Visualization of oxygen profile in reconstructed human epidermis by phosphorescence-lifetime imaging microscopy using...

Area of Science:

• Cell Biology
• Biophysics
• Skin Physiology

Background:

• Reconstructed human epidermis (RHE) models skin for stimulus evaluation.
• Understanding oxygen dynamics is crucial for skin research.

Purpose of the Study:

• To develop and apply a method for visualizing oxygen partial pressure in RHE at cellular resolution.
• To investigate the relationship between oxygen gradients, mitochondrial respiration, and epidermal differentiation.

Main Methods:

• Confocal phosphorescence-lifetime imaging microscopy (PLIM) using the BTPDM1 probe.
• Z-stack imaging to capture 3D oxygen distribution.
• Pharmacological manipulation with FCCP and Antimycin A to probe mitochondrial function.
• Analysis of differentiation markers (filaggrin, loricrin) and calcium-induced differentiation in keratinocytes.

Main Results:

• PLIM revealed an oxygen partial pressure gradient in RHE, decreasing from the basal to the cornified layer.
• FCCP treatment reduced oxygen levels, implicating mitochondrial respiration in the gradient.
• Antimycin A inhibited mitochondrial respiration and decreased epidermal differentiation markers.
• Calcium induction increased mitochondrial oxygen consumption and oxidative phosphorylation in keratinocytes.

Conclusions:

• Oxygen imaging via PLIM is effective for assessing mitochondrial respiration and epidermal differentiation status in RHE.
• Mitochondrial respiration is essential for epidermal maturation.
• Oxygen dynamics play a significant role in RHE physiology.