Since the early development of Fluorescence Correlation Spectroscopy (FCS), it has been recognized that background intensity can lead to artifacts in the amplitude of the autocorrelation function (ACF) and, consequently, to inaccurate estimates of particle numbers. Here, we present a protocol for quantitative background evaluation and amplitude correction in FCS experiments, applicable to different sources of background such as detector noise, autofluorescence, and light scattering. We demonstrate the performance of our approach through three representative case studies: (i) FCS measurements of a bright fluorophore at low concentration, (ii) FCS of dim nanoparticles affected by solvent Raman scattering, and (iii) FCS performed using a confocal setup equipped with a SPAD array, where background originates from detector hot pixels. These examples represent typical experimental conditions in which background signals compromise quantitative interpretation, illustrating how our protocol restores accuracy and reproducibility in FCS analysis. By systematically identifying and correcting these effects, the proposed protocol addresses a long-standing limitation of FCS and provides a robust framework for improving the accuracy and reproducibility of quantitative fluorescence measurements.

Two-pore channels (TPCs) are evolutionarily conserved intracellular cation channels found in both plants and animals, where they mediate ion fluxes across endomembrane compartments. While historically the plant channel was among the first plant ion channels to be characterized, thanks to the relative ease of applying the patch-clamp technique to isolated plant vacuoles, where it is localized, the functional properties of the two main human isoforms, HsTPC1 and HsTPC2, expressed in endosomal and lysosomal membranes, were elucidated much later. In plants, TPCs are typically represented by a single isoform, exemplified by AtTPC1 in the model plant Arabidopsis thaliana, which functions as a voltage-dependent, Ca²⁺-regulated channel. The physiological role of plant TPCs is not yet fully clarified, although evidence suggests that they may contribute to systemic signaling and stress responses. In humans, two main isoforms, HsTPC1 and HsTPC2, are expressed in endosomal and lysosomal membranes. Human TPCs are primarily regulated by the phosphoinositide PI(3,5)P₂ and display a high selectivity for Na⁺. However, these channels also appear as a non-selective cationic conductance when activated by the potent Ca²⁺-mobilizing messenger NAADP, likely through interaction with an accessory protein. Functionally, human TPCs are involved in endolysosomal trafficking, membrane fusion, and intracellular signaling, with emerging roles in immunity, metabolism, and disease. Overall, TPCs represent key components of intracellular ion homeostasis and cellular physiology; however, their precise regulatory mechanisms and integrated physiological roles remain only partially understood and, in several respects, are still elusive.