Methods for modulation and synchronous detection in a flow cytometer and systems for same
Methods for determining a parameter of a particle in a flow stream (e.g., in a particle analyzer of a flow cytometer) from scattered light are described. Methods according to certain embodiments include irradiating a particle in a flow stream with a frequency-modulated beam of laser light modulated at a reference frequency, detecting scattered light from the particle with a photodetector, generating a frequency-encoded data signal from the detected scattered light, synchronizing the frequency-encoded data signal with the reference frequency and determining one or more parameters of the particle based on the synchronized frequency-encoded data signal. Systems and non-transitory computer readable storage medium with instructions for practicing the subject methods are also provided.
1 . A particle analyzer for determining one or more parameters of a particle in a flow stream, the particle analyzer comprising:
a light source comprising:
a laser;
an input polarizer; and
an electro-optical modulator,
wherein the light source is configured to generate a polarized frequency-modulated beam of laser light at a reference frequency;
a flow cell for conveying the particle through the flow stream;
an output polarizer;
a light detection system comprising a photodetector configured to detect scattered light from an irradiated particle in the flow stream conveyed through the output polarizer; and
a processor comprising memory operably coupled to the processor wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to:
generate a reference frequency signal comprising a reference waveform;
generate a frequency-encoded data signal from the detected polarized scattered light;
synchronize the frequency-encoded data signal with the reference frequency to generate a synchronized frequency-encoded data signal by multiplying the frequency-encoded data signal with the reference waveform; and
determine one or more parameters of the particle based on the synchronized frequency-encoded data signal.
2 . The particle analyzer according to claim 1 , wherein the particle analyzer is incorporated into a flow cytometer.
3 . The particle analyzer according to claim 1 , further comprising a particle sorter for sorting the particle based on one or more of the determined parameters of the particle.
4 . The particle analyzer according to claim 1 , wherein the electro-optical modulator is a piezo optic modulator.
5 . The particle analyzer according to claim 1 , wherein the photodetector comprises a forward scatter detector.
6 . The particle analyzer according to claim 1 , wherein the photodetector comprises a side scatter detector.
7 . The particle analyzer according to claim 1 , wherein the output polarizer comprises a quarter waveplate.
8 . The particle analyzer according to claim 6 , wherein the light detection system comprises two side scatter detectors.
9 . The particle analyzer according to claim 8 , wherein the light detection system comprises:
a first side scatter detector comprising a first polarizer having a first polarization; and
a second side scatter detector comprising second polarizer having a second polarization that is perpendicular to the first polarization.
10 . The particle analyzer according to claim 1 , wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to detect the amplitude of light scattered by the particle that is oscillating at the reference frequency.
11 . The particle analyzer according to claim 1 , wherein the particle analyzer is in communication with a lock-in amplifier configured to synchronize the frequency-encoded data signal with the reference frequency.
12 . The particle analyzer according to claim 11 , further comprising an FPGA configured to synchronize the frequency-encoded data signal with the reference frequency by lock-in amplification.
13 . The particle analyzer according to claim 1 , wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to digitally synchronize the frequency-encoded data signal with the reference frequency.
14 . The particle analyzer according to claim 13 , wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to apply a low-pass filter to the synchronized frequency-encoded data signal to generate a data signal profile.