Flowcell, sheath fluid, and autofocus systems and methods for particle analysis in urine samples
The present disclosure relates to apparatus, systems, compositions, and methods for analyzing a sample containing particles. A particle imaging system or analyzer can include a flowcell through which a urine sample containing particles is caused to flow, and a high optical resolution imaging device which captures images for image analysis. A contrast pattern for autofocusing is provided on the flowcell. 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. Cell or particle 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.
1. A particle analysis system that performs geometric hydrofocusing for imaging particles in a body fluid sample, the system comprising:
a flowcell having a flowpath, the flowpath comprising a first channel and a second channel;
a sheath fluid injection system, wherein an output of the sheath fluid injection system is fluidly connected to the first channel,
a sample fluid injection system in fluid communication with the flowpath, the sample fluid injection system comprising an injection tube, wherein:
an outlet of the injection tube is fluidly connected to the second channel,
the first channel and the second channel are coaxial about a longitudinal axis, and
a cross-sectional area of the second channel decreases from the outlet of the injection tube to an image capture site, the cross sectional area orthogonal to the longitudinal axis;
an image capture device aligned with the image capture site along an imaging axis;
an imaging target fixed to the flowcell;
a focusing module comprising a focusing mechanism to move the image capture device; and
a processor operatively coupled to the image capture device, wherein the processor is programmed to:
control the image capture device to acquire a first image of the imaging target,
analyze the first image for a level of contrast,
determine, using the level of contrast, a first position of the image capture device from the imaging target where an image of the imaging target is focused,
control the focusing mechanism to move the image capture device to a second position a predetermined distance from the first position along the imaging axis, wherein the predetermined distance is nonzero, and
control the image capture device at the second position to acquire a second image, wherein the second image is focused at the predetermined distance from the imaging target.
2. The system of claim 1 , further comprising the body fluid sample, wherein the body fluid sample is a urine fluid sample.
3. The system of claim 1 , wherein the decrease in the cross-sectional area is defined by opposed walls of the flowpath angling radially inwardly along the flowpath generally symmetric about the longitudinal axis.
4. The system of claim 1 , wherein the imaging target is fixed on the flowcell.
5. The system of claim 1 , further comprising the body fluid sample and a sheath fluid, wherein the body fluid sample has a sample viscosity, and the sheath fluid has a sheath fluid viscosity that is different from the sample viscosity.
6. A particle analysis system that performs geometric hydrofocusing for imaging particles in a body fluid sample, the system comprising:
a flowcell having a flowpath with an injection tube and an imaging window with an imaging axis therethrough, wherein:
the flowpath is characterized by a first cross-sectional area parallel to the imaging axis at the injection tube,
the flowpath is characterized by a second cross-sectional area parallel to the imaging axis at an imaging target,
the second cross-sectional area is less than the first cross-sectional area,
the injection tube is characterized by a third cross-sectional area, and
the third cross-sectional area is less than the first cross-sectional area;
an image capture device aligned with the imaging window along the imaging axis;
an imaging target fixed to the flowcell;
a focusing module comprising a focusing mechanism to move the image capture device; and
a processor operatively coupled to the image capture device, wherein the processor is programmed to:
control the image capture device to acquire a first image of the imaging target,
analyze the first image for a level of contrast,
determine, using the level of contrast, a first position of the image capture device from the imaging target where an image of the imaging target is focused,
control the focusing mechanism to move the image capture device to a second position a predetermined distance from the first position along the imaging axis, wherein the predetermined distance is nonzero, and
control the image capture device at the second position to acquire a second image, wherein the second image is focused at the predetermined distance from the imaging target.
7. The system of claim 6 , further comprising the body fluid sample, wherein the body fluid sample is a urine sample.
8. A system for imaging a plurality of particles in a body fluid sample having a sample fluid viscosity, the system comprising:
a flowcell having a flowpath and a sample fluid injection tube, the flowpath comprising a first channel and a second channel;
a sheath fluid input in fluid communication with the flowpath of the flowcell, the sheath fluid input fluidly connected to the first channel;
a body fluid sample input in fluid communication with the injection tube of the flowcell, wherein:
the body fluid sample input fluidly connected to the second channel,
the first channel and the second channel are coaxial about a longitudinal axis, and
a cross-sectional area of the second channel decreases from an outlet of the injection tube to an imaging site, the cross sectional area orthogonal to the longitudinal axis;
an imaging device that images the plurality of particles at the imaging site;
the body fluid sample;
a sheath fluid, the sheath fluid having a sheath fluid viscosity that differs from the sample fluid viscosity by a viscosity difference in a predetermined viscosity difference range;
an imaging target fixed to the flowcell,
a focusing module comprising a focusing mechanism to move the imaging device, and
a processor operatively coupled to the imaging device, wherein the processor is programmed to:
control the imaging device to acquire a first image of the imaging target,
analyze the first image for a level of contrast,
determine, using the level of contrast, a first position of the imaging device from the imaging target where an image of the imaging target is focused,
control the focusing mechanism to move the imaging device to a second position a predetermined distance from the first position, wherein the predetermined distance is nonzero, and
control the imaging device at the second position to acquire a second image of the cells, wherein the second image is focused at the predetermined distance from the imaging target, wherein:
the body fluid sample is disposed within the sheath fluid in the second channel,
the plurality of particles comprise cells that extend from the body fluid sample into the sheath fluid.
9. The system of claim 8 , wherein the body fluid sample is a urine sample.
10. The system of claim 1 , wherein the processor is further programmed to:
control the sheath fluid injection system to inject a sheath fluid, and
control the sample fluid injection system to inject the body fluid sample.
11. The system of claim 1 , wherein the predetermined distance depends on temperature.
12. The system of claim 1 , wherein the focusing mechanism comprises a stepper motor.
13. The system of claim 1 , wherein the imaging axis is perpendicular to the longitudinal axis.
14. The system of claim 1 , wherein the image capture device comprises a charge coupled device (CCD).
15. The system of claim 1 , wherein the image capture device comprises a photosensor array.