The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging

Area of Science:

• Cell Biology
• Biophysics
• Physiology

Background:

• Ionic gradients are fundamental to physiological processes across various tissues.
• Calcium (Ca2+) gradients in the epidermis regulate critical functions like cell differentiation and barrier formation.
• Previous methods for studying ionic gradients faced limitations with tissue thickness, dye artifacts, and calibration.

Purpose of the Study:

• To quantitatively study ionic concentrations in tissues using phasor analysis of fluorescence lifetime imaging data.
• To overcome technical challenges in measuring ionic gradients in complex biological systems like the epidermis.
• To elucidate the sources and distribution of calcium in epidermal cells and their role in physiological processes.

Main Methods:

• Application of phasor representation of fluorescence lifetime imaging data.
• Utilizing two-photon microscopy for high spatial resolution imaging.
• Employing the epidermis as a model system to study calcium (Ca2+) gradients.

Main Results:

• The study successfully quantified ionic concentrations, overcoming issues of tissue thickness and dye artifacts.
• Identified intracellular stores (Golgi, endoplasmic reticulum) as the primary source of epidermal free Ca2+, with a minor contribution from extracellular Ca2+.
• Revealed significant cell-to-cell heterogeneity in basal keratinocyte calcium concentrations, challenging existing models of epidermal differentiation.

Conclusions:

• The developed imaging and analytical approach provides superior localization of Ca2+ stores in the epidermis.
• Findings suggest that intracellular calcium dynamics, rather than extracellular changes, are key drivers of epidermal Ca2+ gradients.
• The heterogeneity observed in basal keratinocytes necessitates a re-evaluation of the current understanding of calcium-mediated epidermal differentiation.