Autofocus systems and methods for particle analysis in blood samples

Inventors

Wanders, Bart J. • Jordan, Brett • Farrell, Gregory A. • Adams, Thomas H. • Groner, Warren

Assignees

Iris International Inc

Abstract

Particles such as blood cells can be categorized and counted by a digital image processor. A digital microscope camera can be directed into a flowcell defining a symmetrically narrowing flowpath in which the sample stream flows in a ribbon flattened by flow and viscosity parameters between layers of sheath fluid. A contrast pattern for autofocusing is provided on the flowcell, for example at an edge of a rear illumination opening. The image processor assesses focus accuracy from pixel data contrast. A positioning motor moves the microscope and/or flowcell along the optical axis for autofocusing on the contrast pattern target. The processor then displaces microscope and flowcell by a known distance between the contrast pattern and the sample stream, thus focusing on the sample stream. Blood cell images are collected from that position until autofocus is reinitiated, periodically, by input signal, or when detecting temperature changes or focus inaccuracy in the image data.

Core Innovation

The invention relates to a method for imaging particles in a blood fluid sample using a particle analysis system configured for geometric hydrofocusing. The blood fluid sample is injected into a flowpath of a flowcell so the blood fluid sample flows in a sample flowstream with a flowstream width greater than a flowstream thickness, where the sample flowstream traverses an imaging axis through a decrease in flowpath size. This flow arrangement enables imaging of particles suitable for particle characterization and counting.

Focusing is performed by imaging an imaging target with an image capture device, where the imaging target has a position fixed relative to the flowcell and is outside of the flowpath. The imaging target has a different distance from the image capture device than the sample flowstream, and the imaging axis includes an imaging axis relationship to the objective lens direction for focusing. The image capture device is adjusted by a displacement distance that is equal to a distance between the sample flowstream and the imaging target, with the displacement distance having a value greater than zero, to focus the image capture device on the sample flowstream.

The disclosure further links geometric hydrofocusing and optionally viscosity hydrofocusing using a sheath fluid and, optionally, a viscosity agent to maintain cell viability and improve in-focus alignment of cells and intracellular organelles. The autofocus workflow can be re-initiated periodically or based on detected temperature changes or focus-quality degradation, and focusing can alternatively be adjusted directly using temperature sensors and a known relationship between temperature and focus. This enables reliable imaging with reduced manual review, including WBC differential counting.

Claims Coverage

The independent claim covers a geometric hydrofocusing particle-imaging method that uses a fixed imaging target outside the flowpath to focus the image capture device by moving the device toward a sample flowstream by a positive, predetermined displacement distance equal to the offset between the target and the sample. Dependent claims further refine inventive features such as sheath fluid viscosity difference handling with a viscosity agent, iris-based imaging targets and aperture usage, axis-specific rotational adjustment for focusing, autofocus pattern structure, and an autofocusing approach based on measuring focal distances from test-fluid flows.

Overall, the claim coverage centers on geometric hydrofocusing and a focus method using a fixed imaging target outside the flowpath, where a positive displacement distance equal to the offset between the target and the sample flowstream focuses the image capture device for acquiring focused images for particle characterization and counting. Dependent claims add viscosity-handling with a viability-preserving viscosity agent, iris-based targeting for aperture and autofocus edge imaging, axis-specific rotational focusing, structural autofocus target features, and an autofocus displacement derivation based on measured focal distance differences from test-fluid flows.

Stated Advantages

Documented Applications