Live cell imaging has revealed that calcium ions (Ca(2+)) pass in and out of many cellular organelles. However, technical hurdles have limited measurements of Ca(2+) in acidic organelles, such as endosomes. Although evidence hints that endosomes play a role in Ca(2+) signaling, direct measurements within endosomal lumina represent one of the final frontiers in organelle imaging. To measure Ca(2+) in a TiVAMP-positive endosome sub-population, the pH-resistant ratiometric Ca(2+) biosensor GEM-GECO1 and the ratiometric pH biosensor mKeima were used. A positive correlation between acidic endosomal pH and higher Ca(2+) was observed within these Rab5a- and Rab7-positive compartments. Ca(2+) concentration in most endosomes was estimated to be below 2μM, lower than Ca(2+) levels in several other intracellular stores, indicating that endosomes may take up Ca(2+) during physiological stimulation. Indeed, endosomes accumulated Ca(2+) during glucose-stimulation, a condition where endosomal pH did not change. Our biosensors permitted the first measurements revealing a role for endosomes in cellular Ca(2+) homeostasis during physiological stimulation.

Gold nanoparticles have been widely utilized to achieve colorimetric detection for various diagnostic applications. One of the most frequently used methods for DNA detection involves the aggregation of DNA-modified gold nanoparticles driven by target DNA hybridization. This process, however, is intrinsically slow, limiting its use in rapid diagnostics. Here we take advantage of the reverse process: the disassembly of preformed aggregates triggered by the addition of target DNA via a strand displacement mechanism. A systematic study of the dependence of the disassembly rate on temperature, with and without toeholds, has delivered a system that produces an extremely rapid colorimetric response. Furthermore, using an optimal toehold length of 5 nucleotides, target triggered disassembly is rapid over a wide range of ambient temperatures. Using this overhang system, simple visualization of low picomole amounts of target DNA is possible within 10 min at room temperature.