Method and apparatus for analysis of particles in a liquid sample
Inventors
Lindberg, Stellan • Olesen, Tom • Valvik, Martin
Assignees
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Publication Number
US-8009894-B2
Publication Date
2011-08-30
Expiration Date
Abstract
The invention relates to methods for analysis of particles in a liquid sample, the sample being retained in a sample retaining device, the method comprising: acquiring, by means of an image acquiring device, a plurality of images of said sample at different focus planes within the sample retaining device; analysing said images, by means of an image analyser, for identifying which, if any, of the particles of the sample are imaged in focus in each of the images, and analysing those particles which have been identified as being imaged in focus; wherein said plurality of images are acquired at different, essentially parallel, focus planes, which planes are separated from each other by a distance, said distance being less than 10 micrometers. The invention also relates to apparatuses adapted to be used for the inventive methods.
Core Innovation
The invention relates to a method for analysis of particles in a liquid sample, where the sample is retained in a sample retaining device. A plurality of images of the sample are acquired at different focus planes within the sample retaining device, and the focus planes are essentially parallel and separated by a distance of less than 10 micrometers.
The method analyses the acquired images to identify which particles are imaged in focus in any one of the analysed images. For each identified particle, the method identifies the image or images in which the particle is identified as being imaged in focus, and then analyses the particles using the respective image in which the particle has been identified as being imaged in focus.
Particle in-focus identification is achieved by, for each particle, finding an image in which the particle is distinguishable and then determining a focus metric across images. In one implementation, the metric is based on the greatest difference in light intensity between an area occupied by the particle and an area where no particle is distinguishable; in another implementation, the metric is based on pixel variance of an area comprising the particle and its closest surroundings.
Claims Coverage
The independent claims identified in the provided material are clm-00001, clm-00030, clm-00031, and clm-00034. They share a common inventive approach of acquiring multiple essentially parallel, closely spaced focus-plane images within a sample retaining device and determining, per particle, which image represents that particle being in focus, using either a light-intensity difference metric or a pixel-variance metric.
Parallel closely spaced focus-plane image acquisition in a sample retaining device
Acquiring, by means of an image acquiring device, a plurality of images of the sample at different, essentially parallel, focus planes within the sample retaining device, where the planes are separated by a distance of less than 10 micrometers.
Per-particle in-focus image identification using light-intensity contrast
Identifying particles imaged in focus by, for each particle, finding an image in which the particle is distinguishable; determining the greatest difference in light intensity between an area occupied by the particle and an area where no particle is distinguishable; determining corresponding differences in light intensity in other images where the same particle is distinguishable; and identifying the image where the difference in light intensity is the highest, whereby the particle is regarded as being in focus in that identified image.
Per-particle in-focus image identification using pixel variance
Identifying particles imaged in focus by, for each particle, defining an area of a found image comprising the particle and its closest surroundings; determining the pixel variance of that defined area; determining the pixel variance of the corresponding area of other images in which the same particle is distinguishable; and identifying the image where the pixel variance is the highest, whereby the particle is regarded as being in focus in that identified image.
Movable stepwise focus plane for apparatus-based image acquisition
A measurement apparatus in which a focus plane is movable stepwise within the sample retaining device when held by the holder, whereby the image acquiring device is adapted to acquire a plurality of images of the sample at different essentially parallel focus planes separated by a distance less than 10 micrometers.
Across the independent claims, the core coverage is the same focus-plane imaging framework combined with particle-specific selection of the in-focus image. The claims distinguish between two in-focus identification metrics: choosing the image with the highest light-intensity difference between particle-occupied and non-particle areas, or choosing the image with the highest pixel variance for a defined particle-and-surroundings region.
Stated Advantages
Faster analysis without settling.
Volumetric enumeration.
Depth/discrimination of overlapping particles.
Optional particle typing via physical features (e.g., size/shape).
Documented Applications
Blood sample workflow using dried haemolysing/staining reagents in-device and analysing white blood cells by counting and ratios.
Nucleus imaging based on irradiation at a staining absorption wavelength and detection of black/darker dots at nuclei.
Particle concentration measurement via volumetric enumeration basis derived from imaged area and imaged depth.
Analysis of particles in biological samples including blood, milk, urine, and spinal fluid.
Optional particle typing and counting based on physical features such as size, colour, opalescence, and shape.
General particle analysis including cells (e.g., white blood cells, platelets) and microorganisms such as bacteria and viruses.
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