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 Ca(2+)-free and Ca(2+)-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 Ca(2+)-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 Ca(2+)-free and Ca(2+)-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 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; 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 [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Wokosin DL,Loughrey CM,Smith GLdoi
10.1016/S0006-3495(04)74241-1subject
Has Abstractpub_date
2004-03-01 00:00:00pages
1726-38issue
3eissn
0006-3495issn
1542-0086pii
S0006-3495(04)74241-1journal_volume
86pub_type
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