Photoactivatable MCherry for High-resolution Two-color Fluorescence Microscopy

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2009 Jan 27

PMID 19169259

Citations 292

Authors

Fedor V Subach

George H Patterson

Suliana Manley

Jennifer M Gillette

Jennifer Lippincott-Schwartz

Vladislav V Verkhusha

Affiliations

Soon will be listed here.

Abstract

The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP-tagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified < or =200 nm clusters of transferrin receptor and clathrin light chain at < or =25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.

Citing Articles

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