Photoswitchable cyan fluorescent protein for protein tracking

In recent years diverse photolabeling techniques using green fluorescent protein (GFP)-like proteins have been reported, including photoactivatable PA-GFP, photoactivatable protein Kaede, the DsRed 'greening' technique and kindling fluorescent proteins. So far, only PA-GFP, which is monomeric and gives 100-fold fluorescence contrast, could be applied for protein tracking. Here we describe a dual-color monomeric protein, photoswitchable cyan fluorescent protein (PS-CFP). PS-CFP is capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. We used PS-CFP as a photoswitchable tag to study trafficking of human dopamine transporter in living cells. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and fluorescence resonance energy transfer applications.     

Identification of the amino acid residues responsible for the reversible photoconversion of the monomeric red fluorescent protein TagRFP

The site-directed mutagenesis of the monomeric red fluorescent protein TagRFP and its variants was performed with the goal of generating reversibly photoactivatable fluorescent proteins. Amino acids at positions 69, 148, 165, 179, and 181 (enumeration according to the green fluorescent protein GFP) were shown to play a key role in the manifestation of the photoactivatable properties. A reversibly photoactivatable red fluorescent protein KFP-HC with excitation and emission maxima at 585 and 615 nm, respectively, was generated. The KFP-HC fluorescent intensity was decreased by 5–10 times under green light (530–560 nm) irradiation (due to the fall of the fluorescence quantum yield) and restored under irradiation with blue light (450–490 nm) or after incubation in the dark (recovery half-time of 30 min).     

Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Fluorescent proteins from the green fluorescent protein family have become indispensable imaging tools for cell biology. A wide variety of these proteins were discovered in nonbioluminescent anthozoa in recent years. Some of them feature exciting new properties, with the possibility to alter their intensity and/or fluorescence color by irradiation with light of specific wavelengths. Fluorescent highlighter proteins enable many interesting applications based on regional optical marking in live cells and tissues. This review provides an overview of photoactivatable marker proteins, with a focus on EosFP, a protein that can be switched from green to red fluorescence by approximately 400-nm light. A variety of applications are presented to illustrate the versatility of EosFP in live-cell imaging.     

Innovation: Photoactivatable fluorescent proteins

The fluorescence characteristics of photoactivatable proteins can be controlled by irradiating them with light of a specific wavelength, intensity and duration. This provides unique possibilities for the optical labelling and tracking of living cells, organelles and intracellular molecules in a spatio-temporal manner. Here, we discuss the properties of the available photoactivatable fluorescent proteins and their potential applications.     

Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family

Fluorescent proteins from the green fluorescent protein family have become indispensable imaging tools for cell biology. A wide variety of these proteins were discovered in nonbioluminescent anthozoa in recent years. Some of them feature exciting new properties, with the possibility to alter their intensity and/or fluorescence color by irradiation with light of specific wavelengths. Fluorescent highlighter proteins enable many interesting applications based on regional optical marking in live cells and tissues. This review provides an overview of photoactivatable marker proteins, with a focus on EosFP, a protein that can be switched from green to red fluorescence by approximately 400-nm light. A variety of applications are presented to illustrate the versatility of EosFP in live-cell imaging.     

Axial Resolution Enhancement by 4Pi Confocal Fluorescence Microscopy with Two-Photon Excitation

Confocal fluorescence microscopy and two-photon microscopy have become important techniques for the three-dimensional imaging of intact cells. Their lateral resolution is about 200–300 nm for visible light, whereas their axial resolution is significantly worse. By superimposing the spherical wave fronts from two opposing objective lenses in a coherent fashion in 4Pi microscopy, the axial resolution is greatly improved to ～100 nm. In combination with specific tagging of proteins or other cellular structures, 4Pi microscopy enables a multitude of molecular interactions in cell biology to be studied. Here, we discuss the choice of appropriate fluorescent tags for dual-color 4Pi microscopy and present applications of this technique in cellular biophysics. We employ two-color fluorescence detection of actin and tubulin networks stained with fluorescent organic dyes; mitochondrial networks are imaged using the photoactivatable fluorescent protein EosFP. A further example concerns the interaction of nanoparticles with mammalian cells.     

EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy

The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969-973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one-photon confocal and by two-photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.     

Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras

Fluorescent indicators for the real-time imaging of small molecules or metal ions in living cells are invaluable tools for understanding their physiological function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiologically relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concentration range from 10 nM to 1 mM. Size-exclusion chromatography and fluorescence anisotropy were used to provide a detailed picture of the conformational changes associated with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concentration of the fluorescent proteins and could be understood quantitatively by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small molecules.     

Practical three color live cell imaging by widefield microscopy

Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.     

Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

Imaging the environment of green fluorescent protein

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.     

In vivo molecular and cellular imaging with quantum dots

Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degradation need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans.     

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.     

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.     

A Green Fluorescent Protein with Photoswitchable Emission from the Deep Sea

A colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that ∼15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37°C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.     

Spectral imaging and its applications in live cell microscopy

In biological microscopy, the ever expanding range of applications requires quantitative approaches that analyze several distinct fluorescent molecules at the same time in the same sample. However, the spectral properties of the fluorescent proteins and dyes presently available set an upper limit to the number of molecules that can be detected simultaneously with common microscopy methods. Spectral imaging and linear unmixing extends the possibilities to discriminate distinct fluorophores with highly overlapping emission spectra and thus the possibilities of multicolor imaging. This method also offers advantages for fast multicolor time-lapse microscopy and fluorescence resonance energy transfer measurements in living samples. Here we discuss recent progress on the technical implementation of the method, its limitations and applications to the imaging of biological samples.     

Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2

A number of photoactivatable GFP-like fluorescent proteins (PAFPs) have been reported whose fluorescence can be switched on or whose fluorescent state can be modified by relatively intense irradiation at a specific wavelength. The use of these proteins gives unique opportunities to photolabel and track fusion proteins in a living cell. Here, we provide a protocol for the primary visualization, photoactivation and tracking of two monomeric PAFPs recently developed in our lab. Both these proteins, PS-CFP2 and Dendra2, are fluorescent and can be visualized before photoactivation. Upon photoactivation, their excitation and emission spectra undergo a dramatic red shift. The brightness of their initial and photoconverted states, along with the high dynamic ranges of both proteins, make them an attractive tool for protein photolabeling. Excluding genetic constructs cloning, cell culturing and transfection, the whole protocol may take anywhere from 10 min to several hours, depending on motility of the protein being studied.     

A new fluorescence-based, hydrophobic photolabeling technique for analyzing membrane-associated proteins

We introduce a new, fluorescent and photoactivatable fatty acid derivative (SANU) for hydrophobic labelling of membrane-bound proteins. The technique allows fast and highly sensitive screening of hydrophobically inserting proteins analyzed by SDS-PAGE with a detection limit below 0.1 pmol. A reliable calculation of labelling efficiencies is achieved by simultaneous densitometry of fluorescence and protein staining. We have applied the new technique on the membrane inserting protein talin, G-actin, and, as a negative control, on RNase, which only binds electrostatically to negatively charged lipid interfaces. In several ways superior to radiolabelling, we can recommend this technique for all laboratories under any circumstances.     

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.     

Holographic Photolysis for Multiple Cell Stimulation in Mouse Hippocampal Slices

Background

Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells.

Methods/Principal Findings

The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca²⁺ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells.

Conclusions/Significance

We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically encoded photoactivatable proteins, photoactivatable fluorescent proteins, and voltage-sensitive dyes.