Fluorescence lifetime imaging microscopy: homodyne technique using high-speed gated image intensifier

Area of Science:

• Biophysics
• Chemical Imaging
• Microscopy

Background:

• Intracellular Ca2+ imaging using Fluorescence Lifetime Imaging Microscopy (FLIM) has been demonstrated.
• FLIM's potential extends to imaging a broader range of analytes, leveraging the photophysical properties of various probes.

Purpose of the Study:

• To explore and characterize the capability of FLIM for imaging diverse analytes beyond calcium ions.
• To identify suitable fluorescent probes and assess their suitability for FLIM-based imaging of ions and other substances.

Main Methods:

• Characterization of fluorescence lifetimes for various ion indicators, including those for Cl-, pH, Mg2+, K+, and Na+.
• Evaluation of probes like SPQ, MQAE, resorufin, SNAFL, SNARF, Magnesium Green, Mag-quin-2, Mag-quin-1, PBFI, and Sodium Green for FLIM applications.
• Consideration of environmental factors affecting fluorescence decay times, such as quenching phenomena, temperature, and viscosity.

Main Results:

• FLIM successfully images Cl- (SPQ, MQAE), pH (resorufin, SNAFL, SNARF), and Mg2+ (Magnesium Green, Mag-quin-2, Mag-quin-1).
• Probes for K+ (PBFI) are adequate, with newer probes for Na+ and K+ showing promise for effective imaging.
• Oxygen imaging is feasible, and environmental factors like quenching, temperature, and viscosity influence decay times.

Conclusions:

• FLIM is a versatile imaging technique applicable to a wide range of analytes, including ions and oxygen.
• The method's sensitivity to local environmental effects on fluorophore decay kinetics allows for imaging chemical and physical properties.
• Advancements in instrumentation and probe chemistry are expected to make FLIM more compact and accessible.