Fluorometry method and apparatus using a semiconductor laser diode as a light source
First Claim
1. An apparatus for detecting, in the frequency-domain, a change in phase angle and/or modulation, corresponding to a change in a lifetime in a range from about 50 ps to 100 ns, of a fluorescence emission of at least one fluorophore comprising:
- semiconductor laser diode means which outputs modulated light when driven by a first electrical signal, said light being substantially monochromatic and coherent;
first electrical signal generator means (5a), coupled to said semiconductor laser diode means, for generating said first electrical signal to cause said semiconductor laser diode means to output said modulated light having a wavelength which excites said at least one fluorophore to emit fluorescence; and
second electrical signal generator means (5b), having a synchronization port coupled to said first electrical signal generator means or to an auxiliary electrical signal corresponding to the light output of said laser diode means, said second electrical signal generator means generating at a signal output port thereof a second electrical signal that is phase locked to a modulation phase of said light output of said laser diode means;
a first electrical signal mixer (7) and a second electrical signal mixer (9), said first mixer being responsive to said second electrical signal and to a signal derived from said first electrical signal to produce a first difference-frequency signal;
photodetector means for sensing said emitted fluorescence, said photodetector means being coupled via said second mixer to said second electrical signal generator means to produce a second difference-frequency signal representative of a change in phase angle and/or modulation of said emitted fluorescence of said at least one fluorophore, wherein said change corresponds to a change in fluorescence lifetime; and
comparator means for comparing the second difference-frequency signal with the first difference-frequency signal to produce a signal indicative of said change in fluorescence.
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Abstract
A method and apparatus for detecting the change in phase angle and/or modulation of emitted fluorescence of a fluorophore excited by modulated light from a laser diode. The light is both monochromatic and coherent, and can contain harmonic frequency components. The laser diode can be used in frequency-domain or phase-modulation measurements of fluorescence, using either phase or modulation techniques. The present invention is an improvement of the prior use of a frequency-doubled, cavity-dumped, dye laser in fluorometers. The invention provides an inexpensive light excitation source that is small in size, easily manageable, allows for short measurement times, and has lower power requirements.
143 Citations
16 Claims
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1. An apparatus for detecting, in the frequency-domain, a change in phase angle and/or modulation, corresponding to a change in a lifetime in a range from about 50 ps to 100 ns, of a fluorescence emission of at least one fluorophore comprising:
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semiconductor laser diode means which outputs modulated light when driven by a first electrical signal, said light being substantially monochromatic and coherent; first electrical signal generator means (5a), coupled to said semiconductor laser diode means, for generating said first electrical signal to cause said semiconductor laser diode means to output said modulated light having a wavelength which excites said at least one fluorophore to emit fluorescence; and second electrical signal generator means (5b), having a synchronization port coupled to said first electrical signal generator means or to an auxiliary electrical signal corresponding to the light output of said laser diode means, said second electrical signal generator means generating at a signal output port thereof a second electrical signal that is phase locked to a modulation phase of said light output of said laser diode means; a first electrical signal mixer (7) and a second electrical signal mixer (9), said first mixer being responsive to said second electrical signal and to a signal derived from said first electrical signal to produce a first difference-frequency signal; photodetector means for sensing said emitted fluorescence, said photodetector means being coupled via said second mixer to said second electrical signal generator means to produce a second difference-frequency signal representative of a change in phase angle and/or modulation of said emitted fluorescence of said at least one fluorophore, wherein said change corresponds to a change in fluorescence lifetime; and comparator means for comparing the second difference-frequency signal with the first difference-frequency signal to produce a signal indicative of said change in fluorescence. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for detecting, in the frequency-domain, changes in phase angle and/or modulation, corresponding to changes in a lifetime in a range of 50 ps to 100 ns, of emitted fluorescence of at least one fluorophore in a sample, comprising the steps of:
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driving a semiconductor laser diode means with a first electrical signal to cause said semiconductor laser diode means to output modulated light having a wavelength capable of exciting said at least one fluorophore to emit fluorescence, wherein said modulated light is monochromatic and coherent; exciting said at least one fluorophore with said modulated light to produce a fluorescence emission; generating a second electrical signal, coupled to said first electrical signal or to an auxiliary electrical signal corresponding to the light output of said laser diode means, said second electrical signal being phase locked to said light output of said laser diode means; sensing said fluorescence emission to provide a third electrical signal corresponding to said fluorescence emission; and comparing said second electrical signal with said third electrical signal to generate a difference signal representative of said change in phase angle and/or modulation, corresponding to a change in lifetime, of said emitted fluorescence of said at least one fluorophore. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16)
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Specification