Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy

Abstract

By developing a wide-field scheme for spectral measurement and implementing photoswitching, we synchronously obtained the fluorescence spectra and positions of ∼10(6) single molecules in labeled cells in minutes, which consequently enabled spectrally resolved, 'true-color' super-resolution microscopy. The method, called spectrally resolved stochastic optical reconstruction microscopy (SR-STORM), achieved cross-talk-free three-dimensional (3D) imaging for four dyes 10 nm apart in emission spectrum. Excellent resolution was obtained for every channel, and 3D localizations of all molecules were automatically aligned within one imaging path.

Covalent Protein Labeling and Improved Single-Molecule Optical Properties of Aqueous CdSe/CdS Quantum Dots

“…This improvement in total emission may enable bioimaging with improved localization accuracy, 6 longer tracking times, 7 highly localized biosensing, multicolor super-resolution imaging, 49 and other experiments requiring ultrasensitive probes.…”

Perspectives in Super-Resolved Fluorescence Microscopy: What Comes Next?

“…of the constituting proteins [50]. On the other hand, multi-or hyperspectral detection of individual dye molecule emission-while providing great potential to extract additional information, e.g., about the nanoenvironment and multiplying the number of simultaneously usable fluorescent probes-is strictly limited by the number of photons emitted per dye molecules [51][52][53].…”

Chip-based nanoscopy: towards integration and high-throughput imaging

“…TIRF lens also comes with high magnification (typically 60-100X) that limits the f-o-v down to around 100 x 100 μm 2 . Despite rather small f-o-v, simultaneous multi-color imaging has been achieved in various ways [13][14][15] , reducing collection time significantly. In this paper, we show TIRF and super-resolution imaging using a photonic chip waveguide platform that is ideally suited for high-throughput TIRF and super-resolution imaging 16 .…”