Characterization of a range of fura dyes with two-photon excitation

Wokosin, D., Loughrey, C. and Smith, G. (2004) Characterization of a range of fura dyes with two-photon excitation. Biophysical Journal, 86(3), pp. 1726-1738. (doi:10.1016/S0006-3495(04)74241-1)

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Abstract

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca2+-free and Ca2+-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca2+-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca2+-free and Ca2+-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K-eff) and dynamic ranges (R-f) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K-eff = 181 +/- 52 nM, 1.16 +/- 0.016 muM, 5.18 +/- 0.3 muM, 19.2 +/- 1 muM, and 58.5 +/- 2 muM; and R-f = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca2+] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 muM) extracellular [Ca2+]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca2+ imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Smith, Professor Godfrey and Loughrey, Professor Christopher
Authors: Wokosin, D., Loughrey, C., and Smith, G.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Medical Veterinary and Life Sciences > Institute of Cardiovascular and Medical Sciences
College of Medical Veterinary and Life Sciences
Journal Name:Biophysical Journal
ISSN:0006-3495

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