In vitro optoacoustic flow cytometry with light scattering referencing

Area of Science:

• Biomedical Imaging
• Molecular Biology
• Biophysics

Background:

• Optoacoustic imaging relies on transgene reporters for enhanced contrast, similar to fluorescence methods.
• High-throughput screening is crucial for developing new optoacoustic reporters via protein engineering and directed evolution.
• Current methods struggle with accurate optoacoustic contrast characterization due to cell size and position variability.

Purpose of the Study:

• To develop an optoacoustic flow cytometer (OA-FCM) for precise measurement of intracellular optoacoustic signals.
• To enable high-throughput screening of genetically encoded optoacoustic contrast agents.
• To advance the discovery of novel optoacoustic reporters.

Main Methods:

• Developed an optoacoustic flow cytometer (OA-FCM) for in-flow measurement of optoacoustic signals.
• Integrated light-scattering detection to reference optoacoustic signals, correcting for cell size, position, and excitation flux.
• Calibrated the OA-FCM using micrometer-sized particles for single-cell size object assessment.

Main Results:

• Achieved precise measurement of intracellular optoacoustic signals from genetically encoded chromoproteins in flow.
• Demonstrated accurate referencing of optoacoustic signals using light-scattering for enhanced precision.
• Successfully identified distinct subpopulations in a mixture of E. coli strains with over 90% precision.

Conclusions:

• The OA-FCM provides a robust platform for precise, high-throughput screening of intracellular optoacoustic signals.
• This technology facilitates the identification and development of novel genetically encoded optoacoustic reporters.
• The OA-FCM enables the transfer of fluorescence-based screening strategies, like directed evolution, to the optoacoustic field.