Fluorescence lifetime imaging microscopy in the medical sciences

The steady improvement in the imaging of cellular processes in living tissue over the last 10–15 years through the use of various fluorophores including organic dyes, fluorescent proteins and quantum dots, has made observing biological events common practice. Advances in imaging and recording technology have made it possible to exploit a fluorophore's fluorescence lifetime. The fluorescence lifetime is an intrinsic parameter that is unique for each fluorophore, and that is highly sensitive to its immediate environment and/or the photophysical coupling to other fluorophores by the phenomenon Förster resonance energy transfer (FRET). The fluorescence lifetime has become an important tool in the construction of optical bioassays for various cellular activities and reactions. The measurement of the fluorescence lifetime is possible in two formats; time domain or frequency domain, each with their own advantages. Fluorescence lifetime imaging applications have now progressed to a state where, besides their utility in cell biological research, they can be employed as clinical diagnostic tools. This review highlights the multitude of fluorophores, techniques and clinical applications that make use of fluorescence lifetime imaging microscopy (FLIM).     

Fluorescence molecular imaging of small animal tumor models

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.     

Impact on the endoplasmic reticulum and Golgi apparatus of turnip mosaic virus infection

The impact of turnip mosaic virus (TuMV) infection on the endomembranes of the host early secretory pathway was investigated using an infectious clone that has been engineered for tagging viral membrane structures with a fluorescent protein fused to the viral protein 6K(2). TuMV infection led to the amalgamation of the endoplasmic reticulum (ER), Golgi apparatus, COPII coatamers, and chloroplasts into a perinuclear globular structure that also contained viral proteins. One consequence of TuMV infection was that protein secretion was blocked at the ER-Golgi interface. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the perinuclear structure cannot be restocked in viral components but was dynamically connected to the bulk of the Golgi apparatus and the ER. Experiments with 6K(2) fused to photoactivable green fluorescent protein (GFP) showed that production of motile peripheral 6K(2) vesicles was functionally linked to the perinuclear structure. Disruption of the early secretory pathway did not prevent the formation of the perinuclear globular structure, enhanced the clustering of peripheral 6K(2) vesicles with COPII coatamers, and led to inhibition of cell-to-cell virus movement. This suggests that a functional secretory pathway is not required for the formation of the TuMV perinuclear globular structure and peripheral vesicles but is needed for successful viral intercellular propagation.     

Fluorescence microscopy today

Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.     

Fluorescence microscopy below the diffraction limit

Fluorescence imaging with conventional microscopy has experienced numerous advances in almost every limiting factor from sensitivity to speed. But improved resolution beyond the fundamental limitation of light diffraction has been elusive until recent years. Now, techniques are available that surpass this barrier and improve resolution up to 10 times over that of conventional microscopy. This chapter provides an overview of these new “super-resolution” imaging methods.     

In vivo FLIM-FRET measurements of recombinant proteins expressed in filamentous fungi

Fluorescence imaging techniques are extremely powerful tools in cell biology, providing valuable insights into the structure and function of biomolecules in their native environments. In particular, the use of Förster resonance energy transfer (FRET) has become increasingly important to obtain information on interactions on the nanoscale, in turn providing insights into molecular behaviour inside living cells. This review describes the basic principles of FRET and fluorescence lifetime imaging microscopy (FLIM) and their application in analyses of protein interactions inside living fungal cells.     

Airway imaging in disease: Gimmick or useful tool?

Airway remodeling is an important pathophysiological mechanism in a variety of chronic airway diseases. Historically investigators have had to use invasive techniques such as histological examination of excised tissue to study airway wall structure. The last several years has seen a proliferation of relatively noninvasive techniques to assess the airway branching pattern, wall thickness, and more recently, airway wall tissue components. These methods include computed tomography, magnetic resonance imaging, and optical coherence tomography. These new imaging technologies have become popular because to understand the physiology of lung disease it is important we understand the underlying anatomy. However, these new approaches are not standardized or available in all centers so a review of their validity and clinical utility is appropriate. This review documents how investigators are working hard to correct for inconsistencies between techniques so that they become more accepted and utilized in clinical settings. These new imaging techniques are very likely to play a frontline role in the study of lung disease and will, hopefully, allow clinicians and investigators to better understand disease pathogenesis and to design and assess new therapeutic interventions.     

Recent Advances in Super-Resolution Fluorescence Imaging and Its Applications in Biology

Fluorescence microscopy has become an essential tool for biological research because it can be minimally invasive, acquire data rapidly, and target molecules of interest with specific labeling strategies. However, the diffraction-limited spatial resolution, which is classically limited to about 200 nm in the lateral direction and about 500 nm in the axial direction, hampers its application to identify delicate details of subcellular structure. Extensive efforts have been made to break diffraction limit for obtaining high-resolution imaging of a biological specimen. Various methods capable of obtaining super-resolution images with a resolution of tens of nanometers are currently available. These super-resolution techniques can be generally divided into three primary classes： （1） patterned illumination- based super-resolution imaging, which employs spatially and temporally modulated illumination light to reconstruct sub-diffraction structures; （2） single-molecule localization-based super-resolution imaging, which localizes the profile center of each individual fluo- rophore at subdiffraction precision; （3） bleaching/blinking-based super-resolution imaging. These super-resolution techniques have been utilized in different biological fields and provide novel insights into several new aspects of life science. Given unique technical merits and commercial availability of super-resolution fluorescence microscope, increasing applications of this powerful technique in life science can be expected.     

Emerging functions of the calpain superfamily of cysteine proteases in neuroendocrine secretory pathways

The first calpain protease was discovered over 40 years ago now, yet despite the vast amount of literature that has subsequently emerged detailing their involvement in the pathophysiology of a variety of human diseases, it is only in the last decade that calpain-mediated actions along the secretory pathway have begun to emerge. However, the number of secretory pathway substrates identified and their diversity of function continues to grow. This review summarizes our current knowledge of calpain-mediated mechanisms of action that are pertinent to synaptic vesicle assembly and budding, cytoskeletal organization, endosomal recycling, and exocytotic membrane fusion.     

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

Fluorescence microscopy is an important and extensively utilised tool for imaging biological systems. However, the image resolution that can be obtained has a limit as defined through the laws of diffraction. Demand for improved resolution has stimulated research into developing methods to image beyond the diffraction limit based on far-field fluorescence microscopy techniques. Rapid progress is being made in this area of science with methods emerging that enable fluorescence imaging in the far-field to possess a resolution well beyond the diffraction limit. This review outlines developments in far-field fluorescence methods which enable ultrahigh resolution imaging and application of these techniques to biology. Future possible trends and directions in far-field fluorescence imaging with ultrahigh resolution are also outlined.     

A Single Polar Residue and Distinct Membrane Topologies Impact the Function of the Infectious Bronchitis Coronavirus E Protein

The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection.     

Reshaping the optical dimension in optogenetics

Optogenetics has been revolutionizing circuit neuroscience in the last few years. Optical methods combined with genetics and molecular techniques have provided new tools for stimulation of neurons, which hold great promise to provide a solution to the circuit mapping problem and more generally provide us with the ability to artificially control the natural stimulus space. Nevertheless, until very recently almost all applications of optogenetics have been based on relatively simple optical schemes mainly used for inducing population activity in neuronal assembles. In this context, alternative optical schemes that enhance the spatial or temporal resolution of excitation and allow for flexible and arbitrary generation of light patterns have all synergetic impact on the development of new optogenetic actuators. In the following we discuss and compare the main new optical techniques that have become available in the recent years. Their respective strengths and limitations as well as their application to different biological contexts are illustrated.     

An overview of a wide range of functions of ZnT and Zip zinc transporters in the secretory pathway

Zinc plays essential roles in the early secretory pathway for a number of secretory, membrane-bound, and endosome/lysosome-resident enzymes. It enables the enzymes to fold properly and become functional, by binding as a structural or catalytic component. Moreover, zinc secreted from the secretory vesicles/granules into the extracellular space has a pivotal role as a signaling molecule for various physiological functions. Zinc transporters of the Slc30a/ZnT and Slc39a/Zip families play crucial roles in these functions, mediating zinc influx to and efflux from the lumen of the secretory pathway, constitutively or in a cell-specific manner. This paper reviews current knowledge of the ways these two zinc transporters perform these tasks by manipulating zinc homeostasis in the secretory pathway. Recent questions concerning zinc released into the cytoplasm from the secretory pathway, which then functions as an intracellular signaling molecule, are also briefly reviewed, emphasizing zinc transporter functions.     

An Overview of a Wide Range of Functions of ZnT and Zip Zinc Transporters in the Secretory Pathway

Zinc plays essential roles in the early secretory pathway for a number of secretory, membrane-bound, and endosome/lysosome-resident enzymes. It enables the enzymes to fold properly and become functional, by binding as a structural or catalytic component. Moreover, zinc secreted from the secretory vesicles/granules into the extracellular space has a pivotal role as a signaling molecule for various physiological functions. Zinc transporters of the Slc30a/ZnT and Slc39a/Zip families play crucial roles in these functions, mediating zinc influx to and efflux from the lumen of the secretory pathway, constitutively or in a cell-specific manner. This paper reviews current knowledge of the ways these two zinc transporters perform these tasks by manipulating zinc homeostasis in the secretory pathway. Recent questions concerning zinc released into the cytoplasm from the secretory pathway, which then functions as an intracellular signaling molecule, are also briefly reviewed, emphasizing zinc transporter functions.     

The secretory pathway of protists: spatial and functional organization and evolution.

All cells secrete a diversity of macromolecules to modify their environment or to protect themselves. Eukaryotic cells have evolved a complex secretory pathway consisting of several membrane-bound compartments which contain specific sets of proteins. Experimental work on the secretory pathway has focused mainly on mammalian cell lines or on yeasts. Now, some general principles of the secretory pathway have become clear, and most components of the secretory pathway are conserved between yeast cells and mammalian cells. However, the structure and function of the secretory system in protists have been less extensively studied. In this review, we summarize the current knowledge about the secretory pathway of five different groups of protists: Giardia lamblia, one of the earliest lines of eukaryotic evolution, kinetoplastids, the slime mold Dictyostelium discoideum, and two lineages within the "crown" of eukaryotic cell evolution, the alveolates (ciliates and Plasmodium species) and the green algae. Comparison of these systems with the mammalian and yeast system shows that most elements of the secretory pathway were presumably present in the earliest eukaryotic organisms. However, one element of the secretory pathway shows considerable variation: the presence of a Golgi stack and the number of cisternae within a stack. We suggest that the functional separation of the plasma membrane from the nucleus-endoplasmic reticulum system during evolution required a sorting compartment, which became the Golgi apparatus. Once a Golgi apparatus was established, it was adapted to the various needs of the different organisms.     

Review of acid phosphatase in the diagnosis and prognosis of prostatic cancers

Acid phosphatase is a secretory product frequently utilized as a tumor marker for disseminated, late stage (D2) prostatic cancer. In the 40 years since this association has been recognized, this enzyme has been subjected to extensive biochemical and immunological characterizations. These techniques have also been adapted for rapid and specific determinations of the prostatic isoenzyme levels using a variety of techniques. Since acid phosphatase levels do not become significantly elevated until late stage cancer, newer markers such as prostate-specific antigen have been sought which appear earlier and may be more useful for the screening and monitoring of high risk populations. At this time it is appropriate to review the current and future status of acid phosphatase as a diagnostic aid.     

Protein-specific features of the general secretion pathway in yeast: the secretion of acid phosphatase

The major phosphate-repressible acid phosphatase (APase) of Saccharomyces cerevisiae, a cell wall glycoprotein, has been extensively used as a reporter protein to analyse successive steps in the yeast secretory pathway. In contrast to other yeast secretory proteins, APase can still be translocated into the endoplasmic reticulum (ER) even when it is made without its signal peptide. This property illustrates the permissiveness of targeting to the ER in yeast. Studies on APase-containing hybrid proteins have provided some of the evidence that specific soluble factors must interact with secretory proteins prior to their translocation across the ER membrane. A systematic analysis of mutations affecting the sequence of the APase signal peptide cleavage site demonstrated that cleavage occurs only when the last amino acid of the signal sequence is small and neutral. This was one of the first studies to verify the requirements for signal peptidase cleavage that had previously only been predicted from statistical analysis. Studies performed either with inhibitors of glycosylation or with mutant APases demonstrated the critical role of core glycosylation for APase folding, which is essential for efficient transport beyond the ER. Following the fate of particular modified APases along the secretory pathway provided insights into some general properties of the secretory apparatus and illustrated the specific requirements for a given protein during its intracellular traffic.     

Use of Kaede fusions to visualize recycling of G protein-coupled receptors

The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.     

Multiple pathways of exocytosis, endocytosis, and membrane recycling: validation of a Golgi route

A number of pathways for intracellular membrane traffic have been detected in various cell types. The major established routes are: 1) the lysosomal pathway, which is the major route utilized in phagocytic and cultured cells; 2) the transcellular route, which represents the major type of traffic in nonfenestrated, capillary endothelial cells and which also appears to be the preferred route for the transport of immunoglobulins (intact) across cells; 3) the exocytosis pathway, utilized in secretory cells for discharge of secretory products, and which is also believed to be used for delivery of intrinsic membrane glycoproteins; 4) the plasmalemma to Golgi route, also highly developed in secretory cells, which is believed to be utilized for the recycling of secretory granule membranes; and 5) the biosynthetic pathways for transport of secretory products, lysosomal enzymes, and membrane proteins from the endoplasmic reticulum to the Golgi complex and for transport of lysosomal enzymes from the Golgi complex to lysosomes. It has become clear that cells repeatedly reutilize or recycle the membranes used in these various transport operations. Clathrin-coated vesicles have been found to be involved in transport along all these routes, which suggests that there are multiple populations of coated vesicles with different transport functions in every cell. It has become clear that the Golgi complex is the site where the membrane and product traffic converges and is sorted and directed to its correct destinations. The validation of a transport route from the cell surface to the Golgi complex raises the possibility that bound ligands and membrane constituents could be modified or repaired in transit during recycling through the Golgi complex, which is a biosynthetic compartment.     

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary.2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques.3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.