The basis of ratiometric imaging is the sensitivity of certain fluorophores to specific environmental factors. These environmental factors can include pH, the concentration of certain ions like Ca2+ or the proximity of other fluorophore species (as in FRET). In the case of calcium imaging, the fluorophore Fura-2 has an absorption spectrum that changes with the concentration of Ca2+, while the fluorophore Indo-1 has an emission spectrum that changes based on the level of Ca2+.
Fura-2 is ideal for multiplexing applications because the UV excitation wavelength leaves the green channels are open for any of the many popular green fluorophores. By measuring the ratio of intensities on digital images captured at 340 and 380 nm (the wavelengths at which the difference in fluorescence emission signals is at a maximum) the variation of calcium concentration at various locations can be tracked.
The BrightLine® FURA2-C filter set (figures 1 and 2) offers outstanding brightness and contrast, acheiving a superb balance of the saturated-to-free signal intensities so you can get most accurate ratio calculations with minimal adjustments to camera settings.
|Figure 1. High (saturated Ca2+) concentration||Figure 2. Low (Ca2+-free) concentration|
Learn more about ratiometric imaging:
Optical Filters Impact Fluorescence Fidelity (November 2003)
Read our application note on FRET