Calcium imaging using fluorescence lifetimes and long-wavelength probes

Area of Science:

• Biophysics
• Cell Biology
• Spectroscopy

Background:

• Accurate measurement of intracellular calcium (Ca2+) is crucial for understanding cellular processes.
• Existing calcium indicators often suffer from spectral limitations or phototoxicity.
• Long-wavelength calcium indicators offer potential advantages for in vivo imaging.

Purpose of the Study:

• To develop and validate a fluorescence lifetime imaging (FLIM) method for quantifying calcium concentrations.
• To utilize long-wavelength calcium indicators (Calcium Green, Orange, Crimson) with FLIM.
• To enable calcium imaging in spectral regions with minimal autofluorescence and photobleaching.

Main Methods:

• Frequency-domain fluorescence lifetime measurements of calcium probes.
• Development of a novel fluorescence lifetime imaging (FLIM) apparatus.
• Correction procedures for image intensifier artifacts in FLIM data.
• Application of FLIM for two-dimensional calcium concentration imaging.

Main Results:

• Calcium probes exhibited increased fluorescence lifetimes (50% to severalfold) in response to calcium.
• FLIM successfully generated two-dimensional images of calcium concentration.
• The FLIM method demonstrated independence from probe spectral shifts.
• Effective calcium imaging was achieved using excitation wavelengths from 488 nm to 620 nm.

Conclusions:

• Fluorescence lifetime imaging (FLIM) provides a robust method for calcium concentration measurement.
• Long-wavelength calcium probes coupled with FLIM minimize autofluorescence and photochemical damage.
• FLIM is suitable for calcium imaging in single cells, including applications in lifetime-based flow cytometry.