Asante Calcium Green and Asante Calcium Red--novel calcium indicators for two-photon fluorescence lifetime imaging

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 Ca(2+)-free and Ca(2+)-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca(2+)-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 Ca(2+)-free and Ca(2+)-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K(D) of 180 nM was determined. Thus, quantitative [Ca(2+)]i recordings were realized, unraveling a reversible dopamine-induced [Ca(2+)]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca(2+) indicator dye for 2P-FLIM recordings applicable in diverse biological systems.

Figure 1. Steady-state absorption and fluorescence measurements for ACR and ACG.

Chemical structures of (A) ACR and (B) ACG , . Absorption (black) and relative fluorescence (red) spectra in Ca²⁺-free (solid line, [Ca²⁺]_free  = 0 nM) and Ca²⁺-saturated (dashed line, [Ca²⁺]_free  = 40 µM) buffers. (C) ACR (c = 2.5 µM, λ_ex = 540 nm). (D) ACG (c = 0.9 µM, λ_ex = 517 nm). (E) Fluorescence enhancement factor (FEF) of ACR (dashed line) and ACG (solid line) as a function of excitation wavelength. FEF is the ratio of the fluorescence intensities of the excitation spectra under Ca²⁺-saturated conditions and Ca²⁺-free conditions. The curves are the smoothed means of two measurements.

Figure 2. 2P fluorescence excitation spectra for ACR and ACG.

Logarithmic plot of 2P fluorescence excitation action cross-sections Φ_Fσ₂ as a function of excitation wavelength. (A) ACR (means ± SEM, N = 6). (B) ACG (means ± SEM, N = 6). The black circles correspond to the 2P-reference rhodamine B in methanol and data were taken from the literature . Ca²⁺-free ([Ca²⁺]_free  = 0 nM) and Ca²⁺-saturated ([Ca²⁺]_free  = 40 µM) conditions are depicted by blue and red squares, respectively. (C) Double logarithmic plot of the measured fluorescence intensity I _F as a function of 2P-excitation power P for rhodamine B (black circles) in methanol, ACR (red squares) and ACG (green triangles) in a Ca²⁺-saturated buffer (λ_ex,2P  = 780 nm; N = 3). The data points were fitted by a linear function (r²≥0.98).

Figure 3. Time-resolved fluorescence recordings of ACR and ACG after 2P-excitation at 780 nm.

Fluorescence decay curves of (A) ACR and (B) ACG in Ca²⁺-free (blue, [Ca²⁺]_free  = 0 nM) and Ca²⁺-saturated (red, [Ca²⁺]_free  = 40 µM) buffer solutions and the corresponding global biexponential deconvolution fits with values; IRF: instrument response function (black). The black arrow indicates increasing [Ca²⁺]_free. The residuals and individual values of the global biexponential fits are shown below. Normalized amplitudes α_i (squares, solid lines) and amplitude-weighted average fluorescence decay time τ_av,amp (triangles, dashed line) as a function of [Ca²⁺]_free for (C) ACR and (D) ACG. The blue squares correspond to the normalized amplitudes of the short decay time component (ACR: 0.12 ns±0.006 ns and ACG: 0.25 ns±0.04 ns; Ca²⁺-free species), whereas the normalized amplitudes of the long decay time component (ACR: 0.57 ns±0.003 ns and ACG: 2.38 ns±0.02 ns; Ca²⁺-bound) are depicted by red squares, (means ± SEM, N = 5).

Figure 4. Spectroscopic properties of ACR in 1% BSA in vitro buffer solutions.

(A) Absorption (black) and relative fluorescence (red) spectra of ACR (c = 2.5 µM, λ_ex  = 550 nm) under Ca²⁺-free (solid lines) and Ca²⁺-saturated (dashed lines) conditions. (B) Fluorescence enhancement factor (FEF) of ACR as a function of excitation wavelength (means ± SEM, N = 3). The FEF is the ratio of the fluorescence intensities of the excitation spectra under Ca²⁺-saturated and Ca²⁺-free conditions. (C) Fluorescence decay curves of ACR in Ca²⁺-free (blue) and Ca²⁺-saturated (red) buffers (λ_ex = 550 nm, 1P-excitation, λ_em = 640 nm) and the corresponding global biexponential deconvolution fits with the value; IRF: instrument response function (black). The black arrow indicates increasing [Ca²⁺]_free. The residuals and individual values of the global biexponential fits are shown below.

Figure 5. Behavior of ACR in cockroach salivary glands.

2P-excited (780 nm) fluorescence intensity images of (A) unloaded (1 luminal cuticule, 2 ductal lumen, 3 apically located, point-shaped structures) and (B) ACR-loaded salivary gland ducts (median optical sections). The graphs below the images display the fluorescence intensity traces along the white lines in the images.

Figure 6. Determination of in situ K _D,t for ACR in salivary duct cells.

(A) Fluorescence decay curves extracted from 2P-FLIM images of ACR-loaded salivary duct cells under Ca²⁺-free (blue, [Ca²⁺]_free  = 0 nM) and Ca²⁺-saturated (red, [Ca²⁺]_free  = 68 µM) conditions and the corresponding global biexponential deconvolution fits with the value. The residuals and individual values of the global biexponential fits are shown below. (B) Normalized amplitudes α_i (squares, solid lines) and amplitude-weighted average fluorescence decay time τ_av,amp (triangles, dashed line). The blue squares correspond to the normalized amplitudes of the short decay time component (0.56 ns, Ca²⁺-free species), whereas the normalized amplitudes of the long decay time component (2.44 ns, Ca²⁺-bound species) are depicted by red squares (N = 11–23). The dotted line marks the determined K _D,t.

Figure 7. Analysis of dopamine-induced [Ca²⁺]_i changes in ACR-loaded salivary duct cells by 2P-FLIM recordings.

(A) Normalized amplitudes α_i (squares, solid lines) and amplitude-weighted average fluorescence decay time τ_av,amp (triangles, dashed line). The blue squares correspond to the normalized amplitudes of the short decay time component (0.56 ns, Ca²⁺-free species), whereas the normalized amplitudes of the long decay time component (2.44 ns, Ca²⁺-bound species) are depicted by red squares (means, N = 15). Black bars indicate the periods of 1 µM dopamine presence. (B) Statistical analysis of the dopamine effect; repeated-measures ANOVA and Holm-Sidak's multiple comparison tests (* P<0.05, *** P<0.001). (C) Data converted to [Ca²⁺]_i. The error bars shown were calculated by error propagation of τ_av,amp. Black bars indicate the periods of dopamine presence and (D) corresponding false color-coded 2P-FLIM images at distinct time points.