One type of calcium indicator, known as ratiometric, shifts excitation or emission wavelengths when
calcium is present, allowing the concentration of intracellular calcium to be determined from the ratio of
fluorescence emission or excitation at distinct wavelengths.
Fura-2, one of the most common ratiometric fluorescent Ca2+ indicators, has an emission maximum at
510 nm and an excitation maximum that changes from 380 to 340 nm in response to calcium binding.
Historically, a combination of arc lamp and monochromators have been used as the excitation light
source for calcium indicator, but recently different combinations of LEDs such as 350 and 380 nm or 360
and 380 nm have also been used to excite Fura-2.
Using optimal excitation wavelengths
To precisely match Fura-2 excitation, the researchers used a new 340/385-nm excitation light source
based on Mic-LED-340A and Mic-LED-385B LEDs from Prizmatix Ltd., Israel. The LEDs feature high
intensity and stability as well as fast switching between wavelengths. One LED source had peak
wavelength of 343.63 nm with a full width at half maximum of 12.41 nm and the other had a peak
wavelength at 383.80 nm and full width at half maximum of 13.60 nm.
Prior to performing an actual experiment, the researchers used the Prizmatix Spectra-Viewer online
software to simulate the excitation and emission spectra of Fura-2 with Ca²⁺ in free or bound state by
inputting the optics used in the actual experiments. The software can be used to simulate any dualLED/ratiometric system.
Dynamic imaging of skeletal muscle cells.