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

Star Formation from Galaxies to Globules

The empirical laws of star formation suggest that galactic-scale gravity is involved, but they do not identify the actual triggering mechanisms for clusters in the final stages. Many other triggering processes satisfy the empirical laws too, including turbulence compression and expanding shell collapse. The self-similar nature of the gas and associated young stars suggests that turbulence is more directly involved, but the small scale morphology of gas around most embedded clusters does not look like a random turbulent flow. Most clusters look triggered by other nearby stars. Such a prominent local influence makes it difficult to understand the universality of the Kennicutt and Schmidt laws on galactic scales. A unified view of multi-scale star formation avoids most of these problems. Ambient self-gravity produces spiral arms and drives much of the turbulence that leads to self-similar structures, while localized energy input from existing clusters and field supernovae triggers new clusters in pre-existing clouds. The hierarchical structure in the gas made by turbulence ensures that the triggering time scales with size, giving the Schmidt law over a wide range of scales and the size-duration correlation for young star fields. The efficiency of star formation is determined by the fraction of the gas above a critical density of around 10^5 m(H2)/cc. Star formation is saturated to its largest possible value given the fractal nature of the interstellar medium.Comment: accepted for ApJ, 42 pages, Dannie Heineman prize lecture, January 200

Development of a Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM) system using a Streak Camera

We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (0.2 micron) and temporal (50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging

The Metamorphosis of Tidally Stirred Dwarf Galaxies

We present results from high-resolution N-Body/SPH simulations of rotationally supported dwarf irregular galaxies moving on bound orbits in the massive dark matter halo of the Milky Way.The dwarf models span a range in disk surface density and the masses and sizes of their dark halos are consistent with the predictions of cold dark matter cosmogonies. We show that the strong tidal field of the Milky Way determines severe mass loss in their halos and disks and induces bar and bending instabilities that transform low surface brightness dwarfs (LSBs) into dwarf spheroidals (dSphs) and high surface brightness dwarfs (HSBs) into dwarf ellipticals (dEs) in less than 10 Gyr. The final central velocity dispersions of the remnants are in the range 8-30 km/s and their final $v/\sigma$ falls to values $< 0.5$, matching well the kinematics of early-type dwarfs. The transformation requires the orbital time of the dwarf to be \simlt 3-4 Gyr, which implies a halo as massive and extended as predicted by hierarchical models of galaxy formation to explain the origin of even the farthest dSph satellites of the Milky Way, Leo I and Leo II. Only dwarfs with central dark matter densities as high as those of Draco and Ursa Minor can survive for 10 Gyr in the proximity of the Milky Way: this is naturally achieved within hierarchical models, where the densest objects should have small orbital times due to their early formation epochs. Part of the gas is stripped and part is funneled to the center due to the bar, generating one strong burst of star formation in HSBs and smaller, multiple bursts in LSBs. Extended low-surface brightness stellar and gaseous streams originate from LSBs and, when projected along the line of sight, can lead to overestimate the mass-to-light ratio of the bound remnant by a factor \simlt 2,Comment: 29 pages, 34 figures, submitted to ApJ. Figures 5,11 and 32 are given as separate GIF files. Other figures and the movies of the simulations can be found at http://pcblu.mib.infn.it/~lucio/LG/LG.htm

Optical Imaging and Spectroscopy of the Edge-On Sbc Galaxy UGC10043: Evidence for a Galactic Wind and a Peculiar Triaxial Bulge

We present new optical imaging and spectroscopy of the peculiar, edge-on Sbc galaxy UGC10043. B & R imaging reveals that the inner bulge of UGC10043 is elongated perpendicular to the major axis. At larger r, the bulge isophotes twist to become oblate and nearly circular, suggesting the bulge is triaxial. Based on stellar and ionized gas kinematics, the bulge shows no clear evidence for rotation about either its major or minor axis. The southwestern quadrant of the bulge is girdled by a narrow dust lane parallel to the minor axis that may be part of an inner polar ring. The stellar disk of UGC10043 has a low optical surface brightness, a small scale height, a mild integral sign warp, and a dusty, inner region that appears tilted relative to the outlying disk. The HA and [NII] emission lines in UGC10043 resolve into multiple velocity components, indicating the presence of a large-scale galactic wind with an outflow velocity of ~104 km/s. HA+[NII] imaging reaffirms this picture by revealing ionized gas extended to |z|~3.5kpc in a biconical structure. The [NII]/HA line intensity ratio increases with increasing distance from the plane, reaching values as high as 1.7. Unlike most galaxies with large-scale winds, UGC10043 has only a modest global star formation rate (~1M_sun/yr), implying the wind is powered by a rather feeble central starburst. We discuss evolutionary scenarios that could account for both the structural complexities of UGC10043 and its large-scale wind. [Abridged]Comment: accepted to the Astronomical Journal (July 2004); version with full-resolution and color figures available at http://cfa-www.harvard.edu/~lmatthe

Control of star formation by supersonic turbulence

Understanding the formation of stars in galaxies is central to much of modern astrophysics. For several decades it has been thought that stellar birth is primarily controlled by the interplay between gravity and magnetostatic support, modulated by ambipolar diffusion. Recently, however, both observational and numerical work has begun to suggest that support by supersonic turbulence rather than magnetic fields controls star formation. In this review we outline a new theory of star formation relying on the control by turbulence. We demonstrate that although supersonic turbulence can provide global support, it nevertheless produces density enhancements that allow local collapse. Inefficient, isolated star formation is a hallmark of turbulent support, while efficient, clustered star formation occurs in its absence. The consequences of this theory are then explored for both local star formation and galactic scale star formation. (ABSTRACT ABBREVIATED)Comment: Invited review for "Reviews of Modern Physics", 87 pages including 28 figures, in pres

Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-015) in human malignant glioma xenografts

In spite of aggressive surgery, irradiation and/or chemotherapy, treatment of malignant gliomas remains a major challenge in adults and children due to high treatment failure. We have demonstrated significant cell lysis and antitumour activity of the E1B-55 kDa-gene-deleted adenovirus ONYX-015 (dl1520, CI-1042; ONYX Pharmaceuticals) in subcutaneous human malignant glioma xenografts deriving from primary tumours. Here, we show the combined efficacy of this oncolytic therapy with radiation therapy. Total body irradiation (5 Gy) of athymic nude mice prior to intratumoral injections of ONYX-015 1 x 10(8) PFU daily for 5 consecutive days yielded additive tumour growth delays in the p53 mutant xenograft IGRG88. Radiation therapy was potentiated in the p53 functional tumour IGRG121 with a 'subtherapeutic' dose of 1 x 10(7) PFU daily for 5 consecutive days, inducing significant tumour growth delay, 90% tumour regression and 50% tumour-free survivors 4 months after treatment. These potentiating effects were not due to increased adenoviral infectivity or replication. Furthermore, cell lysis and induction of apoptosis, the major mechanisms for adenoviral antitumour activity, did not play a major role in the combined treatment strategy. Interestingly, the oncolytic adenovirus seemed to accelerate radiation-induced tumour fibrosis. Potentiating antitumour activity suggests the development of this combined treatment for these highly malignant tumours

Splinting or surgery for carpal tunnel syndrome? Design of a randomized controlled trial [ISRCTN18853827]

BACKGROUND: Carpal tunnel syndrome is a common disorder, which can be treated with surgery or conservative options. However, there is insufficient evidence and no consensus among physicians with regard to the preferred treatment for carpal tunnel syndrome. Therefore, a randomized controlled trial is conducted to compare the short- and long-term efficacy of surgery and splinting in patients with carpal tunnel syndrome. An attempt is also made to avoid the (methodological) limitations encountered in earlier trials on the efficacy of various treatment options for carpal tunnel syndrome. METHODS: Patients of 18 years and older, with clinically and electrophysiologically confirmed idiopathic carpal tunnel syndrome, are recruited by neurologists in 13 hospitals. Patients included in the study are randomly allocated to either open carpal tunnel release or wrist splinting during the night for at least 6 weeks. The primary outcomes are general improvement, waking up at night and severity of symptoms (main complaint, night and daytime pain, paraesthesia and hypoesthesia). Outcomes are assessed up to 18 months after randomization

Measurement of overall quality of life in nursing homes through self-report: the role of cognitive impairment

Measuring quality of life is a necessity for adequate interventions. This paper concerns the usefulness of six self-report measures for overall quality of life for nursing home residents with various levels of cognitive impairment. It was investigated which proportion of residents from four cognition groups could complete a scale, and internal consistency and construct validity of the scales were studied. Data collection took place in ten Dutch nursing homes (N = 227). The proportion of residents that could complete each scale varied. The Depression List could be administered most often to the cognitively most impaired group (43%; Mini Mental State Examination-scores 0–4). In the three cognition groups with MMSE-score >5, internal consistency of the Depression List, Geriatric Depression Scale and Negative Affect Scale was adequate in all three groups (alpha ≥.68). Intercorrelation was highest for the Philadelphia Geriatric Center Morale Scale, the Depression List, and the Geriatric Depression Scale (rho ≥.65). Nonetheless, self-report scales were not strongly correlated with two observational scales for depression, especially in cognitively severely impaired residents (rho ≤.30). In conclusion, it may not be possible to measure overall quality of life through self-report, and possibly also through observation, in many nursing home residents