Choosing The Right Dye For Imaging Intracellular Calcium

Increases in intracellular calcium (Ca 2+) control a diverse set of cellular processes. A variety of intracellular sources and plasma membrane Ca 2+-permeable channels contribute to changes in calcium concentrations. Moreover, the endogenous proteins and organelles that detect or buffer these changes in Ca 2+ levels have differing affinities for calcium. As a result, the magnitude and duration of a change in the level of Ca 2+ can vary greatly, and thus observing these concentration changes requires a diverse portfolio of detection reagents. The most suitable Ca 2+ dye for an imaging experiment depends on both the application being performed and the nature of the Ca 2+ concentration change.

Choosing a Dye With the Optimal Affinity for Calcium

It’s important to match the dye affinity for Ca2+ to the anticipated magnitude of the increase in calcium levels being imaged. For example, a high-affinity dye used to detect a large increase in Ca2+ will likely become saturated, resulting in an incorrect measurement of the magnitude of change [1]. Conversely, a low-affinity dye will likely be unable to detect small changes in Ca2+.

Dye emission intensities are also important to consider when choosing a dye for a particular imaging application. The Fluo family of dyes has a very low emission at rest, resulting in a large increase in emission intensity upon Ca2+ binding but making it difficult to resolve the cell at rest [1,2]. Members of the Oregon Green® 488 BAPTA dye family have a higher resting fluorescence and can therefore be used to locate cells and structures; however, the maximal change in emission intensity is lower upon Ca2+ binding.

High-affinity dyes such as Fluo-4, Fluo­-3, and Oregon Green® 488 BAPTA-1 and BAPTA-2 are best suited for detecting a response to low-intensity stimulation, whereby a small number of plasma membrane or intracellular Ca2+ channels are recruited. A response to medium-intensity stimulation of cells—whereby many channels are recruited for prolonged periods of time—is best detected using medium-affinity dyes such as Fluo-5F. For high-intensity stimulation, low-affinity dyes such as Oregon Green® 488 BAPTA-5N and Fluo-4FF are most useful (Table 1).

Choose red Ca2+ dyes for imaging cells or tissues with high autofluorescence, for simultaneous imaging of a green fluorophore (Figure 1), or for studies of calcium signaling (Figure 2). As with green-emitting dyes, there are both high-affinity (Rhod-3 and X-Rhod-1) and low-affinity (X-Rhod-5F) red-emitting dyes.

Table 1. Properties and Applications of Calcium Dyes Excited by Visible Light.
 Filter Kd Applications
Oregon Green® 488 BAPTA-1 and BAPTA-2
345 nM/
390 nM/
170 nM/
580 nM
Spectrally compatible with most plate readers. Imaging of calcium signals derived from low-level stimulation. Can be multiplexed with red fluorophores such as TagRFP or mCherry.
Suitable for small Ca2+ changes; signal can saturate, resulting in a plateau.
700 nM/
570 nM
Imaging of calcium signals derived from low-level stimulation. Can be multiplexed with GFP or other green fluorophores.
Suitable for small Ca2+ changes; signal can saturate, resulting in a plateau.
Oregon Green® 488 BAPTA-6F
2.3 µM/
3 µM
Imaging of calcium signals derived from medium-level stimulation.
Suitable for large Ca2+ changes, thus difficult to detect small changes in cytosolic calcium.
1.6 µM
Imaging of calcium signals derived from medium-level stimulation. Can be multiplexed with GFP or other green fluorophores.
Suitable for large Ca2+ changes, thus difficult to detect small changes in cytosolic calcium.
9.7 µM
Imaging of calcium signals arising from maximum stimulation.
Suitable for very large Ca2+ changes, thus difficult to detect small changes in cytosolic calcium.
Oregon Green® 488 BAPTA-5N
22 µM/
20 µM
Can be loaded into ER to monitor release from internal stores.
Often necessary to wash out cytosolic dye component by mild permeabilization or via patch pipette.
570 nM
Used to monitor mitochondrial calcium transients.
Beneficial to confirm mitochondrial localization of Rhod-2 with mitochondrial probes such as CellLight® Mitochondria-GFP.
Figure 1.
Simultaneous imaging of Ca2+ and a GFP chimera. HeLa cells were transduced with CellLight® Mitochondria-GFP. The following day, cells were loaded with X-Rhod-1, AM (5 µM) for 30 min at room temperature. The image, taken with standard FITC/TRITC filter sets, demonstrates the compatibility of red Ca2+ dyes with GFP.

Imaging Intracellular Calcium Signaling
Figure 2. Imaging intracellular Ca2+ signaling. tSA-201 cells expressing CellLight® Tubulin-GFP and CaV2.1 (P Type) calcium ion channels were loaded with Rhod-3 according to instructions. The cells were rinsed once and placed in a laminar flow system and imaged at 100 msec intervals with a standard TRITC filter. When the solution was switched from control (time 0; A) to buffer containing high (50 mM) potassium substituted for sodium, the cells rapidly depolarized, allowing calcium influx through the opened CaV ion channels (B, C). The FITC filter image (D) shows GFP tubulin expressed in the same cells.

Loading Calcium Dyes Into Cells

Acetoxymethyl ester (AM) derivatives of fluorescent Ca2+ indicators are the most common dye form used for loading into cells. The AM derivative can cross the plasma membrane, followed by cleavage of the AM moiety by endogenous esterases to produce a charged molecule that is trapped within the cell. For single-cell imaging or recording of electrophysiological data, the cell-impermeant salt form of the dye can be loaded through intracellular injection or dialysis.

Ca2+ concentration changes can be imaged in locations other than the cytoplasm. For example, the low-affinity Mag-Fluo-4 dye can record high Ca2+ concentrations, such as those in the endoplasmic reticulum (ER) [3], without becoming saturated. In contrast, the Ca2+ affinity of this dye is too low to report changes in cytoplasmic Ca2+ concentration. When using Mag-Fluo-4 to report changes in ER Ca2+ concentration, the dye can be washed out of the cytoplasm via mild permeabilization or patch pipette dialysis to ensure that the detected signal is due to Ca2+ located in the ER.

The combination of Mag-Fluo-4 AM with a red cytoplasmic dye such as Rhod-3 AM can be used to simultaneously image cytoplasmic and ER Ca2+ concentration changes. Rhod-2 localizes to mitochondria, so it can be used to report mitochondrial Ca2+ transients [4].  Lysosomal Ca2+ can be measured by bathing cells in the dextran-conjugated version of certain Ca2+ dyes [5]. Ratiometric Ca2+ reporters, such as the organic dyes Indo-1 and Fura-2, and the genetically encoded Premo™ Cameleon Ca2+ sensor, are useful for imaging Ca2+ changes localized in small, subcellular regions, or in cases where movement of the cell or tissue may give rise to false changes in dye emission.

Complete Range of Reagents for Calcium Imaging

For more information on the complete range of reagents available for calcium imaging applications, see Introduction to Ca2+ Measurements with Fluorescent Indicators in The Molecular Probes® Handbook.

  1. Maravall M, Mainen ZF, Sabatini BL et al. (2000) Biophys J 78:2655–2667.
  2. Gee K, Brown KA, Chen W-N U et al. (2000) Cell Calcium 2:97–106.
  3. Park MK, Tepikin AV, Petersen OH et al. (2000) EMBO J 19:5729–5739.
  4. Quintanilla RA, Matthews-Roberson TA, Dolan PJ et al. (2009) J Biol Chem 284:18754–18766.
  5. Lloyd-Evans E, Morgan AJ, He X et al. (2008) Nat Med 11:1247–1255.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.