Multicolor Nanoscopy using a New Class of Low-Power Probes

Abstract

STED/RESOLFT approaches provide the most promising technology for real-time video nanoscopy. Unfortunately, STED has sometimes been hobbled by two practical problems: large laser powers that (especially in the visible) damage cells and Stokes' shift similarities among dyes that inhibit multicolor imaging. The former occurs because emission must be separated from the powerful STED beam, pushing deactivation into a redder portion of the emission where the cross section is reduced. Our new probes, in contrast, all have very strong cross sections, and they all deactivate at the same NIR wavelength. Hence, we are able to obtain simultaneous superresolved multicolor imaging at the convenient (and less damaging) wavelength of 780nm.We constructed a home-built microscope to test our probes.Our system generates a green excitation pulse with Gaussian profile in space of just 4-8μW and a more powerful “donut” beam at ∼780nm, using several picosecond wide pulses at 80MHz repetition.We achieve resolutions of ∼50nm when coating 20nm aliphatic-amine beads with NHS-ester versions of our dyes. In fixed cells, microtubules coated with a secondary dye-anti mouse antibody bound to a primary mouse antibody specific to α-tubulin demonstrated ∼90nm resolution with only a few mW applied to the donut beam. We also directly labeled microtubules with orange and red versions of our dye; in this case, we achieve ∼100nm resolution with a single depleting beam.These dyes are essentially normal fluorophores modified to contain “quenching antennae” at 780nm. Unlike STED, these antennae are best with many ps to hundreds of ps wide laser pulses; thus, inexpensive ns-pulsed 780nm diodes may be helpful in disseminating the technology. We are currently pursuing alternative antennae and additional dye colors appropriate to in vivo nanoscopy.