Surpassing the Background Barrier for Multidimensional Single-Molecule Localization Super-Resolution Imaging: A Case...

The background barrier restricts the dimensionality of live-cell single-molecule localization super-resolution imaging. Ideally, a probe exclusively turned on by its target, without any nonspecific fluorescence signals from off-target molecules, constitutes a practical solution to surpass this barrier. Yet, few such fluorophores have been developed. A lysosome with a unique acidic lumen was chosen as the target for demonstrating the concept advantage. A representative lyso-tracker Lyso-R (piperazine rhodamine) with high brightness has been spirocyclized with o-phenylenediamine to form Lyso-Rpa. This probe shifted its bright-dark spirocyclization balance to a strong acidity domain (pK_a = -0.18). Consequently, under no-wash conditions, Lyso-Rpa showed almost undetectable background photons (only one-sixtieth of that of Lyso-R) in a neutral cellular environment, and it formed sparsely brightened molecules at a low ratio (∼1 × 10^-3%) in lysosomes. This background-free probe enabled super-resolution imaging and modeling of live-cell lysosomes in four dimensions at 2 s resolution, with quantitative determination of lysosomal volume expansion and deformation at starvation. Our molecular approach sheds new light on surpassing the background barrier for multidimensional super-resolution imaging.