Elsevier

Cell Calcium

Volume 58, Issue 6, December 2015, Pages 638-648
Cell Calcium

A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

https://doi.org/10.1016/j.ceca.2015.10.003Get rights and content

Highlights

  • We evaluated multiple Ca2+ sensitive dyes to image local Ca2+ signals.

  • Cal-520 is the best performing green-emitting dye.

  • Rhod-4 is the best performing red-emitting dye.

  • GCaMP6 protein sensors are not suitable for imaging local Ca2+ signals.

Abstract

Localized subcellular changes in Ca2+ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca2+ signaling have been greatly facilitated by the availability of fluorescent Ca2+ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca2+ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca2+ signals is lacking. Here, we evaluated several fluorescent Ca2+ indicators in the context of local Ca2+ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP3) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca2+ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca2+-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca2+ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca2+ signals.

Introduction

The calcium ion (Ca2+) is a ubiquitous second messenger that regulates a multitude of different physiological pathways including, secretion, fertilization, gene transcription and apoptosis. This single element is able to regulate so many diverse functions because cells have developed an elaborate toolkit of Ca2+ channels, pumps, exchangers and buffering proteins that enable changes in cytosolic [Ca2+] to be generated with precise control in magnitude, space and time [1]. An excellent example is seen in smooth muscle where transient, spatially restricted microdomains of Ca2+ promote relaxation by specifically activating plasmalemmal K+ channels, whereas waves and global Ca2+ signals that engulf the whole cell cause contraction [2], [3]. Our understanding of cellular Ca2+ signals has largely derived from progressive improvements in fluorescent Ca2+ indicator probes, coupled with advances in optical imaging technology. Indeed, it is now possible to monitor Ca2+ flux through individual channels in intact cells with millisecond temporal fidelity and sub-micrometer spatial resolution [4], [5].

The ability to image cellular Ca2+ signals dates back to the initial development of small molecule fluorescent Ca2+ indicator dyes by Tsien [6], [7], together with a strategy for facile loading of these indicators into intact cells via membrane-permeant ester forms [8]. These dyes consist of a Ca2+ chelating moiety conjugated to a fluorescence reporter. In the absence of Ca2+, photo-induced electron transfer from the Ca2+ chelator quenches fluorescence of the conjugated fluorophore. As Ca2+ levels rise this phenomenon is inhibited, resulting in a change in fluorescence intensity or shift in spectral properties (change in peak excitation or emission wavelengths). The first widely used indicator, fura-2 displays a shift in excitation spectra with Ca2+, enabling absolute calibration of [Ca2+] in terms of the ratio of fluorescence emitted at two different excitation wavelengths. However, with the exception of the newly developed indicator Asante Calcium Red (ACR) [9], all available ratiometric probes require excitation by phototoxic UV wavelengths, and the need to alternate excitation or emission wavelengths (as is the case for fura-2 and indo-1, respectively) severely limits temporal resolution. Instead, most studies imaging rapid, subcellular Ca2+ transients have utilized single wavelength Ca2+ indicators such as Fluo-4 and Oregon Green BAPTA-1 (OGB1), which produce a change in fluorescence intensity with [Ca2+] without any appreciable shifts in excitation or emission spectra. These indicator dyes are bright, exhibit large changes in fluorescence (30-fold or more) on binding Ca2+, and can be normalized for factors such as differences in dye loading by calculating a ‘pseudo-ratio’ of fluorescence relative to that at the same location either at rest before stimulation or after raising cytosolic [Ca2+] to saturating levels.

The single-wavelength indicators operate within the visible light spectrum, with the most popular and those available with the greatest range of affinities utilizing blue excitation and green emission. Recently, there has been increased interest in red-shifted indicators [10]. Longer wavelengths (red and near IR) have inherent advantages of reduced phototoxicity and scattering, and leave the short end of the visible spectrum available for applications including simultaneous use of green or yellow fluorescent protein tags and optogenetic control of membrane potential using channel rhodopsin. Red-emitting Ca2+ dyes conjugated to BAPTA such as rhod-2 have long been available, but their use for monitoring cytosolic Ca2+ signals is hampered by their propensity to accumulate in mitochondria. Newer dyes such as Rhod-4 and ACR are reported to show improved properties. However, to date only a few reports have utilized these probes [9], [11], [12], [13].

In parallel to the use of small-molecule synthetic indicators, the past decade has seen significant advances in the development of genetically encoded fluorescent Ca2+ indicators (GECIs). This has been motivated in large part by their promise as in vivo sensors of neuronal activity, employing changes in cytosolic [Ca2+] resulting from opening of voltage-gated Ca2+ channels as a surrogate readout of action potential spiking. For this purpose, GECIs have some major advantages over synthetic indicators. They can be incorporated into the genome of transgenic mice, obviating any need for loading with exogenous indicator and, in contrast to the indiscriminate uptake of membrane-permeant dye esters, can be targeted to distinct populations of cells and/or subcellular locations using cell specific promoters and targeting sequences. A currently popular GECI is the single-fluorophore sensor GCaMP, consisting of the circularly permuted green fluorescent protein (GFP) fused to the calmodulin (CaM) binding region of chicken myosin light kinase (M13) at its N terminus and to a vertebrate CaM at its C terminus. Binding of Ca2+ causes the M13 and CaM domains to interact, leading to an increase in fluorescence. Several iterations of the original GCaMP sensor [14] have now been developed, with the most recent, GCaMP6, yielding three variants (slow, medium and fast) which have been reported to outcompete synthetic indicator dyes in terms of their sensitivity and dynamic range [15]. Nevertheless, the requirements for detecting bulk neuronal signals arising from spike-evoked opening of voltage-gated Ca2+ channels differ appreciably from those for monitoring subcellular Ca2+ transients from individual and small clusters of Ca2+ channels. Most notably, bulk cytosolic [Ca2+] in neurons decays relatively slowly over tens or hundreds of ms [16], whereas local Ca2+ microdomains collapse much more rapidly [17].

Several studies have evaluated the ability of various GECIs to monitor Ca2+ activity in the cell body, spines and dendrites of neurons, but none have compared GECI responses to synthetic Ca2+ dyes in the context of subcellular changes in cytosolic [Ca2+] [15], [18], [19], [20]. An earlier report did present a systematic comparison of small-molecule indicators for visualizing IP3-mediated, subcellular Ca2+ puffs [21]. However, that study utilized slow, confocal laser scanning microscopy, before the advent of approaches including total internal reflection (TIRF) microscopy and fast EMCCD and sCMOS cameras that have greatly improved the spatial and temporal resolution of local Ca2+ signals. Moreover, new indicator dyes have since become available with improved Ca2+ binding properties, enhanced fluorescence brightness, and extended spectral range. A more recent report assessed the utility of green-emitting dyes for detecting local Ca2+ transients in cardiomyocytes [22] but was limited in scope, focusing only on three, similar fluo indicators (fluo-2, -3, -4).

Motivated by recent developments in both small-molecule and protein-based fluorescent Ca2+ probes, we describe here a systematic study of different indicators to determine optimum choices for imaging IP3-mediated local Ca2+ signals in cultured mammalian cells using high-speed (∼420 frames per second) camera-based fluorescence microscopy. We tested six green-emitting (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520) and three red-emitting (Rhod-4, X-Rhod-1, and Asante Calcium Red) synthetic Ca2+ dyes; as well as the slow, medium and fast GCaMP6 variants. Among these, we find, Cal-520 is the optimal indicator for detecting and faithfully tracking local Ca2+ puffs; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca2+ signals.

Section snippets

Cell culture

Human neuroblastoma SH-SY5Y cells (ATCC; #CRL-2266) were cultured on cell culture-grade plastic tissue flasks in a 1:1 mixture of Ham's F12 (Gibco; #11765) and Eagle's minimal essential media (Gibco; #12360) supplemented with 10% fetal bovine serum (Gibco; #26140-095), 1% nonessential amino acids (Gibco; #11140-050), and 1% penicillin-streptomycin (Gibco; #15070-063). Cells were maintained at 37° C in a humidified environment with 95% air and 5% CO2. For experimentation, cells were harvested by

Experimental procedure

Ca2+ puffs in response to photo-liberation of ci-IP3 were recorded using each Ca2+ indicator individually loaded into human neuroblastoma SH-SY5Y cells; a cell line well characterized for the study of local Ca2+ signals [5], [25], [27], [28], [29]. Puffs were evoked using photolysis flashes with a fixed intensity and duration (75 ms) selected to generate a measurable local response without producing a global rise in cytosolic [Ca2+] that would obscure the detection and analysis of puffs. As

Disclosures

All authors declare that they have no competing financial interests.

Author contributions

Conception and design of the research by I.P. and I.F.S. Data was collected by J.T.L and analyzed by J.T.L., I.P. and I.F.S. Manuscript was written by J.T.L, I.P. and I.F.S. All authors have read and approved the published manuscript.

Acknowledgements

The authors thank Kyle L. Ellefsen for assistance with the algorithm used to detect and analyze local Ca2+ signals. This work was supported by National Institutes of Health grants GM 100201 to I.F.S, and GM 048071 and GM 065830 to I.P.

References (29)

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