Real-time quantification method of cell viability through supravital dye uptake using lens-free imaging system
Inventors
LEE, Jong Muk • Reyes-Hernandez, Darwin R. • Nablo, Brian J.
Assignees
Government Of United States, Represented By Secretary Of Commerce Of National Institute Of Standards And Technology AS • SOL Inc Korea • United States Department of Commerce
Publication Number
US-12158410-B2
Publication Date
2024-12-03
Expiration Date
2040-11-30
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Abstract
The present disclosure relates to a real-time quantification method of cell viability through a supravital dye uptake using a lens-free imaging system. The method includes a step of incubating a sample cell in a cell culture medium, steps of detecting light penetrating the cell culture medium and identifying a boundary region of the sample cell at a preset time interval based on the detected light, a step of staining the incubated sample cell with the supravital dye, a step of detecting intensity of light penetrating the cell culture medium at a preset time interval, a step of calculating absorbance of the sample cell included in the cell culture medium at a preset time interval based on the boundary region and the detected intensity of light and a step of analyzing a viability of the sample cell based on the calculated absorbance.
Core Innovation
The invention relates to a real-time quantification method of cell viability through supravital dye uptake using a lens-free imaging system. This method incubates a sample cell in a culture medium, detects light penetrating the medium to identify a boundary region of the cell at preset intervals, stains the sample with a supravital dye, detects light intensity penetrating the medium, calculates absorbance based on the boundary and intensity, and analyzes cell viability accordingly.
The problem addressed is that conventional methods for evaluating cell viability using supravital dyes require destructive post-treatment procedures such as extracting dye from stained cells, which complicates and prolongs the viability quantification process. Additionally, conventional optical microscopes used for this purpose are relatively large, require preset optical paths, and necessitate removing cells from incubators for imaging, which delays analysis and reduces throughput.
The inventive concept enables time series quantification of absorbance without destructive post-treatment, allowing real-time absorbance measurement during incubation and staining. The method also reduces equipment volume by employing a lens-free imaging system that can be operated inside a standard incubator, enhancing convenience, speed, and efficiency in cell viability quantification.
Claims Coverage
The patent includes one independent claim that defines a method with several inventive features related to cell viability quantification using a lens-free imaging system.
Real-time cell viability quantification using lens-free imaging
A method that incubates a sample cell, detects light penetrating the culture medium, identifies the cell boundary based on detected light at preset intervals, stains the cell with a supravital dye, detects light intensity, calculates absorbance based on the boundary and detected intensity at preset intervals, classifies boundary regions based on absorbance into overlapping and non-overlapping, and analyzes cell viability accordingly.
Boundary region identification using pinhole-filter arranged system
The boundary region identification is performed while sequentially arranging a light source, a pinhole filter with a light-penetrating pinhole, the cell culture medium, and a lens-free image sensor. A shadow image of the sample cell is received and used to specify the boundary region, with coordinates of corresponding pixels identified.
Absorbance calculation based on multiple individual regions and pixel-level data
When the boundary region is detected as multiple individual regions, the method calculates light intensity for each region and computes absorbance for each pixel of the lens-free image sensor.
Viability analysis correction based on overlapping regions
Cell viability analysis includes correcting viability based on overlap degree in overlapping regions, exemplified by multiplying viability by ½ for regions with two overlapping cells, with overlap degree calculated based on absorbance.
Light intensity detection using collimated light arrangement
Detection of intensity of light penetrating the culture medium is performed with a sequential arrangement of a light source, a collimator converting light to collimated light, the culture medium, and the lens-free image sensor.
Light intensity detection distinguishing inside and outside of boundary region
Detection includes measuring first light intensity inside the boundary region and second light intensity outside the boundary region by the lens-free image sensor.
Absorbance calculation based on intensity ratio
Absorbance is calculated using the ratio of the first light intensity to the second light intensity, with absorbance decreasing as the ratio increases and increasing as the ratio decreases.
Classification of overlapping and non-overlapping regions based on shape and area
Distinguishing boundary regions by classifying oblong boundary regions with internal area not greater than a first preset area as non-overlapping, and circular boundary regions with internal area not greater than a smaller second preset area as overlapping, where the first area is greater than the second.
Performing incubation, staining, and analysis within incubator environment
The incubating, staining, and analyzing steps are performed while the lens-free imaging system and cell culture medium are inside an incubator with a preset environmental condition.
The claims define a comprehensive method for real-time cell viability quantification using a lens-free imaging system that incorporates innovative boundary region identification, absorbance calculation based on pixel-level intensity measurements, correction for overlapping cells, and integration within an incubator environment to enable efficient and non-destructive analysis.
Stated Advantages
The method quantifies absorbance without destructive post-treatment, shortening the time required for viability measurement.
Using a lens-free image sensor reduces the equipment volume needed for absorbance quantification, enabling more convenient and efficient procedures.
Real-time absorbance quantification is enabled at each moment during incubation and staining, allowing time-series analysis of cell viability.
Based on relative differences in absorbance, the method can derive the degree of cell overlap to facilitate 3D imaging of sample cells.
Documented Applications
Evaluating the health of cells, harmfulness of compounds, and effects of treatments in tissue culture.
Quantifying cell viability in real time during incubation and staining processes within an incubator environment.
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