Applying first & second harmonic spectral phasor analysis on a single-wavelength calcium fluorophore

Area of Science:

• Cellular Biology
• Biophysics
• Spectroscopy

Background:

• Ratiometric and single-wavelength fluorophores have limitations in assessing baseline cellular activity.
• Characterizing basal cellular calcium activity is crucial for understanding cellular responses.

Purpose of the Study:

• To develop and validate a novel approach for characterizing cell-wide basal calcium activity.
• To utilize first and second harmonic spectral phasor analysis for enhanced cellular microenvironment insights.

Main Methods:

• Cells were stained with the calcium fluorophore Oregon Green™ BAPTA 1-AM.
• First and second harmonic spectral phasor analysis was applied to characterize cellular regions.
• Large and small cursor analysis identified specific intracellular structures.

Main Results:

• Significant differences in wavelength and spectral width were observed in the nucleus, cytoplasm, and membrane under basal conditions.
• Smaller cursors revealed structures resembling the sarcoplasmic reticulum.
• The first harmonic was more sensitive to wavelength (λ) changes, while the second harmonic was more sensitive to spectral width.

Conclusions:

• Combining first and second harmonic frequencies with phasor analysis provides a more comprehensive understanding of fluorophore microenvironments.
• This method enhances the characterization of cellular microenvironments under basal conditions and treatment.
• The approach offers improved insights into cellular activity compared to using the first harmonic alone.