Photo-activated raster scanning thermal imaging at sub-diffraction resolution.

Active thermal imaging is a valuable tool for the nondestructive characterization of the morphological properties and the functional state of biological tissues and synthetic materials. However, state-of-the-art techniques do not typically combine the required high spatial resolution over extended fields of view with the quantification of temperature variations. Here, we demonstrate quantitative far-infrared photo-thermal imaging at sub-diffraction resolution over millimeter-sized fields of view. Our approach combines the sample absorption of modulated raster-scanned laser light with the automated localization of the laser-induced temperature variations imaged by a thermal camera. With temperature increments ∼0.5-5 °C, we achieve a six-time gain with respect to our 350-µm diffraction-limited resolution with proof-of-principle experiments on synthetic samples. We finally demonstrate the biological relevance of sub-diffraction thermal imaging by retrieving temperature-based super-resolution maps of the distribution of Prussian blue nanocubes across explanted murine skin biopsies.

Author Correction: µMAPPS: a novel phasor approach to second harmonic analysis for in vitro-in vivo investigation of collagen microstructure.

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µMAPPS: a novel phasor approach to second harmonic analysis for in vitro-in vivo investigation of collagen microstructure.

Second Harmonic Generation (SHG) is a label-free imaging method used to monitor collagen organization in tissues. Due to its sensitivity to the incident polarization, it provides microstructural information otherwise unreachable by other intensity based imaging methods. We develop and test a Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG (µMAPPS) that maps the features of the collagen architecture in tissues at the micrometer scale. µMAPPS retrieves pixel-by-pixel the collagen fibrils anisotropy and orientation by operating directly on two coupled phasor spaces, avoiding direct fitting of the polarization dependent SHG signal. We apply µMAPPS to fixed tissue sections and to the study of the collagen microscopic organization in tumors ex-vivo and in-vivo. We develop a clustering algorithm to automatically group pixels with similar microstructural features. µMAPPS can perform fast analyses of tissues and opens to future applications for in-situ diagnosis of pathologies and diseases that could assist histo-pathological evaluation.

Diffusion-photodynamics coupling in Fluorescence Correlation Spectroscopy studies of photoswitchable Green Fluorescent Proteins: an analytical and simulative study.

The photodynamics of the Green Fluorescent Protein (GFP) has been addressed in detail, particularly by means of Fluorescence Correlation Spectroscopy (FCS), a technique that provides direct information when the diffusion and the photodynamics time scales are well separated. Efficient photoswitchable GFPs, a crucial component for applications in nanoscopy imaging, have long residence times in the dark state, typically longer than the diffusion time of the protein through the observation volume. In these cases, the effect of the coupling between photodynamics and the diffusion process on the analysis of the FCS measurements cannot be disregarded, and the use of FCS methods becomes therefore critical. This work deals with the analytical and simulative study of such coupling and indicates that the corrections to be applied to the conventional decoupled FCS model scale as the square root of the ratio between the diffusion and the dark state relaxation times. We discuss the possibility to estimate the extent of the diffusion/photodynamics coupling from the analysis of the inverse of the fluorescence autocorrelation function g(t), defined as G(-1)(g(t)) = g(0)/g(t) - 1. The function G(-1)(g(t)) is analyzed in terms of a parabolic expansion in which the curvature term directly provides the desired measure of the coupling. We validate the analytical prediction and the graphical estimate of the coupling on simulations of FCS experiments that are based on a coupled Monte Carlo-Brownian Dynamics algorithm. The analysis of the curvature of G(-1)(g(t)), applied to experimental FCS data of the photoswitchable E222Q mutant of GFPMut2 (Mut2Q), indicates that the trapping rate for this chromophore is 3 orders of magnitude underestimated when the diffusion/photodynamics coupling is not taken into account and sheds some additional light on the complex energy diagram for this protein.

Synthesis of branched Au nanoparticles with tunable near-infrared LSPR using a zwitterionic surfactant.

Asymmetric branched gold nanoparticles are obtained using for the first time in the seed-growth approach a zwitterionic surfactant, laurylsulfobetaine, whose concentration in the growth solution allows to control both the length to base-width ratio of the branches and the LSPR position, that can be tuned in the 700-1100 nm near infrared range.

Protonation and conformational dynamics of GFP mutants by two-photon excitation fluorescence correlation spectroscopy.

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.

Voltage regulation of fluorescence emission of single dyes bound to gold nanoparticles.

An organic dye, SAMSA, bound to gold nanoparticles, displays random photoactivated fluorescence blinking whose rate depends on the size of the nanoparticles. We report experiments indicating that (1) the dye emission wavelength is red-shifted (10-30 nm) by applying an external low voltage (1-10 V) and that (2) the fluorescence emission of single dyes can be resonantly driven by tuning the alternating external bias frequency from 1 to 3 Hz, depending on the nanoparticle size. These properties appear highly valuable and promising for devising light emitting nanostructures.

Temporal myofascial flap in reconstructive surgery of the oral cavity.

AIM: The temporal myofascial flap is a simple, rapid and reliable surgical method for immediate reconstruction of facial defects: indications in the light of modern anatomical knowledge and personal experience, with the accent on achieving an appropriate access route without damaging the facial nerve. METHODS: Our series covers the period from January 1999 to December 2004, during which time myofascial flaps of temporal muscle were used for immediate reconstruction in 20 surgical oncological cases involving the face and neck. RESULTS: Postoperative progress was regular; no lesions of the facial nerve were observed, nor any cases of flap necrosis, including partial. Epithelialisation could already be observed as early as 15 days postsurgery without skin grafting being employed. CONCLUSIONS: Application of a Medpor prosthesis eliminates the only negative outcome from the aesthetic standpoint, related to harvesting the muscle from the fossa.

Selective fluorescence recovery after bleaching of single E2GFP proteins induced by two-photon excitation.

We report the two-photon excitation and emission or a recently developed green fluorescent protein (GFP) mutant, E(2)GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 400 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 +/- 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.

Treatment of lower lip cancer: an experience of 48 cases.

The article reports results obtained in 48 cases of lower lip cancer. Tumor classified as T1 or T2, requiring a resection up to 60% of the lower lip, were treated with the stair-case technique. Nine patients were treated with the bilateral symmetrical stair-case technique since their lesions were located medially, while 23 were treated with the bilateral method using two asymmetrical flaps because their lesions were in paramedian position but larger than 2 cm. Ten patients required a unilateral flap. The cases classified as T3, in which the lesion required resection of more than 60% of the lip, were treated with the Bernard-Freeman-Fries technique.

Rotational dynamics of curved DNA fragments studied by fluorescence polarization anisotropy.

The rotational dynamics of short DNA fragments with or without intrinsic curvature were studied using time-resolved phase fluorimetry of intercalated ethidium with detection of the anisotropy. Parameters determined were the spinning diffusion coefficient of the DNA fragments about the long axis and the zero-time ethidium fluorescence anisotropy. We find a significant decrease in the spinning diffusion coefficient for all curved fragments compared to the straight controls. This decrease is likewise evident in rotational diffusion coefficients computed from DNA structures obtained by a curvature prediction program for these sequences. Using a hinged-cylinder model, we can identify the change in rotational diffusion coefficient with a permanent bend of 13-16 degrees per helix turn for the sequences studied. Moreover, for some of the curved fragments an increased flexibility has to be assumed in addition to the permanent bend in order to explain the data.

New insight on beta-lactoglobulin binding sites by 1-anilinonaphthalene-8-sulfonate fluorescence decay.

The fluorescence time decay parameters of the beta-lactoglobulin-1-anilinonaphthalene-8-sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a "surface" site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye-Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the "surface" site mainly responds to the presence of these local titrating charges while the "internal" site more closely follows the overall protein net charge.

[Synovial chondromatosis of the temporomandibular joint]. / Condromatosi sinoviale dell'articolazione temporo-mandibolare.

Synovial chondromatosis is an uncommon monoarticular proliferative disease of the synovium characterized by loose bodies developed in the synovial membrane. The literature is reviewed and two cases of synovial chondromatosis of the temporomandibular joint characterized by swelling, pain and limitation of mandibular movement are reported. Radiographic evidence of loose bodies may or may not be present. Computed tomography (CT) or magnetic resonance (MR) might be helpful in the diagnosis of synovial chondromatosis. The treatment of choice of synovial chondromatosis is surgical and the loose bodies should be removed by arthrotomy with examination of the joint cavity. In our patients, no recurrence had occurred according to other authors' experience.

Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to beta-lactoglobulin from fluorescence spectroscopy.

Steady-state and dynamic fluorescence titrations show that: (a) the complex between beta-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH approximately 2) to the native state (pH approximately 6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are a major contribution to ANS binding to BLG.

Metabolites of the angiotensin II antagonist tasosartan: the importance of a second acidic group.

Described in this paper is the synthesis and pharmacological activity of five metabolites of the angiotensin II antagonist tasosartan (1). Of particular interest is the effect of the additional acidic group of the enol metabolite (8) on activity. As suggested by the structural-activity relationship of other angiotensin II antagonist series, a second acidic group can improve receptor binding activity but decrease in vivo activity after oral dosing. The metabolic introduction of a second acidic group in tasosartan bypasses this problem and contributes to the excellent profile of the compound. A molecular modeling study provides a rationale for the role of the enol group of 8 in AT1 receptor binding.

Radical retropubic prostatectomy: limited benefit of autologous blood donation.

PURPOSE: We determine whether autologous blood donation significantly decreases the need for homologous transfusions after radical prostatectomy. MATERIALS AND METHODS: The effects of estimated blood loss and autologous donation on the rate of homologous transfusions were analyzed in 3 groups of 100 consecutive patients treated between 1983 and 1992. RESULTS: Overall, donors were less likely than nondonors to receive homologous blood. As median estimated blood loss decreased from 1,200 to 800 cc from groups 1 to 3 (p < 0.05), the incidence of nondonors requiring homologous blood decreased from 62 to 11% and that of autologous units transfused decreased from 96 to 19%. CONCLUSIONS: With decreasing blood loss, safe but stringent criteria for transfusion and improved safety of the blood supply, autologous donation is an inefficient method to lower the slight risk of complications following homologous transfusion during radical prostatectomy.

Conformation of short DNA fragments by modulated fluorescence polarization anisotropy.

The technique of fluorescence polarization anisotropy (FPA) decay of intercalated ethidium has been used to study DNA conformation and dynamics, which are being recognized as primary determinants in transcription control and other cellular processes. Frequency modulated FPA when applied to two DNA molecules, a "straight" 50 base-pairs duplex fragment, and a bent fragment of similar length, has yielded different rotational diffusion coefficients for the two fragments. The data have been processed with an analytical model and with Brownian dynamics simulations, obtaining a good fit and a quantitative agreement between the two models. Both analyses have confirmed that one fragment can be described as a straight cylinder, while the other fragment is bent, with an angle estimated to be 45 degrees +/- 3 degrees. FPA has proved to be very powerful in determining simple conformations of short DNA duplexes and also particularly apt to probe the dynamical features of DNA fragments where conventional methods are either too cumbersome or fail to give quantitative results. In addition, the ligand no longer behaves ideally due to its complex structure and charge distribution. Thus for the protein the slope is no longer related simply to the net ligand charge, and the PB model gives a much larger slope than the LL model.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of ligands on the fluorescence polarisation anisotropy of ethidium bound to DNA.

The decay of the fluorescence polarisation anisotropy (FPA) of the ethidium-DNA complex has been measured by multifrequency phase fluorometry, in order to study the perturbations induced by the presence of different ligands on the torsional dynamics of DNA, a moderately flexible polymer that undergoes bending (flexure of the helix axis) and torsional (twisting of base pairs) motions. Two probes have been used together with ethidium: an intercalator, chloroquine, and a minor groove binding dye: hoechst 33258. Chloroquine is found to substantially modify the DNA torsional dynamics both in linear and in circularly closed DNAs only at high binding ratios, in agreement with previous reports [Wu et al. Biochem. 27 (1988) 8128]. The effective elastic constant becomes approximately three times larger when the dye/base pairs binding ratio is higher than 0.14. The minor groove ligand hoechst 33258, on the other hand, greatly increases the effective elastic constant to the point that at dye/base pairs ratios larger than 0.5, the effective elastic constant becomes stiffer by several orders of magnitude, suggesting a progressive hindering of internal motions. The results reported here show that DNA torsions are more effectively influenced by groove-binding molecules than by intercalators and it is expected that the large perturbation of the former ligand may be useful when describing the change in the dynamical properties induced by DNA binding proteins. FPA in the frequency domain, the technique adopted throughout this work, has proved to be very sensitive to changes of the elastic constant that describes DNA torsional dynamics. Several computer simulations performed in order to predict the FPA time decay of intercalated ethidium have led to good agreement with the observed results.

Pyrido[2,3-d]pyrimidine angiotensin II antagonists.

A series of pyrido[2,3-d]pyrimidine angiotensin II (A II) antagonists was synthesized and tested for antagonism of A II. Compounds with a biphenylyltetrazole pharmacophore and small alkyl groups at the 2- and 4-positions of the pyridopyrimidine ring were found to be the most potent in an AT1 receptor binding assay and in blocking the A II pressor response in anesthetized, ganglion-blocked A II-infused rats. 5,8-Dihydro-2,4-dimethyl-8-[(2'-(1H-tetrazol-5-yl) [1,1'-biphenyl]-4-yl)methyl]pyrido[2,3-d]pyrimidin-7(6H)-one (4a) was one of the more potent compounds in the binding assay and was the most efficacious compound in the A II-infused rat model. Further study of 4a in Goldblatt (2K-1C) rats showed the compound to have oral bioavailability and to be an efficacious and potent compound in a high renin form of hypertension.