Fluorometry method and apparatus using a semiconductor laser diode as a light source

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.