The discovery and development, over the past decade, of fluorescent proteins from a wide variety of marine organisms has initiated a revolution in the study of cell behavior by providing convenient markers for gene expression and protein targeting in living cells and organisms. The most widely used of these fluorescent proteins, the green fluorescent protein (GFP) first isolated from the jellyfish Aequorea victoria, can be attached to virtually any protein of interest and still fold into a fluorescent molecule. The resulting GFP fusion product can be used to localize previously uncharacterized proteins or to visualize and track known proteins to further understand cellular events. The use of fluorescent proteins as a minimally invasive tool for studying protein dynamics and function has been stimulated by the engineering of genetic variants with improved brightness, photostability and expression properties (see Figure 1). Cells that express gene products tagged with fluorescent proteins can be imaged with low light intensities over many hours to provide useful information about changes in the steady-state distribution of a protein over time.

Introduction to Fluorescent Proteins - The discovery of green fluorescent protein in the early 1960s ultimately heralded a new era in cell biology by enabling investigators to apply molecular cloning methods, fusing the fluorophore moiety to a wide variety of protein and enzyme targets, in order to monitor cellular processes in living systems using optical microscopy and related methodology. When coupled to recent technical advances in widefield fluorescence and confocal microscopy, including ultrafast low light level digital cameras and multitracking laser control systems, the green fluorescent protein and its color-shifted genetic derivatives have demonstrated invaluable service in many thousands of live-cell imaging experiments.

The Fluorescent Protein Color Palette - A broad range of fluorescent protein genetic variants have been developed over the past several years that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Extensive mutagenesis efforts in the original jellyfish protein have resulted in new fluorescent probes that range in color from blue to yellow and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone Discosoma striata and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, red, and far-red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.

Imaging Parameters for Fluorescent Proteins - The wide spectrum of fluorescent proteins and derivatives uncovered thus far are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These unique probes have proven extremely useful as reporters for gene expression studies in both cultured cells and entire animals. In living cells, fluorescent proteins are most commonly utilized to track the localization and dynamics of proteins, organelles, and other cellular compartments, as well as a tracer of intracellular protein trafficking. Quantitative imaging of fluorescent proteins is readily accomplished with a variety of techniques, including widefield, confocal, and multiphoton microscopy, to provide a unique window for exposing the intricacies of cellular structure and function.

Optical Highlighter Fluorescent Proteins - Protein chromophores that can be activated to initiate fluorescence emission from a quiescent state (a process known as photoactivation), or are capable of being optically converted from one fluorescence emission bandwidth to another (photoconversion), represent perhaps the most promising approach to the in vivo investigation of protein lifetimes, transport, and turnover rates. Appropriately termed molecular or optical highlighters, photoactivated fluorescent proteins generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength. Photoconversion optical highlighters, on the other hand, undergo a change in the fluorescence emission bandwidth profile upon optically-induced changes to the chromophore. These effects result in the direct and controlled highlighting of distinct molecular pools within the cell.

Choosing Filter Combinations for Fluorescent Proteins - Fluorescence filter combinations designed to image fluorescent proteins must be carefully chosen to maximize the level of emission intensity presented to the detector while simultaneously reducing the number of unwanted photons from autofluorescence or bleed-through by other fluorophores. The broad absorption and emission spectral profiles exhibited by most fluorescent proteins offer a wide range of choice in filters, which are usually optimized for use with a specific detection system (human eye, digital camera, or photomultiplier). This interactive tutorial is designed to enable the identification of critical filter parameters, including the center wavelength, bandwidth region, and dichromatic mirror cut-on wavelength, which are necessary for imaging fluorescent proteins.

Choosing Fluorescent Proteins for Dual Labeling Experiments - The broad excitation and emission spectral profiles exhibited by fluorescent proteins and their color-shifted genetic variants often require specialized considerations when designing live-cell imaging experiments using two or more of these unique probes simultaneously. Of primary concern are potential bleed-through artifacts resulting from the significant degree of emission spectral overlap usually exhibited by fluorescent protein combinations. This interactive tutorial explores matching fluorescent proteins for dual labeling investigations with regards to spectral bandwidth and overlap, excitation efficiency, emission window dimensions, and other parameters necessary to design logical experiments.

Fluorescence Resonance Energy Transfer with Fluorescent Proteins - Fluorescent proteins are increasingly being applied as non-invasive probes in living cells due to their ability to be genetically fused to proteins of interest for investigations of localization, transport, and dynamics. In addition, the spectral properties of fluorescent proteins are ideal for measuring the potential for intracellular molecular interactions using the technique of Förster (or fluorescence) resonance energy transfer (FRET) microscopy. Because energy transfer is limited to distances of less than 10 nanometers, the detection of FRET provides valuable information about the spatial relationships of fusion proteins on a sub-resolution scale. This interactive tutorial explores various combinations of fluorescent proteins as potential FRET partners and provides information about critical resonance energy transfer parameters, as well as suggestions for microscope optical filter and light source configuration.

Optical Highlighter Fluorescent Proteins - Photoactivated fluorescent proteins generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength. Photoconversion optical highlighters, on the other hand, undergo a change in the fluorescence emission bandwidth profile upon optically-induced changes to the chromophore. This interactive tutorial explores the optical conversion of several useful highlighter probes and simulates how these proteins would be viewed in an actual confocal microscope.

Fluorescent Protein Fluorophore Maturation Mechanisms - Autocatalytic formation of the fluorophore (also referred to as a chromophore) within the shielded environment of the polypeptide backbone during fluorescent protein maturation follows a surprisingly unified mechanism, especially considering the diverse natural origins of these useful biological probes. Shortly after synthesis, most fluorescent proteins slowly mature through a multi-step process that consists of folding, initial fluorophore ring cyclization, and subsequent modifications of the fluorophore. The spectral properties of fluorescent proteins are dependent upon the structure of the fluorophore as well as the localized interactions of amino acid residues in the immediate vicinity, and in some cases, residues far removed from the fluorophore. The interactive tutorials in this section explore fluorophore formation in a wide variety of spectrally diverse fluorescent proteins deduced from crystallographic studies.

General Fluorescent Protein References - The disciplines of cellular and molecular biology are being rapidly and dramatically transformed by the application of fluorescent proteins developed from marine organisms as fusion tags to track protein behavior in living cells. The most widely used of these probes, green fluorescent protein, can be attached to virtually any target of interest and still fold into a viable fluorescent species. The resulting chimera can be employed to localize previously uncharacterized proteins or to visualize and track known proteins to further understand critical events at the cellular and molecular levels. This section features a bibliography of literature sources for review articles and original research reports on the discovery, applications, and continued development of fluorescent proteins.

Biosensor Fluorescent Protein References - By directly coupling the advanced microscopic technique of fluorescence resonance energy transfer (FRET) to the ubiquitous green fluorescent protein and its multispectral variants fused with selected biopolymers, ingenious researchers have created a new class of biosensors capable of elucidating mechanisms of signaling, enzymatic cleavage, and other critical functions in living cells. This section features a bibliography of literature sources for review articles and original research reports on the construction, applications, and continued development of biosensor fluorescent proteins.

Optical Highlighter Fluorescent Protein References - The ability to selectively initiate or alter fluorescence emission profiles in photoconversion optical highlighter proteins renders these probes as excellent tools for exploring protein behavior in living cells. As the fluorescence intensity (or color spectrum) of highlighters occurs only after photon-mediated conversion, newly synthesized non-photoactivated protein pools remain unobserved and do not complicate experimental results. This section lists sources for review articles and original research reports on optical highlighter fluorescent proteins.

Fluorescent Protein Photobleaching References - The field of cell biology is rapidly being transformed by the application of fluorescent proteins as fusion tags to track dynamic behavior in living cells. In this regard, fluorescence recovery after photobleaching (FRAP) is often employed to selectively destroy fluorescent molecules within a region of interest with a high-intensity laser, followed by monitoring the recovery of new fluorescent molecules into the bleached area over a period of time with low-intensity laser light. The resulting information can be used to determine kinetic properties, including the diffusion coefficient, mobile fraction, and transport rate of the fluorescently labeled molecules. The selected references in this section point to important literature sources for information on FRAP with fluorescent proteins.

Fluorescent Protein FRET References - Understanding the dynamic interactions between proteins within living cells is fundamental to a basic knowledge of the underlying concepts that guide molecular and cellular biology. Over the past few years, the rapid development of fluorescent proteins and their application as fusion products and biosensors has significantly expanded the molecular toolkit available for probing the mysteries of cellular physiology and pathology. In this regard, fluorescence (or Förster) resonance energy transfer (FRET) is emerging as a powerful optical microscopy technique for examining physiological processes with high temporal and spatial resolution. The references listed in this section highlight important literature sources for review articles and original research reports on the construction and applications of fluorescent proteins for resonance energy transfer experiments.

Internet-Based Education on Fluorescent Proteins - Despite the explosive growth of the Internet (in terms of the World Wide Web) as an informational resource for the original scientific literature pertaining to fluorescent protein investigations, there remains an obvious void in educational Websites targeted at beginning students and novices in the field. To address this issue, educational sites dedicated to optical microscopy and digital imaging being constructed and hosted at The Florida State University are turning their attention to the increasing application of fluorescent proteins for live-cell imaging studies. The primary focus of this effort is to create new sections of the sites that address the structure and properties of fluorescent proteins as well as optimizing their utility in imaging experiments.

Fluorescent Protein Vector Commercial Sources - A variety of fluorescent proteins, available as recombinant DNA plasmid vectors designed for transfection of mammalian cells or transformation of bacteria, are commercially available from a number of distributors. Most of the vectors containing fluorescent protein DNA sequences have been codon-optimized for expression in mammalian cells and contain antibiotic genes for selection of stable mutants having relatively constant expression levels. The vectors often contain multiple cloning sequences that enable researchers to easily insert their gene of interest for fusion to the fluorescent protein. Other common features in fluorescent protein vectors include a human cytomegalovirus (CMV) promoter, a Kozak translation initiation site, an early mRNA polyadenylation signal, and a bacterial antibiotic gene.

Live Cell Imaging - One of the foremost targets in the life sciences is to understand the structure, function, and behavior of living organisms, and with evolving advances in technology, such as the development of confocal microscopy and fluorescent probes, it has become possible to pursue this goal at the cellular and subcellular levels. Still, working with and imaging live cells can be a complex, if not daunting, task to microscopists unfamiliar with the techniques and tools that are available. The following is a compilation of resources that offer overviews, background information, interactive forums, frequently asked questions, protocols, and hints that should aid any microscopist attempting to enter into this important, burgeoning field.

Microscopy Specimen Chambers - The demands of modern confocal microscopy, especially those involving imaging of living cells and tissues, require that researchers take special precautions with their specimens. Indeed, simple microscope slides are unsuitable for many applications, resulting in the development of a broad range of specimen chambers, which can often supply the flexibility a microscopist needs. The list of resources in this section exemplifies the great variety of specimen chambers that are available today and should help visitors locate the products that are best suited for their specific scientific pursuits.

Fluorescence Filters - Fluorescence microscopy relies heavily on the ability to select a specific wavelength region for excitation of the specimen and gathering secondary emission during image formation. Recent advances in interference filter design have resulted in highly accurate filters that cover a wide range of bandpass profiles, ranging from just a few to tens and hundreds of nanometers. Listed below is a compilation of manufacturers that design, manufacture, and supply fluorescence filters to the microscopy community. The products that they offer will meet most system requirements, but if a specialized application necessitates a filter with an unusual spectral range or other unique specifications, many of the companies will fabricate custom filters.

Fluorescent Protein Principle Investigators - Many of the scientists involved with research targeting various aspects of cell biology are using fluorescent proteins as imaging probes for cell structure, function, and dynamics. Several of the principle investigators have built extensive websites detailing their laboratories, and these sites are quite useful to visitors interested in learning more about this exciting and rapidly evolving research arena. Included in the information on a majority of the websites linked below are the current research interests, curriculum vitas, publications, lists of laboratory personnel, contact information, educational tutorials, image galleries, and digital videos.

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