Optical flow cytometer for fluorescence and scattering measurements by segmentation of beam emitted by a single non-coherent light source
An optical flow cytometer for fluorescence and scattering measurement includes a non-coherent light source generating an illumination beam; and a beam truncation device having: a first passage accommodating a first part of the illumination beam with a first divergence, the first part performing scattering measurements; a second passage accommodating a second part of the illumination beam with a second divergence, the second part being dedicated to fluorescence measurements; the second divergence being greater than the first divergence; the first and second passages separated by a beam truncation device area that stops illumination beam; a focalization lens focusing the first part and the second part of the illumination beam on a flowcell having an interrogation zone; a scattering detector receiving light scattered from the first illumination beam part as a particle crosses the interrogation zone; and a fluorescent detector receiving fluorescent light emitted by the particle crossing the interrogation zone.
1 . An optical flow cytometer for fluorescence measurement and scattering measurement comprising:
a non-coherent light source intended to generate an illumination beam; and
a beam truncation device comprising at least:
a first passage to let pass a first part of the illumination beam with a first divergence, the first part being dedicated to scattering measurements;
a second passage to let pass a second part of the illumination beam with a second divergence, the second part being dedicated to fluorescence measurements;
the second divergence being greater than the first divergence; the first and second passages being separated by an area of the beam truncation device that stops the illumination beam;
at least a focalization lens to focus said first part and said second part of the illumination beam on a flowcell comprising an optical interrogation zone;
the flowcell intended to contain particles flowing through the optical interrogation zone; the beam truncation device and the at least a focalization lens are optically positioned between the light source and the flowcell;
a scattering detector for receiving light that is scattered from the first part of the illumination beam as a particle crosses the optical interrogation zone; and
a fluorescent detector for receiving fluorescent light emitted by the particle crossing the optical interrogation zone.
2 . The optical flow cytometer according to claim 1 , characterized in that the non- coherent light source is an LED, a filament lamp or an arc lamp.
3 . The optical flow cytometer according to claim 1 , characterized in that the second passage has a cross section larger than the cross section of the first passage.
4 . The optical flow cytometer according to claim 1 , characterized in that the first passage is centered along the optical axis of the illumination beam.
5 . The optical flow cytometer according to claim 1 , characterized in that a second beam truncation device is disposed after the flowcell to stop the first and/or the second part of the illumination beam.
6 . The optical flow cytometer according to claim 1 , characterized in that, when the first passage has a circular cross section with a radius R, the radial distance between the first passage and the second passage is greater than or equal to 0.2*R.
7 . The optical flow cytometer according to claim 1 , characterized in that the beam truncation device comprises at least one filter to be placed on the first passage and/or on the second passage in order to change the spectral characteristic of the first part of the illumination beam and/or the second part of the illumination beam respectively.
8 . The optical flow cytometer according to claim 1 , characterized in that the beam truncation device comprises at least one polarizer to be placed on the first passage and/or on the second passage in order to change the polarization of the first part of the illumination beam and/or the second part of the illumination beam respectively.
9 . The optical flow cytometer according to claim 1 , characterized in that the beam truncation device comprises at least one another lens to be placed on the first passage and/or on the second passage in order to change the direction of the first part of the illumination beam and/or the second part of the illumination beam respectively.
10 . The optical flow cytometer according to claim 1 , characterized in that the beam truncation device comprises a diaphragm where the first passage and the second passage are holes.
11 . The optical flow cytometer according to claim 1 , characterized in that the cross section of the first passage and/or of the second passage is circular, square, rectangular or random.
12 . The optical flow cytometer according to claim 1 , characterized in that a fluorescence lens is disposed at 90° with respect to the optical axis in order to converge fluorescence light coming from the flowcell to the fluorescence detector for a 90° fluorescence measurement.
13 . The optical flow cytometer according to claim 1 , characterized in that the scattering detector is arranged to detect light scattered by the flowing particle at angles within a range of 0°-5° measured from the divergence of the first part of the illumination beam.
14 . The optical flow cytometer according to claim 1 , characterized in that a detection lens is disposed after the flowcell to collect and focus light scattered by the flowing particle to the scattering detector.