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Main Menu - Block
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Fly Facility
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Janelia Experimental Technology
- Mass Spectrometry
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
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- Scientific Computing Systems
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- Vivarium
Note: Research in this publication was not performed at Janelia.
Abstract
Traditional small-molecule fluorophores are always fluorescent. This attribute can obscure valuable information in biological experiments. Here, we report on a versatile "latent" fluorophore that overcomes this limitation. At the core of the latent fluorophore is a derivative of rhodamine in which one nitrogen is modified as a urea. That modification enables rhodamine to retain half of its fluorescence while facilitating conjugation to a target molecule. The other nitrogen of rhodamine is modified with a "trimethyl lock", which enables fluorescence to be unmasked fully by a single user-designated chemical reaction. An esterase-reactive latent fluorophore was synthesized in high yield and attached covalently to a cationic protein. The resulting conjugate was not fluorescent in the absence of esterases. The enzymatic activity of esterases in endocytic vesicles and the cytosol educed fluorescence, enabling the time-lapse imaging of endocytosis into live human cells and thus providing unprecedented spatiotemporal resolution of this process. The modular design of this "fluorogenic label" enables the facile synthesis of an ensemble of small-molecule probes for the illumination of numerous biochemical and cell biological processes.
