The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging.
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The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging. / Celli, A; Sanchez, S; Behne, Martin; Hazlett, T; Gratton, E; Mauro, T.
in: BIOPHYS J, Jahrgang 98, Nr. 5, 5, 2010, S. 911-921.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging.
AU - Celli, A
AU - Sanchez, S
AU - Behne, Martin
AU - Hazlett, T
AU - Gratton, E
AU - Mauro, T
PY - 2010
Y1 - 2010
N2 - Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.
AB - Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.
KW - Adult
KW - Humans
KW - Time Factors
KW - Imaging, Three-Dimensional methods
KW - Calcium metabolism
KW - Extracellular Space metabolism
KW - Biophysical Phenomena
KW - Calibration
KW - Epidermis cytology
KW - Fluorescence
KW - Organic Chemicals metabolism
KW - Tissue Fixation
KW - Adult
KW - Humans
KW - Time Factors
KW - Imaging, Three-Dimensional methods
KW - Calcium metabolism
KW - Extracellular Space metabolism
KW - Biophysical Phenomena
KW - Calibration
KW - Epidermis cytology
KW - Fluorescence
KW - Organic Chemicals metabolism
KW - Tissue Fixation
M3 - SCORING: Zeitschriftenaufsatz
VL - 98
SP - 911
EP - 921
JO - BIOPHYS J
JF - BIOPHYS J
SN - 0006-3495
IS - 5
M1 - 5
ER -
