Method and apparatus for analysis of particles in a liquid sample

A method 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;
• and
• analysing said images, by means of an image analyser, for identifying particles of the sample which are imaged in focus in any one of the analysed images, and for identifying, for each of the identified particles, in which of said analysed images the particle is identified and analysing those particles which have been identified as being imaged in focus in any one of the analysed images using respective image in which respective particle has 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;
• wherein said identification of particles which are imaged in focus is achieved by, for each of the particles:
• finding an image in which the particle is distinguishable;
• defining an area of that found image, said area 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.

A measurement apparatus for analysis of particles in a liquid sample, the apparatus comprising an image acquiring device, an image analyser, a holder arranged to hold a sample retaining device retaining the liquid sample, and a light refractor positioned between said image acquiring device and said holder;

• wherein a focus plane is movable stepwise within the sample retaining device when it is held by the holder, whereby the image acquiring device is adapted to acquire a plurality of images of said sample at different focus planes within the sample retaining device, the different focus planes being essentially parallel to each other and being separated from each other by a distance, said distance being less than 10 micrometers;
• wherein the image analyser is arranged to analyse at least one acquired image for identifying which of the particles are imaged in focus and analysing those particles which have been identified as being imaged in focus;
• wherein said identification of particles which are imaged in focus is achieved by, for each of the particles:
• finding an image in which the particle is distinguishable;
• determining the greatest difference in light intensity between an area of the image which is occupied by the particle and an area of the image where no particle is distinguishable;
• determining the corresponding differences in light intensity in other images in which 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.

A measurement apparatus for analysis of particles in a liquid sample, the apparatus comprising an image acquiring device, an image analyser, a holder arranged to hold a sample retaining device retaining the liquid sample, and a light refractor positioned between said image acquiring device and said holder;

• wherein a focus plane is movable stepwise within the sample retaining device when it is held by the holder, whereby the image acquiring device is adapted to acquire a plurality of images of said sample at different focus planes within the sample retaining device, the different focus planes being essentially parallel to each other and being separated from each other by a distance, said distance being less than 10 micrometers;
• wherein the image analyser is arranged to analyse at least one acquired image for identifying which of the particles are imaged in focus, and analysing those particles which have been identified as being imaged in focus;
• wherein said identification of particles which are imaged in focus is achieved by, for each of the particles:
• finding an image in which the particle is distinguishable;
• defining an area of that found image, said area 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.