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<p>Rapid developments in the field of organotypic cultures have generated a growing need for effective and non-invasive methods for quality control during tissue development. In this study, we correlate metabolic changes with epidermal differentiation and demonstrate that multiphoton microscopy with fluorescence lifetime imaging (MPM-FLIM) can be applied to monitor epidermal differentiation of keratinocytes with respect to proliferative and differentiated states. In a 2D keratinocyte tissue culture model, increased expression of differentiation markers keratin-1 and keratin-10 was induced with calcium supplementation. An accompanying shift from glycolysis to mitochondrial respiration was detected in metabolic flux assays. Analysis of MPM-FLIM images acquired at 750&#xa0;nm and 900&#xa0;nm excitation revealed a decreased relative fraction of intracellular NADH and FAD after high calcium treatment, consistent with increased oxidative phosphorylation. Epidermal differentiation could be monitored over a 96&#xa0;h period. Discrimination analysis based on k-means clustering generated clusters that correlated well with the duration of high Ca<sup>2+</sup> treatment, suggesting that MPM-FLIM can provide useful parameters for monitoring keratinocyte differentiation.</p>

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Insights into metabolic changes during epidermal differentiation as revealed by multiphoton microscopy with fluorescence lifetime imaging

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年2月21日

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  • Monika Malak,
  • Chen Qian,
  • Jeemol James,
  • Syam Nair,
  • Julie Grantham,
  • Marica B. Ericson

摘要

Rapid developments in the field of organotypic cultures have generated a growing need for effective and non-invasive methods for quality control during tissue development. In this study, we correlate metabolic changes with epidermal differentiation and demonstrate that multiphoton microscopy with fluorescence lifetime imaging (MPM-FLIM) can be applied to monitor epidermal differentiation of keratinocytes with respect to proliferative and differentiated states. In a 2D keratinocyte tissue culture model, increased expression of differentiation markers keratin-1 and keratin-10 was induced with calcium supplementation. An accompanying shift from glycolysis to mitochondrial respiration was detected in metabolic flux assays. Analysis of MPM-FLIM images acquired at 750 nm and 900 nm excitation revealed a decreased relative fraction of intracellular NADH and FAD after high calcium treatment, consistent with increased oxidative phosphorylation. Epidermal differentiation could be monitored over a 96 h period. Discrimination analysis based on k-means clustering generated clusters that correlated well with the duration of high Ca2+ treatment, suggesting that MPM-FLIM can provide useful parameters for monitoring keratinocyte differentiation.