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Cell Viability and DNA Denaturation Measurements by Two-Photon Fluorescence Excitation in CW Al:GaAs Diode Laser
Journal of Biomedical Optics
|September 28, 2012
Summary
This study shows a safe, single-beam method using two-photon fluorescence to monitor cell viability and DNA changes in optically trapped cells. It effectively tracks cellular necrosis and thermal damage without harming cells.
Area of Science:
- Biophysics
- Cell Biology
- Optical Trapping
Background:
- Assessing cell viability and DNA integrity is crucial in biological research.
- Optical trapping offers precise manipulation of single cells but requires careful consideration of laser-induced damage.
- Simultaneous trapping and excitation methods are needed for efficient cellular analysis.
Purpose of the Study:
- To develop and validate a single diode laser beam method for simultaneous optical trapping and two-photon fluorescence excitation.
- To assess cell viability and monitor DNA denaturation in optically trapped cells.
- To evaluate the safety and efficacy of the method under intense laser illumination and for studying cellular responses like necrosis and thermal damage.
Main Methods:
- Utilized a single diode laser beam for simultaneous optical trapping and two-photon fluorescence excitation.
- Measured cell viability and DNA denaturation in Chinese Hamster Ovary (CHO) cells and T lymphocytes.
- Analyzed two-photon fluorescence emission spectra from cells loaded with various fluorescent probes, including acridine orange for nucleic acid analysis.
Main Results:
- Demonstrated that the single beam method is safe for monitoring optically trapped cells, even under high laser power density (∼106 W/cm²).
- Successfully monitored the dynamics of cellular necrosis induced by ethanol addition during optical trapping.
- Assessed thermal damage in heat-treated samples by observing spectral shifts in acridine orange fluorescence.
Conclusions:
- The developed single diode laser beam technique provides a safe and effective tool for real-time monitoring of cell viability and DNA status in optical traps.
- This method allows for the study of cellular responses to various stimuli, including necrosis induction and thermal stress.
- The approach is suitable for analyzing cells labeled with different fluorescent probes, offering versatility in biological applications.
