Electrooptic measurement device and method intended for classifying and counting microscopic elements
The invention relaters to a device ( 100 ) for biological analysis by measurement of photoluminescence in a fluid in a measurement tank ( 111 ). This device ( 100 ) comprises at least two light sources ( 121, 131 ) adapted to emit in different spectral areas respectively appropriate for measurement of absorption and fluorescence, and a sensor device ( 140 ) comprising a sensor ( 141 ), an optical system ( 142 ), and filter means ( 144 ), which three elements are mutualized in accordance with the invention to enable absorption and/or fluorescence to be measured. In accordance with the invention the internal gain of the sensor ( 141 ) is configurable to enable the fluorescence and absorption measurements to be executed sequentially.
1. A device ( 100 ) for biological analysis by electro-optical measurements on a fluid in a measurement tank ( 111 ), said device ( 100 ) comprising at least two light sources ( 121 , 131 ) configured to emit in different spectral areas respectively appropriate for measurement of absorption and fluorescence, a means for measuring the impedance associated with each absorption and fluorescence measurement, and a shared detection means ( 140 ) comprising three elements being mutualized to enable absorption and/or fluorescence to be measured, said three elements being a sensor ( 141 ), an optical system ( 142 ), and filter means ( 144 ), the internal gain of the sensor ( 141 ) being configurable to enable the fluorescence and absorption measurements to be executed sequentially.
2. A device according to claim 1 , characterized in that the sensor ( 141 ) is an avalanche photodiode connected to a bias circuit ( 143 ) adapted to inhibit the internal avalanche gain.
3. A device according to claim 2 , characterized in that the inhibited internal gain is 1, the avalanche photodiode ( 141 ) behaving as a simple photodiode.
4. A device according to claim 2 , characterized in that the bias circuit ( 143 ) comprises a two-position switch providing two different voltages, a high voltage and a low voltage, and electrically controlled to provide the high voltage or the low voltage for biasing the photodiode ( 141 ).
5. A device according to claim 2 , characterized in that the bias circuit ( 143 ) comprises a programmable voltage generator controlled by digital or analog means to control the bias voltage applied by this generator to the avalanche photodiode.
6. A device according to claim 1 , characterized in that the two light sources ( 121 , 131 ) have emission optical axes coplanar with the optical axis of the light sensor ( 141 ).
7. A device according to claim 6 , characterized in that the optical axis of the light source ( 131 ) emitting at the absorption wavelengths is aligned with that of the light sensor ( 141 ) and the optical axis of the light source for exciting fluorescence ( 121 ) is perpendicular to the optical axes of the sensor ( 141 ) and the other light source ( 131 ).
8. A device according to claim 1 , characterized in that the sensor ( 141 ) is connected to an electronic amplification stage ( 203 ) with electronic gain variable as a function of the measurement effected.
9. A device according to claim 8 , characterized in that the internal gain of the sensor ( 141 ) and the electronic gain are configurable to enable discriminating absorption and fluorescence measurements for a plurality of cell populations chosen from at least basophils, lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, platelets, erythroblasts, reticulocytes, and reticulated platelets.
10. A device according to claim 8 , characterized in that the internal gain of the sensor ( 141 ) and the electronic gain are configurable to enable, with the sensor device, discriminating absorption and fluorescence measurements for each analysis cycle, a cycle enabling the classification and the absolute counting of a family of cells present in the sample.
11. A device according to claim 10 , characterized in that the internal gain of the sensor ( 141 ) and the electronic gain are configurable to enable, with the sensor device, discriminating absorption and fluorescence measurements for at least the analysis cycles of basophils and reticulocytes.
12. A device according to claim 1 , characterized in that, for the fluorescence measurement, a fluorescence marker being used is Thiazole Orange, the fluorescence excitation source emits at around 470 nm and the filter means of the sensor device comprise a colored glass-type filter of cut-off wavelength situated in a vicinity of 495 nm.
13. A device according to claim 1 , characterized in that the two sources may be turned on simultaneously to obtain biochemical and morphometric data.
14. A method for classifying and counting cell populations present in the biological sample, wherein the method uses the device of claim 1 for classifying and counting the cell populations and comprises the steps of:
placing a fluid in the measurement tank, the fluid comprising cell populations present in a biological sample,
emitting light in different spectral areas respectively appropriate for measurement of absorption and/or fluorescence with the at least two light sources,
measuring the impedance of the cell populations, and
measuring associated absorption and/or fluorescence of the cell populations.