Abstract
The great demand for long-wavelength and high signal-to-noise Ca2+ indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments.DOI: http://dx.doi.org/10.7554/eLife.05808.001
Highlights
In recent years fluorescence imaging has been one of the fastest growing methods in physiology, cell biology and neuroscience, constantly driving the need for improved fluorescent probes (Wilt et al, 2009; Miyawaki, 2011; Looger and Griesbeck, 2012)
We recently introduced a family of red emitting calcium indicators based on X-Rhodamine: Calcium Ruby (CaRuby) (Collot et al, 2012), which bears an azido side arm for click chemistry and Collot et al eLife 2015;4:e05808
To increase the affinity of CaRuby, we modified the structure of the probe (Figure 1A), focusing on the Ca2+ chelating BAPTA moiety, as increasing the electron density of BAPTA lowers its KD for calcium (Tsien, 1980)
Summary
In recent years fluorescence imaging has been one of the fastest growing methods in physiology, cell biology and neuroscience, constantly driving the need for improved fluorescent probes (Wilt et al, 2009; Miyawaki, 2011; Looger and Griesbeck, 2012). A major problem of red-shifted fluorophores is that they are significantly more lipophilic than fluorescein-like dyes This leads to more leakage through cell membranes as well as to intracellular compartmentalization. These effects can be minimized by using the probes as conjugates of inert hydrophilic compounds such as dextrans This conjugation commonly uses one of the carboxylic groups of the BAPTA moiety, affecting the calcium affinity of BAPTA-based indicators (Tsien, 1980). These indicator-dextran conjugates are strongly shifted to lower affinities making them all but useless for sensitive and quantitative [Ca2+] measurements. Such a strategy would lead to a disproportionally large increase in the calcium buffering capacity of the indicator (Neher, 2005), resulting in a stronger disruption of cellular signaling than for a low concentration of high affinity indicator (Markram et al, 1998)
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