through the calibration channel;
measuring the phase delay between the fluorescence from the sample and the signal used to modulate a source of the excitation light and an output of the detector; and
using the drift to calculate a corrected phase delay of the fluorescence measured from the sample. - View Dependent Claims (16)
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Specification