A user-friendly two-color super-resolution localization microscope

Abstract

We report a robust two-color method for super-resolution localization microscopy. Two-dye combination of Alexa647 and Alexa750 in an imaging buffer containing COT and using TCEP as switching regent provides matched and balanced switching characteristics for both dyes, allowing simultaneous capture of both on a single camera. Active sample locking stabilizes sample with 1nm accuracy during imaging. With over 4,000 photons emitted from both dyes, two-color superresolution images with high-quality were obtained in a wide range of samples including cell cultures, tissue sections and yeast cells.

Cdk5-dependent phosphorylation of liprinα1 mediates neuronal activity-dependent synapse development

“…The superresolution images were acquired with a custom-built STORM system designed specifically for the dual-channel imaging of Alexa Fluor 647-and Alexa Fluor 750-immunolabeled samples as described previously (55).…”

A Mitochondrion‐Specific Photoactivatable Fluorescence Turn‐On AIE‐Based Bioprobe for Localization Super‐Resolution Microscope

“…Super‐resolution imaging techniques which break the diffraction limit (Abby limit of 200 nm for visible light microscopy) have become a powerful tool for visualizing intracellular structures in nanometer scale, and led to numerous important discoveries in biology . Among these methods, single‐molecule localization microscopy (SLM), such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), is gaining increasing attention. The SLM technique relies on the controllable switching of fluorophores between their bright and dark states, and the quality of its output image is collaboratively decided by (1) the brightness of the fluorescent molecule during each switching, (2) the on/off ratio that represents the ratio between the quantity of molecules in bright state and dark state, as well as (3) the accuracy and efficiency of a certain probe to specifically tag the specified protein or cellular structures without introducing artifacts or interfering the normal functionality.…”

Visualizing the inner life of microbes: practices of multi-color single-molecule localization microscopy in microbiology

“…Cellular components other than proteins are usually reliant on non-genetic targeting tools and thus are mostly investigated by dSTORM and PAINT studies. In case of dual dSTORM experiments relying on organic dyes, it becomes apparent that the membrane-permeable, spectrally distinct red/far-red dye combination TMR (-Star)/ATTO655 (Figure 2m, four out of six red/far-red pairings [35,38,39]) were preferred over AF647 paired with spectrally close dyes in a spectral demixing imaging mode (AF700 (Figure 2k) [53] or AF750 [55]) or green/red [30], green/far-red [15,37] or STORM activator/acceptor (Figure 2l) [41] dye combinations.…”

“…Parallel imaging, on the other hand, reduces imaging times and allows for parallel drift correction. Chromatic aberrations can also be avoided by spectral demixing [53,55]. Next to the extremes of sequential and parallel imaging modes also an alternating read-out mode by orthogonal activation schemes can be applied, either using one [41] or several color channels [31,79].…”