Imaging Ca2+ with a Fluorescent Rhodol

Abstract

Ca²⁺ mediates a host of biochemical and biophysical signaling processes in cells. The development of synthetic, Ca²⁺-sensitive fluorophores has played an instrumental role in our understanding of the temporal and spatial dynamics of Ca²⁺. Coupling Ca²⁺-selective ligands to fluorescent reporters has provided a wealth of excellent indicators that span the visible excitation and emission spectrum and possess Ca²⁺ affinities suited to a variety of cellular contexts. One underdeveloped area is the use of hybrid rhodamine/fluorescein fluorophores, or rhodols, in the context of Ca²⁺ sensing. Rhodols are bright and photostable and have good two-photon absorption cross sections (σ_TPA), making them excellent candidates for incorporation into Ca²⁺-sensing scaffolds. Here, we present the design, synthesis, and application of rhodol Ca²⁺ sensor 1 (RCS-1), a chlorinated pyrrolidine-based rhodol. RCS-1 possesses a Ca²⁺ binding constant of 240 nM and a 10-fold turn response to Ca²⁺. RCS-1 effectively absorbs infrared light and has a σ_TPA of 76 GM at 840 nm, 3-fold greater than that of its fluorescein-based counterpart. The acetoxy-methyl ester of RCS-1 stains the cytosol of live cells, enabling observation of Ca²⁺ fluctuations and cultured neurons using both one- and two-photon illumination. Together, these results demonstrate the utility of rhodol-based scaffolds for Ca²⁺ sensing using two-photon illumination in neurons.

Figure 1

Spectroscopy characterization of rhodol Ca²⁺ sensor 1. a) Absorbance spectrum of rhodol Ca²⁺ sensor 1 (10 μM) in the absence (black dotted line) and presence (black solid line) of 39 μM Ca²⁺. Fluorescence emission spectra of rhodol Ca²⁺ sensor 1 (10 μM) in the absence (green dotted line) and presence (green solid line) of 39 μM Ca²⁺. Excitation provided at 515 nm. b) Ca²⁺ titration of rhodol Ca²⁺ sensor 1. The fluorescence emission spectra of rhodol Ca²⁺ sensor 1 was recorded at increasing concentrations of Ca²⁺ from 0 μM to 39 μM, with intermediate concentrations of 17 nM, 38 nM, 65 nM, 100 nM, 150 nM, 225 nM, 351 nM, 602 nM, and 1.35 μM. Excitation provided at 515 nm. c) Two-photon absorption spectra of rhodol Ca²⁺ sensor 1 with Ca²⁺ (39 μM). Error bars are ± standard deviation for n = 3 separate determinations.

Figure 2

Live-cell imaging of histamine-evoked Ca²⁺ fluctuations with rhodol Ca²⁺ sensor 1. a) Confocal fluorescence microscopy images (one-photon) of HeLa cells incubated with rhodol Ca²⁺ sensor tetra-AM (1.7 μM). Scale bar is 20 μm. b) Quantification of intracellular [Ca²⁺] fluctuations measured in response to stimulation with histamine (5 μM). c) Two photon laser scanning fluorescence microscopy images of HeLa cells incubated with rhodol Ca²⁺ sensor tetra-AM (1.7 μM). Scale bar is 20 μm. d) Quantification of intracellular [Ca²⁺] fluctuations measured in response to stimulation with histamine (5 μM).

Figure 3

Visualization of Ca²⁺ transients in rat hippocampal neurons using rhodol Ca²⁺ sensor 1. a) Widefield fluorescence micrograph of cultured rat hippocampal neurons incubated with rhodol Ca²⁺ sensor 1 tetra AM ester (1.7 μM). Scale bar is 20 μm. b) Relative changes rhodol Ca²⁺ sensor 1 fluorescence vs. time for neurons in panel a. c) Two-photon laser scanning microscopy image of rat hippocampal neuron stained with rhodol Ca²⁺ sensor 1 tetra AM ester (1.7 μM). Scale bar is 20 μM. d) Intracellular Ca²⁺ transients recorded as relative changes in rhodol Ca²⁺ sensor 1 fluorescence vs. time for the neuron in panel c.

Scheme 1

Synthesis of rhodol Ca²⁺ sensor 1.