Automated holographic video microscopy assay

A system for characterizing a parameter of a sample by holographic microscopy, comprising:

• a holographic microscope including a laser beam source and an objective lens, the laser beam positioned to interact with a fluidic sample channel having media, probe beads, and target objects, scattering from the sample comprising probe beads having target objects bound to a surface and interacting with an unscattered portion of the laser beam to provide a holographic scattering pattern,
• an image collection device for collecting image data characteristic of the scattering pattern from the holographic microscope;
• and,
• a computer system including a processor and memory, the memory having stored thereon computer readable instructions, the computer readable instructions configured to:
• receive holographic image data of the sample;
• determine a first estimate of the number of coated probe beads in the holographic image data, each object associated with a set of concentric bright and dark rings;
• determine, for each set of concentric bright and dark rings a centroid defined by an approximate x, y position in a plane, with each pixel of the image data voting for pixels in a transformed image that may be centroids;
• determine an estimate of the axial position (z) of each of the coated probe beads;
• determine by Lorenz-Mie analysis an estimate of each of the coated probe beads effective sphere radius and effective sphere refractive index, where each coated probe bead is treated as an effective sphere for Lorenz-Mie analysis;
• determine by Maxwell Garneet effective-medium theory the effective coating diameter, and effective coating refractive index, an effective coating comprising bound target objects and media within an associated effective sphere;
• compare one of the radius and refractive index for each of the objects the same of radius or refractive index of a control sample;
• and
• determine for each one of the objects if that one object is bound to a material relative to the control sample.

A method of characterizing parameters for holographic microscopy of objects comprising the steps of:

• receiving, by a processor, image data from a storage medium;
• transforming, by a processor, the image data using a Hough circular transform to determine by a voting algorithm a first estimate of the number of objects in the image data and the objects approximate x, y position in a plane;
• applying, by a processor, Lorenz-Mie analysis of the image data for each of the objects to determine an estimate of the radius and refractive index of each of the objects;
• applying, by a processor, Maxwell Garnett effective-medium theory analysis of the image data for each of the objects to determine the effective diameter of a coating associated with the object;
• identifying, by a processor, from one or both of estimated radii and estimated refractive indices those particles having a coating;
• and
• generating a signal to the user indicating completed process.