Differentiation of flow cytometry pulses and applications
View Patent ↗A method of analyzing pulses from a flow cytometer in which particles in a fluid pass through an excitation volume of an electromagnetic radiation and interact with the electromagnetic radiation to generate signals in the form of pulses includes generating a time-dependent pulse indicative of the characteristics of one or more particles passing through the excitation volume of the electromagnetic radiation, determining a measurement window by selecting a portion of the pulse with a starting point and an ending point above a predetermined value, and calculating a first derivative of the pulse with respect to time over the measurement window.
1. A method of analyzing pulses of a capillary flow cytometer comprising:
passing a fluid containing particles through an excitation volume in a capillary flow cytometer, said excitation volume being defined at least partially by a light beam and a portion of a capillary illuminated by the light beam;
generating a time-dependent pulse as one or more particles pass through the excitation volume;
computing a first derivative of the pulse with respect to time;
calculating a velocity of the one or more particles passing through the excitation volume using the computed first derivative; and
correcting the time-dependent pulse using the calculated velocity.
2. The method of claim 1 wherein said passing step comprises passing a liquid sample containing the particles through the excitation volume.
3. The method of claim 1 wherein said excitation volume is defined at least partially by a focused light beam having a single peak.
4. The method of claim 1 wherein the generating step comprises generating a time-dependent pulse indicative of scatter light from the one or more particles interacting with the light beam when passing through the excitation volume.
5. The method of claim 1 wherein the generating step comprises generating a time-dependent pulse indicative of fluorescence from the one or more particles interacting with the light beam when passing through the excitation volume.
6. The method of claim 1 wherein said correcting step comprises multiplying an area or a width of the time-dependent pulse by the calculated velocity.
7. The method of claim 1 further comprising the steps of determining a number of times the first derivative has a zero value, and identifying a singlet or doublet pulse using the number of times.
8. The method of claim 1 further comprising identifying a maximum of the first derivative, calculating a ratio of the identified maximum to a width of the pulse, and differentiating a singlet pulse from a doublet pulse using the calculated ratio.
9. An apparatus for analyzing particles comprising:
a capillary tube adapted to be filled by a fluid containing particles to be analyzed;
a source of light;
an optical system operable to direct a light beam from the source of light to a portion of the capillary tube forming an excitation volume;
means for causing the fluid to pass through the excitation volume in which particles to be analyzed interact with the light beam to scatter light and/or emit fluorescence;
means for detecting the fluorescence in form of pulses; and
means for computing a first derivative of the pulses with respect to time over a measurement window.
10. The apparatus of claim 9 further comprising means for detecting scatter light in form of pulses, and means for calculating a first derivative of the scatter light pulses with respect to time over a measurement window.
11. The apparatus of claim 9 further comprising means for calculating a velocity of particles using the computed first derivative of the pulses.
12. The apparatus of claim 9 wherein said optical system is adapted to direct a focused light beam to the portion of the capillary tube.
13. The method of claim 1 wherein the particle containing fluid is a liquid fluid.
14. The method of claim 1 wherein the light beam is a single Gaussian beam produced by a laser.