Patent ID: 8681215
Filing Date: 2014-03-25
Classification: G01N

Abstract:
1. A method, comprising: a) holding a liquid sample in a sample cell of a particle analyzer, the liquid sample including particles of a lower size range that are resolvable by dark-field imaging, particles of an intermediate size range that are resolvable by dark-field imaging and by bright-field imaging, and particles of an upper size range that are resolvable by bright-field imaging, some of the particles in the liquid sample being fluorescent particles that emit light in a first wavelength band after absorbing light in a second wavelength band, b) illuminating the liquid sample in the sample cell with light in the first wavelength band with the dark-field light source in a dark-field geometry to yield scattered light in the first wavelength band; c) collecting, focusing, and detecting the scattered light in the first wavelength band with the imaging system to capture a dark-field image frame in which the particles of the lower size range and the particles of the intermediate size range are resolved, d) analyzing the dark-field image frame with an analysis module of the processing system to locate images of the particles of the lower size range and the particles of the intermediate size range; e) analyzing the images of the particles of the lower size range and the particles of the intermediate size range from the dark-field image frame with the analysis module of the processing system to determine relative sizes of the particles of the lower size range and the particles of the intermediate size range; f) illuminating the liquid sample in the sample cell with light in the first wavelength band with the bright-field light source in a bright-field geometry to yield transmitted light in the first wavelength band; g) collecting, focusing, and detecting the transmitted light in the first wavelength band with the imaging system to capture a bright-field image frame in which the particles of the intermediate size range and the particles of the upper size range are resolved, h) analyzing the bright-field image frame with the analysis module of the processing system to locate images of the particles of the intermediate size range and the particles of the upper size range; i) analyzing the images of the particles of the intermediate size range and the particles of the upper size range from the bright-field image frame with the analysis module of the processing system to determine absolute sizes of the particles of the intermediate size range and the particles of the upper size range; j) comparing the dark-field image frame and the bright field image frame with the analysis module of the processing system to identify corresponding images of the particles of the intermediate size range located in both the dark-field image frame and the bright-field image frame; k) comparing the relative sizes and the absolute sizes of the particles of the intermediate size range that were determined by analyzing the corresponding images with the analysis module of the processing system to determine a calibration curve; l) applying the calibration curve to the relative sizes of the particles of the lower size range with the analysis module of the processing system to determine calibrated sizes of the particles of the lower size range; m) illuminating the liquid sample in the sample cell with light in the second wavelength band with the fluorescence light source to yield fluorescent light in the first wavelength band and scattered light in the second wavelength band; n) passing the scattered light, the transmitted light, and the fluorescent light in the first wavelength band to the detector array with the wavelength selective filter of the imaging system, while blocking the scattered light in the second wavelength band with the wavelength selective filter; o) collecting and focusing the fluorescent light in the first wavelength band onto the detector array with the magnification sub-system; p) detecting the fluorescent light in the first wavelength band with the detector array to capture a fluorescence image frame; q) analyzing the fluorescence image frame with the analysis module of the processing system to locate images of the fluorescent particles; and r) comparing the fluorescence image frame, the dark-field image frame, and the bright-field image frame with the analysis module of the processing system to identify corresponding images of the fluorescent particles located in the fluorescence image frame and in either or both of the dark-field image frame and the bright-field image frame; s) determining a particle size distribution of the liquid sample from the calibrated sizes of the fluorescent particles of the lower size range, and the absolute sizes of the fluorescent particles of the intermediate size range and the fluorescent particles of the upper size range with the analysis module of the processing system.