Advances in fluorescence microscopy and the engi-neering of the green fluorescent protein (GFP) from Aequorea victoria into mutants with improved properties and altered colours have provided the basic tools that allow the investigation of complex processes in live cells. Fluorescent protein-based indicators can be designed to respond to various biological events and signals, targeted to subcellular compartments and introduced into various tissues and intact organisms.
Recent advances in fluorescent proteins include the engineering or discovery of variants that have enhanced brightness, improved pH resistance, the ability to undergo photochemical colour conversion, or red fluorescent emission.
Among the alternatives to fluorescent proteins, the tetracysteine–biarsenical labelling system is one of the most promising owing to its minimal steric bulk, fast rate of labelling, the availability of several colours and its ability to provide staining that is suitable for electron microscopy.
The barrel-like structures of Aequorea fluorescent proteins (AFPs), which effectively shield the chromophore from the external environment, make them well suited to more 'passive' applications such as monitoring the spatio-temporal (location, translocation, accumulation and degradation) dynamics of appropriate fusion proteins.
In the more 'active' applications of fluorescent proteins, biochemical parameters such as metabolite concentration, enzyme activity, or protein–protein interactions can be detected by their effects on the fluorescence properties of the designed indicators. Such indicators can be divided further into molecules with single chromophores versus composites that are based on fluorescence resonance energy transfer (FRET) between two chromophores.
Single-fluorophore fluorescent protein-based indicators have been engineered to respond to various cellular parameters including pH, halides, free Ca2+ and redox potentials.
Intramolecular FRET-based indicators have been engineered to monitor intracellular Ca2+, cyclic GMP, GTPase and kinase activities. Protein–protein interactions, cyclic AMP dynamics and clustering in lipid rafts have been analysed by using intermolecular FRET-based probes.
The applications of fluorescent probes will continue to expand and provide exciting new insights into the biology of living cells. Particularly exciting areas include high-throughput screening, single-molecule spectroscopy and whole-body in vivo imaging.
Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein–protein interactions.
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We thank S. R. Adams and J. Babendure for their comments on the manuscript. This work was supported by a National Institutes of Health grant to R.Y.T. and postdoctoral fellowship to A.Y.T, a grant from the Alliance for Cellular Signaling (to J.Z. and R.Y.T.) and the Howard Hughes Medical Institute. J.Z. is supported in part by a postdoctoral fellowship from La Jolla Interfaces in Science and Burroughs Wellcome Fund, and R.E.C. is supported in part by a postdoctoral fellowship from the Canadian Institutes of Health Research.
The irreversible destruction, by any one of a number of different mechanisms, of a fluorophore that is under illumination.
- FLUORESCENCE RESONANCE ENERGY TRANSFER
(FRET).The non-radiative transfer of energy from a donor fluorophore to an acceptor fluorophore that is typically < 80 Å away. FRET will only occur between fluorophores in which the emission spectrum of the donor has a significant overlap with the excitation of the acceptor.
- QUANTUM YIELD
The probability of luminescence occurring in given conditions — expressed by the ratio of the number of photons that are emitted by the luminescing species to the number of photons that are absorbed.
The pH at which a molecule, or a particular site within a molecule, carries an ionizable H+ 50% of the time.
Proteins from blue-green algae and red algae that exhibit intense fluorescence owing to the presence of multiple bilin chromophores that are covalently attached to the protein.
The core portion of a molecule that is directly responsible for absorbing photons. Chromophores usually contain alternating single and double bonds.
A chromophore that can re-emit photons.
- MUSHROOM BODIES
Two prominent bilaterally symmetrical structures in the fly brain that are crucial for olfactory learning and memory.
- FLUORESCENCE-LIFETIME IMAGING MICROSCOPY
(FLIM). An imaging technique in which the lifetime, rather than the intensity, of the fluorescent signal is measured. This approach can be used to measure FRET.
- FLUORESCENCE-ACTIVATED CELL SORTING
(FACS). A flow cytometry application in which live fluorescent cells are excited at a specific wavelength and then sorted into physically separated subpopulations on the basis of their fluorescence emission.
- POSITRON EMISSION TOMOGRAPHY
(PET). Positron emission tomography is an imaging technique that is used to detect decaying nuclides, such as 15O, 13N, 11C, 18F, 124I and 94mTc.
- MAGNETIC RESONANCE IMAGING
The use of radio waves in the presence of a magnetic field to extract information from certain atomic nuclei (most commonly hydrogen, for example, in water). This technique is used to show certain types of tissue damage and the presence of tumours.
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Zhang, J., Campbell, R., Ting, A. et al. Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3, 906–918 (2002). https://doi.org/10.1038/nrm976
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