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.
Diagnostics (Basel, Switzerland)
|December 16, 2020
Summary
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.
