Imaging system

A method of determining a position or a movement of a particle in a sample, comprising the steps of:

• a. providing a sample having a particle, wherein a size of the particle is less than about 20 microns;
• b. optionally illuminating the sample;
• c. optionally positioning the sample along an optical axis of a lens at a distance from a focal plane of the lens that is greater than a depth of field of the lens;
• d. generating a blurred image of the particle with the lens having a magnification ratio of from about 1:5 to about 5:1, wherein a dimension of the blurred image of the particle is greater than the dimension of the particle multiplied by the magnification ratio of the lens, and wherein a field of view in an image plane of the lens is larger than twice the size of the particle;
• e. sensing the blurred image of the particle on at least two pixels of an image sensor having a pixel size of less than about the dimension of the blurred image of the particle wherein an intensity of the blurred image of the particle is distributed on at least two pixels of the image sensor to form an intensity distribution;
• and
• f. processing an image of the sample, including finding a center of an intensity distribution for at least two pixels within a high precision, sub-pixel particle position measurement resolution, to measure the position or the movement of the particle, wherein a size of the particle is less than a dimension of about five pixels and wherein finding the center of the intensity distribution comprises at least one of finding a two dimensional fit to the intensity distribution and finding a geometric center of a bead image based on a threshold of the intensity distribution.