Systems and methods for distinguishing optical signals of different modulation frequencies in an optical signal detector
Systems and method for detecting optical signals, and for discriminating optical signals emitted by an emission moiety that is excited by an associated excitation signal from background signals and other optical noise, employing digital techniques for determining the portion of a detected optical signal having a modulation frequency corresponding to a modulation of the associated excitation signal.
1. A method for determining a concentration of an analyte of interest in a sample contained in a receptacle by detecting an optical signal emitted from the contents of the receptacle in the presence of multiple, different optical signals originated from the same receptacle or from different receptacles, said method comprising:
(a) forming amplification products from the analyte of interest contained within the receptacle;
(b) hybridizing a detectably labeled probe to the amplification products;
(c) generating an optical excitation signal, wherein the optical excitation signal has a predetermined excitation wavelength that excites an emission moiety of the probe, which emits an optical emission signal that is associated with the excitation wavelength, and wherein the optical excitation signal is modulated at a predetermined modulation frequency;
(d) while forming the amplification products, directing the optical excitation signal at the contents of the receptacle;
(e) with a signal detecting device, detecting an optical signal including an optical emission signal emitted from the contents of the receptacle and converting the detected optical signal to an analog detection signal;
(f) digitizing the analog detection signal using a computer-implemented signal-digitizing algorithm to generate digitized detection data;
(g) determining the amplitude of the digitized detection data that is at the predetermined modulation frequency by mathematically processing the digitized detection data using a computer-implemented digital signal processing technique for determining the amplitude of the optical signal at the predetermined modulation frequency to thereby ascertain the portion of the detected optical signal that corresponds to the associated optical emission signal; and
(h) determining the concentration of the analyte of interest present within the receptacle by executing a computer-implemented algorithm for processing data relating to the portion of the detected optical signal that corresponds to the associated optical emission signal determined in step (g).
2. The method of claim 1 , further comprising determining a concentration of a second analyte of interest in a sample contained in a receptacle by detecting a second optical signal emitted from the contents of the receptacle by:
(i) generating a second optical excitation signal, wherein the second optical excitation signal has a predetermined second excitation wavelength that excites an emission moiety of the probe, which emits a second optical emission signal that is associated with the second excitation wavelength and wherein the second optical excitation signal is modulated at a predetermined second modulation frequency;
(j) while forming the amplification products, directing the second optical excitation signal at the contents of the receptacle;
(k) with the same or a different signal detecting device, detecting a second optical signal including an optical emission signal emitted from the contents of the receptacle and converting the detected second optical signal to a second analog detection signal;
(l) digitizing the second analog detection signal using a computer-implemented signal-digitizing algorithm to generate second digitized detection data;
(m) determining the amplitude of the second digitized detection data that is at the predetermined second modulation frequency by mathematically processing the second digitized detection data using a computer-implemented signal-processing technique for determining the amplitude of the second optical signal at the predetermined second modulation frequency to thereby ascertain the portion of the second detected optical signal that corresponds to the associated second optical emission signal; and
(n) determining the concentration of the second analyte of interest present within the receptacle by executing a computer-implemented algorithm for processing data relating to the portion of the second detected optical signal that corresponds to the associated second optical emission signal determined in step (m).
3. The method of claim 2 , wherein the modulation frequencies are different for at least two optical signals to be detected.
4. The method of claim 1 , wherein the optical emission signal is a fluorescent emission.
5. The method of claim 1 , wherein the emission moiety comprises a fluorescent dye.
6. The method of claim 1 , wherein step (g) comprises executing a Goertzel signal processing technique.
7. The method of claim 1 , wherein the analog detection signal is digitized at a frequency that is at least twice the modulation frequency of the associated optical excitation signal.
8. The method of claim 7 , wherein the modulation frequency is within the range of 200-350 Hz, and the digitization frequency is about 4 kHz.