Abstract
For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca2+-free and Ca2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca2+-dependent way, unraveling in vitro dissociation constants K D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca2+-free and Ca2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K D of 180 nM was determined. Thus, quantitative [Ca2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.
Highlights
The basis of all cellular processes and activities is the precise regulation of intracellular ion homeostasis
The fluorescence quantum yield of Ca2+-bound Asante Calcium Red (ACR) at lex = 515 nm was determined absolutely at WF = 0.0660.01 (N = 6), whereas it was estimated for the Ca2+-free ACR relatively at WF = 0.0017 as its fluorescence quantum yield was below the limit of detection to be determined by absolute measurements
ACR and Asante Calcium Green (ACG) exhibited a change in the fluorescence quantum yield upon Ca2+ binding and met the requirement for time-resolved fluorescence measurements
Summary
The basis of all cellular processes and activities is the precise regulation of intracellular ion homeostasis. Serve as ubiquitous intracellular second messengers and play a pivotal role in numerous processes, such as neuronal signaling, fluid secretion, exocytosis or contraction. Regulation of the intracellular Ca2+ concentration ([Ca2+]i) is maintained by a variety of channels, exchangers and pumps located in the plasma membrane or in internal membranes. This Ca2+ signaling toolkit results in an appropriate stimulus-induced temporal and spatial intracellular Ca2+ pattern [1,2,3]. Quantification of [Ca2+]i and its dynamics is crucial for a better understanding of physiological processes as well as dysfunctions.
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