The study of the function of Ca<sup>2+</sup> ions inside cells is one the most dynamic areas of modern cell biology. Ca<sup>2+</sup> is known to control neurosynaptic transmission, secretion of hormones, muscle contraction, and a myriad of physiological functions. It is suspected to be involved in cell division, movements of non-muscle cells, as well as memory and learned patterns of the nervous system. In all cases, localized fluctuations in cytosolic free Ca<sup>2+</sup> levels inside cells are believed to control these functions. Techniques for the measurement and manipulation of Ca<sup>2+</sup> are therefore crucial and have been advancing rapidly largely as a result of invention of fluorescent ion indicators.
We list our fluorescent calcium indicators by grouping them according to the color of the light they emit on binding calcium, namely Red, Green, and Near UV. We also sell non-fluorescent BAPTA derivatives.
Standard instrumentation is readily available in most laboratories and core imaging facilities for compatibility with the multitude of green fluorescent probes in the market. Photochemical constraints and low quantum yields have contributed to the relatively small amounts of red-emitting probes compared to the green-emitting probes.
Red-Emitting Fluorescent Calcium Indicators
Our new red calcium indicator, Ion Red Calcium 660 (IRC-660) is under development. Upon completion, this product will have properties that exceed the normal criteria for fluorescent calcium indicators:
- Good loading
- Good quantum yield
- Great pKa
- Good extinction coefficient (22,000)
Advantages over green emitting fluorescent indicators1
The Red-emitting wavelength region is attractive for biological research because it affords:
- Higher tissue penetration due to reduced scattering and reduced absorption by endogenous fluorophores (usually green)
- Lower phototoxicity
- Reduced spectral overlap by spectrally broad autofluorescence
- Work in an optical window where water and hemoglobin absorption is reduced
- Monitoring Ca<sup>2+</sup> in cells or tissues expressing yellow or green fluorescent protein or labelled with other green-emitting fluorophores
These advantages will make the dye usable in almost all cells and hence widen the scope of screening tremendously.
Table 4.1 Ion Indicators Calcium Indicators
|ION Calcium Red 660 K+ Salt||1056||580 nm||660 nm||40||0.400||H2O||5.7|
|ION Calcium Red 660 AM||1190||*||*||*||*||DMSO||*|
*The AM is for non-invasive loading and generates the salt inside the cell by non-specific esterases.
Protocol- to be written by Dr. Joe Kao (U. Maryland).
Modifications of IRC-660 can be made for other applications upon request in a CUSTOM SYNTHESIS.
Green-Emitting Fluorescent Calcium Indicators
Most useful and inexpensive lasers operate in the green part of the visible spectrum and that has made green-emitting fluorescent calcium indicators very useful for confocal microscopy, flow cytometry, and screening. The popular green-emitting fluorescent calcium indicators for screening are FLUO-2, FLUO-3 and FLUO-4. They are all derived from the original FLUO compound (Figure 1) invented by Minta and Tsien (1989).2
|FLUO-2||x = H||R = CH3|
|FLUO-3||x = Cl||R = CH3|
|FLUO-4||x = F||R = CH3|
Table of Properties for FLUO-2, FLUO-3, and FLUO-4
|Indicator||MW||Excitation (nm)||Emission (nm)||Kd||Extinction||Solubility|
|FLUO-2 K+ Salt||891||490||515||230||80,000||H2O|
|FLUO-3 K+ Salt||960||506||526||390||80,000||H2O|
|FLUO-4 K+ Salt||927||490||515||345||80,000||H2O|
Near UV (Traditional) Calcium Indicators
We offer Fura-2 (Figure 1) as a traditional calcium indicator which emits near UV. Fura-2 is very useful because it is ratiometric. It displays a shift in fluorescence upon binding calcium and the ratio of the two wavelengths can be used to effect calibration. The ratiometry figure below (Figure 2) makes it useful even though the excitation ratio is in the UV region where the energy is very high and there is great autofluorescence.
Ca2+ indicators that are excited at visible wavelengths have advantages over UV-excited indicators. Whereas UV light can cause cell damage, visible light is generally biologically benign. Moreover, much less cellular autofluorescence is excited by visible light than by UV light. The ready availability of low-cost lasers that emit visible light makes the visible indicators ideal for confocal microscopy and flow cytometry.
- Oheim M, Hoff MV, Feltz A, Zamateeva A, Mallet J-M, Collot M (2014), New red-fluorescent calcium indicators for optogenetic, photoactivation, and multi-color imaging. Biochimica et Biophysica Acta.
- Minta A, Tsien R (1989), JBC 264, 8171.