A note on estimating absolute cytosolic Ca²⁺ concentration in sensory neurons using a single wavelength Ca²⁺...

Ca²⁺ imaging is frequently used in the investigation of sensory neuronal function and nociception. In vitro imaging of acutely dissociated sensory neurons using membrane-permeant fluorescent Ca²⁺ indicators remains the most common approach to study Ca²⁺ signalling in sensory neurons. Fluo4 is a popular choice of single-wavelength indicator due to its brightness, high affinity for Ca²⁺ and ease of use. However, unlike ratiometric indicators, the emission intensity from single-wavelength indicators can be affected by indicator concentration, optical path length, excitation intensity and detector efficiency. As such, without careful calibration, it can be difficult to draw inferences from differences in the magnitude of Ca²⁺ transients recorded using Fluo4. Here, we show that a method scarcely used in sensory neurophysiology - first proposed by Maravall and colleagues (2000) - can provide reliable estimates of absolute cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in acutely dissociated sensory neurons using Fluo4. This method is straightforward to implement; is applicable to any high-affinity single-wavelength Ca²⁺ indicator with a large dynamic range; and provides estimates of [Ca²⁺]_cyt in line with other methods, including ratiometric imaging. Use of this method will improve the granularity of sensory neuron Ca²⁺ imaging data obtained with Fluo4.