Dynamic ratiometric imaging of cytosolic free Ca²⁺ in skeletal muscle cells using 340/385-nm light-emitting diode illuminators

Calcium-sensitive fluorescent indicators fall broadly into two categories, ratiometric (dual-wavelength) or single-wavelength indicators based on their response to a calcium elevation. Ratiometric indicators shift either their excitation or their emission wavelengths in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. Fura-2 is one of the most common ratiometric fluorescent Ca²⁺ indicators, which has an emission maximum at 510 nm whereas its excitation maximum changes from 380 to 340 nm in response to calcium binding. Historically, a combination of arc lamp and monochromators has been used as the light source for Fura-2 ratiomatric fluorescence microscopy. In recent years, different combinations of LEDs such as 350/380 nm or 360/380 nm have been used to excite Fura-2. To precisely match the Fura-2 excitation, in this study, we built a new fast switchable 340/385-nm LED excitation light source (Prizmatix Ltd., Israel). The newly constructed light source has been exploited in Fura-2 ratiometric calcium imaging of skeletal muscle cells. The spontaneously elicited Ca²⁺ transients in the cells were recorded with high temporal resolution. The light source utilized for the demonstrated instrumentation in this report optimally matches the excitation wavelengths of either calcium-free or bound states of Fura-2. The high-intensity stability and fast switching of the 340/385-nm LED illuminators indicate their potential as a preferred light source for Fura-2 ratiometric calcium imaging over the existing light sources.