A Light-Sheet-Based Imaaging Spectrometer to Characterize Acridine Orange Fluorescence within Leukocytes
Amy J Powless1, Sandra P Prieto1, Madison R Gramling2
1Biomedical Engineering Department, University of Arkansas, Fayetteville, AR 72701, USA.
Insights
Low-cost point-of-care (POC) blood analysis using acridine orange (AO) fluorescence requires optimized staining. This study developed a novel imaging spectrometer to analyze AO-stained leukocyte spectra, revealing key factors influencing fluorescence for improved POC diagnostics.
Area of Science:
- Biomedical Engineering
- Spectroscopy
- Cellular Imaging
Background:
- Point-of-care (POC) blood analyzers increasingly use fluorescent dyes like acridine orange (AO) for leukocyte classification.
- Accurate classification of leukocyte subpopulations relies on reproducible colorimetric features, which are sensitive to staining protocols (AO concentration, timing, pH).
Purpose of the Study:
- To develop and utilize a light-sheet fluorescence imaging spectrometer with an automated spectral extraction algorithm.
- To investigate the spectral features of acridine orange (AO)-stained leukocytes and understand factors influencing fluorescence.
Main Methods:
- Developed a light-sheet fluorescence imaging spectrometer with 9 nm spectral resolution.
- Collected and stained whole blood specimens with AO using point-of-care (POC) methods.
- Implemented a post-processing pipeline including image segmentation, quality control, and spectral extraction for cell-by-cell analysis.
Main Results:
- Increased AO concentration caused a red-shift in AO fluorescence; pH variations did not significantly alter fluorescence.
- Observed red-shift trends in AO-stained leukocyte spectra correlated with dye accumulation in acidic vesicles and longer incubation times.
- Successfully extracted and analyzed spectral features from individual AO-stained leukocytes.
Conclusions:
- The developed imaging spectrometer system provides a tool to study spectral features of AO-stained leukocytes.
- Findings guide the optimization of AO staining protocols for future POC systems relying on fluorescence and colorimetric features for leukocyte subpopulation identification.
Abstract:
Low-cost imaging systems that utilize exogenous fluorescent dyes, such as acridine orange (AO), have recently been developed for use as point-of-care (POC) blood analyzers. AO-based fluorescence imaging exploits variations in emission wavelength within different cell types to enumerate and classify leukocyte subpopulations from whole blood specimens. This approach to leukocyte classification relies on accurate and reproducible colorimetric features, which have previously been demonstrated to be highly dependent on the cell staining protocols (such as specific AO concentration, timing, and pH). We have developed a light-sheet-based fluorescence imaging spectrometer, featuring a spectral resolution of 9 nm, with an automated spectral extraction algorithm as an investigative tool to study the spectral features from AO-stained leukocytes. Whole blood specimens were collected from human subjects, stained with AO using POC methods, and leukocyte spectra were acquired on a cell-by-cell basis. The post-processing method involves three steps: image segmentation to isolate individual cells in each spectral image; image quality control to exclude cells with low emission intensity, out-of-focus cells, and cellular debris; and the extraction of spectra for each cell. An increase in AO concentration was determined to contribute to the red-shift in AO-fluorescence, while varied pH values did not cause a change in fluorescence. In relation to the spectra of AO-stained leukocytes, there were corresponding red-shift trends associated with dye accumulation within acidic vesicles and at increasing incubation periods. The system presented here could guide future development of POC systems reliant on AO fluorescence and colorimetric features to identify leukocyte subpopulations in whole blood specimens.
