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Photoluminescence: Applications01:14

Photoluminescence: Applications

Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with...

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Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber
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Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

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Fluorescent measurement of [Ca²⁺]c: basic practical considerations.

Alec W M Simpson1

  • 1Department of Cell and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK. Awms@liverpool.ac.uk

Methods in Molecular Biology (Clifton, N.J.)
|September 26, 2012
PubMed
Summary
This summary is machine-generated.

Choosing the right fluorescent dye for measuring cytosolic calcium is crucial. This guide details dye selection, loading methods, and calibration pitfalls for accurate calcium concentration measurements.

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Last Updated: May 18, 2026

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Analytical Chemistry

Background:

  • Cytosolic calcium (Ca2+) plays a vital role in numerous cellular processes.
  • A wide variety of fluorescent dyes are available for Ca2+ measurement, including single and dual excitation/emission probes.
  • The selection of an appropriate dye is contingent upon specific experimental objectives and available equipment.

Purpose of the Study:

  • To provide a comprehensive overview of fluorescent dyes used for cytosolic calcium measurement.
  • To discuss the theoretical principles and practical considerations for dye selection, loading, and calibration.
  • To highlight potential pitfalls in the process of measuring intracellular Ca2+ concentrations.

Main Methods:

  • Overview of different types of fluorescent calcium indicators (single/dual excitation/emission).
  • Description of common methods for indicator dye loading: incubation with ester derivatives, micropipet injection, and reverse permeabilization.
  • Discussion on targeting indicators to specific intracellular organelles.

Main Results:

  • No single universal approach exists for all investigators due to diverse experimental needs and equipment.
  • Calibration of fluorescent signals to Ca2+ concentration requires meticulous attention to detail.
  • Potential challenges and sources of error in Ca2+ measurement using fluorescent dyes are identified.

Conclusions:

  • Effective measurement of cytosolic calcium requires careful selection of fluorescent probes and appropriate loading techniques.
  • Understanding the theoretical basis and potential pitfalls is essential for accurate Ca2+ concentration determination.
  • This chapter serves as a theoretical guide, with detailed experimental protocols provided elsewhere.