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

## TL;DR

This paper introduces a new method using harmonic spectral phasor analysis to study calcium activity in cells with a single-wavelength fluorophore.

## Contribution

The novel use of first and second harmonic spectral phasor analysis to assess calcium microenvironments in different cell regions.

## Key findings

- First harmonic is more sensitive to λmax, while the second harmonic is more sensitive to spectral width.
- Significant emission differences were observed in the nucleus, cytoplasm, and membrane under basal conditions.
- Combined use of first and second harmonic analysis provides better insight into cellular microenvironments.

## Abstract

Ratiometric and single-wavelength fluorophores are limited in their ability to provide basal level activity prior to treatment. This study presents a novel approach to characterise cell-wide basal activity of calcium using first and second harmonic spectral phasor analysis. Cells stained with the single-wavelength calcium fluorophore Oregon Green™ BAPTA 1-AM exhibited significant differences in wavelength or width in the nucleus, cytoplasm and membrane under basal conditions. Large and small cursor analysis was applied in the first and second harmonic, with smaller cursors revealing a region of interest enveloping and protruding from the nucleus in a structure akin to the sarcoplasmic reticulum. The first harmonic was found to be more sensitive in measurements of λmax, while the second harmonic showed increased sensitivity in measurements of spectral width. The results of this study indicate that first and second harmonic frequencies should be used in conjunction with phasor analysis of fluorophore microenvironments, rather than the first harmonic alone. Use of this approach may provide more insight into the cellular microenvironment under basal activity and treatment responses.

•· First and second harmonic spectral phasor analysis was used to establish the calcium microenvironment at basal activity.•· Significant emission differences of a single-wavelength fluorophore was detected across cellular regions.•· First harmonic showed increased sensitivity in λmax while the second harmonic was more sensitive in spectral width.•· Future studies utilising spectral phasor analysis may benefit from using the first and second harmonic together.

· First and second harmonic spectral phasor analysis was used to establish the calcium microenvironment at basal activity.

· Significant emission differences of a single-wavelength fluorophore was detected across cellular regions.

· First harmonic showed increased sensitivity in λmax while the second harmonic was more sensitive in spectral width.

· Future studies utilising spectral phasor analysis may benefit from using the first and second harmonic together.