Direct frequency domain fluorescence lifetime imaging using field programmable gate arrays for real time processing.

Area of Science:

• Biophotonics
• Fluorescence Spectroscopy
• Imaging Technology

Background:

• Frequency domain fluorescence lifetime imaging (FD-FLIM) traditionally uses complex heterodyne or homodyne detection.
• Existing FD-FLIM systems often require additional components, increasing cost and complexity.
• Alternative methods using light source reflection for phase referencing also add complexity.

Purpose of the Study:

• To develop a novel, simplified, and cost-effective FD-FLIM detection scheme.
• To enable simultaneous probing of nanosecond lifetime fluorophores across multiple emission bands.
• To demonstrate the accuracy and sensitivity of the new detection method.

Main Methods:

• Implementation of light source modulation and emission digitization using Field Programmable Gate Arrays (FPGAs).
• Utilization of fixed gain avalanche photodiodes for fluorescence detection.
• Application of the discrete Fourier transform for real-time computation of modulation and phase lifetimes.

Main Results:

• A novel FD-FLIM system capable of probing nanosecond lifetime fluorophores (2-10 ns) simultaneously in three emission bands (405/20 nm, 440/40 nm, 525/50 nm).
• Excitation using a 375 nm diode laser modulated at frequencies between 1 MHz and 83 MHz.
• Demonstrated accuracy and sensitivity through imaging of standard fluorophores and ex vivo human coronary artery tissue.

Conclusions:

• The developed FPGA-based FD-FLIM system offers a simplified and potentially lower-cost alternative to traditional methods.
• The system effectively performs real-time lifetime calculations and simultaneous multi-band imaging.
• The novel detection scheme shows promise for various applications, including biological tissue analysis.