Quantitative Imaging of Protein-Protein Interactions by Multiphoton Fluorescence Lifetime Imaging Microscopy using a Streak camera

Fluorescence Lifetime Imaging Microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. We review here the recent developments in multiphoton FLIM methods and also present a description of a novel multiphoton FLIM system using a streak camera that was developed in our laboratory. We provide an example of a typical application of the system in which we measure the fluorescence resonance energy transfer between a donor/acceptor pair of fluorescent proteins within a cellular specimen.Comment: Overview of FLIM techniques, StreakFLIM instrument, FRET application

Focusing and Compression of Ultrashort Pulses through Scattering Media

Light scattering in inhomogeneous media induces wavefront distortions which pose an inherent limitation in many optical applications. Examples range from microscopy and nanosurgery to astronomy. In recent years, ongoing efforts have made the correction of spatial distortions possible by wavefront shaping techniques. However, when ultrashort pulses are employed scattering induces temporal distortions which hinder their use in nonlinear processes such as in multiphoton microscopy and quantum control experiments. Here we show that correction of both spatial and temporal distortions can be attained by manipulating only the spatial degrees of freedom of the incident wavefront. Moreover, by optimizing a nonlinear signal the refocused pulse can be shorter than the input pulse. We demonstrate focusing of 100fs pulses through a 1mm thick brain tissue, and 1000-fold enhancement of a localized two-photon fluorescence signal. Our results open up new possibilities for optical manipulation and nonlinear imaging in scattering media

Characterization of the CASZ1-dependent Mechanisms Underlying Vertebrate Blood Vessel Development

The vascular system is the first organ system to form during embryonic development. Its proper establishment is required for embryonic growth and survival as well as tissue homeostasis. Blood vessels are comprised of endothelial cells which must coordinate multiple behaviors such as migration, adhesion, sprouting, and proliferation to properly assemble into cord-like structures and then undergo further remodeling and branching to form a patent vascular network. Disruptions in these processes result in a number of human disease states including tumorigenesis, stroke, and atherosclerosis necessitating a better understanding of the factors and pathways that regulate these key developmental steps. Despite the large number of identified growth factors and proteins that are critical for vascular development, the field still lacks a thorough understanding of how endothelial gene programs are regulated. CASTOR (CASZ1) is an evolutionarily conserved transcription factor expressed in the cardiovascular system. Mutations in the human Casz1 locus have been recently genetically associated with cardiovascular disease risk factors including hypertension and high blood pressure although the mechanisms by which CASZ1 functions in vascular biology are unknown. Here we demonstrate that CASZ1 is critical for proper vascular assembly and morphogenesis during embryonic development. Mechanistically, we have determined that CASZ1 directly binds to and regulates the expression of Epidermal growth factor-like domain 7 (Egfl7), an extracellular matrix (ECM) protein previously implicated in vessel sprouting and vascular lumen formation. We further showed that the CASZ1/Egfl7 transcriptional pathway is required to promote the activation of the RhoA GTPase signaling pathway to directly modulate endothelial cell adhesion, proliferation, and cell shape in order to support proper cellular behavior for vessel development. Moreover, for the first time we have uncovered the molecular and cellular basis for vascular lumen formation in Xenopus and show that EGFL7 is required for the proper cell shape changes and resolution of cell-cell junctions necessary to facilitate the transition of cord-like structures into functional vascular tubes. Collectively, this work provides insight into how transcription factors, ECM proteins, and intracellular signaling pathways are coordinated to achieve timely and appropriate assembly of a hierarchically branched and perfusable vascular network.Doctor of Philosoph

Fluorescent carbon dioxide indicators

Over the last decade, fluorescence has become the dominant tool in biotechnology and medical imaging. These exciting advances have been underpinned by the advances in time-resolved techniques and instrumentation, probe design, chemical / biochemical sensing, coupled with our furthered knowledge in biology. Complementary volumes 9 and 10, Advanced Concepts of Fluorescence Sensing: Small Molecule Sensing and Advanced Concepts of Fluorescence Sensing: Macromolecular Sensing, aim to summarize the current state of the art in fluorescent sensing. For this reason, Drs. Geddes and Lakowicz have invited chapters, encompassing a broad range of fluorescence sensing techniques. Some chapters deal with small molecule sensors, such as for anions, cations, and CO2, while others summarize recent advances in protein-based and macromolecular sensors. The Editors have, however, not included DNA or RNA based sensing in this volume, as this were reviewed in Volume 7 and is to be the subject of a more detailed volume in the near future

A Whole New World - Exploring Emotion in Music

When Disney movies open with a murmur of classi cal music, crescendoing into powerful waves, they immediately transport us to animated lands of princes and princesses, talking animals and evil stepmothers. Or if you haven’t watched a Disney movie in a while, what about the recent allure of the sea shanty? When listening to the now-famil iar rhythm of the folk songs that traditionally accompanied labo rious tasks while at sea, we imagine ourselves on a 19th century ship, helping to raise the sail or hoist up the anchor. How do we create entire worlds for ourselves, whether familiar or from cen turies before, based on the music we hear? Human perception of music is influenced by pitch, key, tempo and other factors, which evoke emotion by activating the limbic and paralimbic systems [1, 2]. That said, the whole story behind music is still being sounded out, and some current theories are explored below.Temple University. College of Liberal ArtsPsychology and Neuroscienc

Glucose Sensing by Time-Resolved Fluorescence of Sol-Gel Immobilized Glucose Oxidase

A monolithic silica gel matrix with entrapped glucose oxidase (GOD) was constructed as a bioactive element in an optical biosensor for glucose determination. Intrinsic fluorescence of free and immobilised GOD was investigated in the visible range in presence of different glucose concentrations by time-resolved spectroscopy with time-correlated single-photon counting detector. A three-exponential model was used for analysing the fluorescence transients. Fractional intensities and mean lifetime were shown to be sensitive to the enzymatic reaction and were used for obtaining calibration curve for glucose concentration determination. The sensing system proposed achieved high resolution (up to 0.17 mM) glucose determination with a detection range from 0.4 mM to 5 mM

Epidemiology of pneumoconiosis in coalminers of Nord-Pas-de-Calais in France

This is a longitudinal study of 3 167 active or retired pneumoconiotic coalminers from Nord - Pas de Calais collieries (HBNPC) recognised through medico-legal decision over three defined periods: 1942-61, 1952-81 and 1982-87

Dendrimer-Based Fluorescent Indicators: In Vitro and In Vivo Applications

BACKGROUND: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. PRINCIPAL FINDINGS: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. CONCLUSION: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy

Cellular Oxygen and Nutrient Sensing in Microgravity Using Time-Resolved Fluorescence Microscopy

Oxygen and nutrient sensing is fundamental to the understanding of cell growth and metabolism. This requires identification of optical probes and suitable detection technology without complex calibration procedures. Under this project Microcosm developed an experimental technique that allows for simultaneous imaging of intra- and inter-cellular events. The technique consists of frequency-domain Fluorescence Lifetime Imaging Microscopy (FLIM), a set of identified oxygen and pH probes, and methods for fabrication of microsensors. Specifications for electronic and optical components of FLIM instrumentation are provided. Hardware and software were developed for data acquisition and analysis. Principles, procedures, and representative images are demonstrated. Suitable lifetime sensitive oxygen, pH, and glucose probes for intra- and extra-cellular measurements of analyte concentrations have been identified and tested. Lifetime sensing and imaging have been performed using PBS buffer, culture media, and yeast cells as a model systems. Spectral specifications, calibration curves, and probes availability are also provided in the report