Sample processing improvements for quantitative microscopy
Inventors
Fine, Alan Marc • Macaulay, Hershel
Assignees
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Publication Number
US-11598699-B2
Publication Date
2023-03-07
Expiration Date
Abstract
Among other things, a diluted sample is generated based on mixing a small sample of blood with a one or more diluents. A thin film of the diluted sample is formed on the surface of a contact optical microscopy sensor. Red blood cells within a portion of the thin film of the diluted sample are illuminated using light of a predetermined wavelength. One or more images of the diluted sample are acquired based on illuminating the red blood cells within the portion of the thin film of the diluted sample. The acquired one or more images of the diluted sample are then processed. The mean corpuscular hemoglobin in the red blood cells within the portion of the thin film of the diluted sample is determined based on processing the acquired images of the diluted sample.
Core Innovation
The invention provides a contact optical microscopy (COM)-based microspectrometry method for determining a mean corpuscular hemoglobin in red blood cells in a diluted blood sample. The method generates the diluted sample by mixing blood with one or more diluents and forms a film of the diluted sample on a microscopy apparatus including a contact optical microscopy sensor with a pixel array underlying the film, so transmitted-light signals are sensed directly through the film.
Red blood cells within a portion of the film are illuminated using light of a predetermined wavelength, and the contact optical microscopy sensor acquires one or more images after the light has transmitted through the diluted sample. The mean corpuscular hemoglobin is determined based on an intensity of the light of the predetermined wavelength sensed by a plurality of pixels of the pixel array and pixel sizes of the plurality of pixels.
The disclosed content further includes computer vision background estimation and segmentation to isolate red blood cell regions for calculating the mean corpuscular hemoglobin. It also discusses selecting the predetermined wavelength to maximize extinction and red blood cell contrast based on hemoglobin absorbance, considering oxygenated hemoglobin, deoxygenated hemoglobin, and isosbestic considerations, and using nitrite to mediate hemoglobin conversion.
Claims Coverage
The document contains one independent claim that defines the contact optical microscopy microspectrometry workflow and the quantitative basis for determining mean corpuscular hemoglobin. Dependent claims further refine the method by specifying nitrite-containing diluent, sphering of red blood cells, apparatus constraints, and image-processing requirements.
Diluted blood film on a contact optical microscopy sensor
Generating a diluted sample by mixing blood with one or more diluents and forming a film of the diluted sample on a microscopy apparatus comprising a contact optical microscopy sensor with a pixel array underlying the film.
Predetermined-wavelength transmitted-light imaging with a pixel array
Illuminating red blood cells within a portion of the film using light of a predetermined wavelength and acquiring one or more images after the light has transmitted through the diluted sample.
Mean corpuscular hemoglobin determination from pixel intensity and pixel sizes
Determining a mean corpuscular hemoglobin in the red blood cells based on an intensity of the light of the predetermined wavelength sensed by a plurality of pixels of the pixel array and pixel sizes of the plurality of pixels.
Nitrite-containing diluent for diluted blood
Generating a diluted blood sample such that at least one diluent comprises a nitrite.
Sphering red blood cells via mixing
Generating a diluted blood sample by mixing a sample of blood with one or more diluents to produce sphering of the red blood cells.
Predetermined height with transparent lid constraint
Selecting a predetermined height so that lowering a transparent lid onto a film surface constrains red blood cells within a portion of the film in relation to a broadest in-plane dimension of the red blood cells and avoids structural damage.
Background estimation and segmentation with exactly one red blood cell per region
Estimating a background pixel value, segmenting image regions each containing exactly one red blood cell, and calculating mean corpuscular hemoglobin from those regions.
Quantitative image feature coverage for red blood cells
Characterizing the method so that the acquired images contain image features of at least one hundred red blood cells.
Claim coverage centers on a contact optical microscopy sensor with a pixel array that acquires transmitted-light images of red blood cells illuminated at a predetermined wavelength, and on determining mean corpuscular hemoglobin from pixel intensity together with pixel sizes. Dependent claim refinements specify nitrite-containing diluents, sphering of red blood cells, a transparent lid at a predetermined height, and image-analysis steps including background estimation and segmentation with exactly one red blood cell per region.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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