MUCIDS: an operative C environment for acquisition and processing of polarized-light scattered from biological specimens

In this work, we describe a software package, MUCIDS, completely developed in our laboratory, for acquisition and processing of differential polarizxition light-scattering data from specimens of biophysical interest. MUCIDS is a C environment that manages the whole activity of an instrument used for measurements of Mueller matrix scattering elements. It allows one to capture, analyse, process and display data from this or from other similar light-scattering experiments. The entire system is suitable for routine measurements in a general biophysical (or microbiological) laboratory because of its easy handling and maintenance. The software was written in C lattice and will run on IBM personal computers and similar. It uses IBM/DAC and GPIB/IBM interface card

AFM-STED correlative nanoscopy reveals a dark side in fluorescence microscopy imaging

It is known that the presence of fluorophores can influence the dynamics of molecular processes. Despite this, an affordable technique to control the fluorophore distribution within the sample, as well as the rise of unpredictable anomalous processes induced by the fluorophore itself, is missing. We coupled a stimulated emission depletion (STED) microscope with an atomic force microscope to investigate the formation of amyloid aggregates. In particular, we studied the in vitro aggregation of insulin and two alloforms of b amyloid peptides. We followed standard methods to induce the aggregation and to label the molecules at different dye-to-protein ratios. Only a fraction of the fibrillar aggregates was displayed in STED images, indicating that the labeled molecules did not participate indistinctly to the aggregation process. This finding demonstrates that labeled molecules follow only selected pathways of aggregation, among the multiple that are present in the aggregation reaction

Two-photon excitation selective plane illumination microscopy (2PE-SPIM) of highly scattering samples: Characterization and application

In this work we report the advantages provided by two photon excitation (2PE) implemented in a selective plane illumination microscopy (SPIM) when imaging thick scattering samples. In particular, a detailed analysis of the effects induced on the real light sheet excitation intensity distribution is performed. The comparison between single-photon and twophoton excitation profiles shows the reduction of the scattering effects and sample-induced aberrations provided by 2PE-SPIM. Furthermore, uniformity of the excitation distribution and the consequent improved image contrast is shown when imaging scattering phantom samples in depth by 2PE-SPIM. These results show the advantages of 2PE-SPIM and suggest how this combination can further enhance the SPIM performance. Phantom samples have been designed with optical properties compatible with biological applications of interest. © 2013 Optical Society of America

From deceased to bioengineered graft: New frontiers in liver transplantation

none6siopenCesaretti M.; Zarzavajian Le Bian A.; Moccia S.; Iannelli A.; Schiavo L.; Diaspro A.Cesaretti, M.; Zarzavajian Le Bian, A.; Moccia, S.; Iannelli, A.; Schiavo, L.; Diaspro, A

Targeted photoimmunotherapy for cancer

Photodynamic therapy (PDT) is a clinically approved procedure that can exert a curative action against malignant cells. The treatment implies the administration of a photoactive molecular species that, upon absorption of visible or near infrared light, sensitizes the formation of reactive oxygen species. These species are cytotoxic and lead to tumor cell death, damage vasculature, and induce inflammation. Clinical investigations demonstrated that PDT is curative and does not compromise other treatment options. One of the major limitations of the original method was the low selectivity of the photoactive compounds for malignant over healthy tissues. The development of conjugates with antibodies has endowed photosensitizing molecules with targeting capability, so that the compounds are delivered with unprecedented precision to the site of action. Given their fluorescence emission capability, these supramolecular species are intrinsically theranostic agents

Fourier ring correlation simplifies image restoration in fluorescence microscopy

Fourier ring correlation (FRC) has recently gained popularity among fluorescence microscopists as a straightforward and objective method to measure the effective image resolution. While the knowledge of the numeric resolution value is helpful in e.g., interpreting imaging results, much more practical use can be made of FRC analysis\u2014in this article we propose blind image restoration methods enabled by it. We apply FRC to perform image de-noising by frequency domain filtering. We propose novel blind linear and non-linear image deconvolution methods that use FRC to estimate the effective point-spread-function, directly from the images. We show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. We also address two important limitations in FRC that may be of more general interest: how to make FRC work with single images (within certain practical limits) and with three-dimensional images with highly anisotropic resolution

3D HDO-CLEM: cellular compartment analysis by correlative light-electron microscopy on cryosections.

Fundamental to obtaining a depth-understanding of the function and structure of cells is the ability to study and correlate their molecular topography with the ultrastructural morphology, for example, to visualize the position of a given protein relative to a given cell compartment and its morphology. Standard fluorescence light microscopy (FLM) relies on simple sample preparations, and localizes proteins in living or fixed cells with a resolution in the range of few hundred nanometers, allowing large field of view. However, FLM is unable to visualize the unlabeled cellular context. On the other hand, electron microscopy (EM) techniques reveal protein topology with the resolution in a range of a few tens of nanometer, retains the cellular context, but can only be applied on a limited field of view. Therefore, both approaches present shortcomings, in terms of field of view, statistical output, resolution, sample preparation, and context analysis, that can likely complement each other. To bridge the gap between FLM imaging and EM, several laboratories have developed methods for correlative light-electron microscopy (CLEM). In a nutshell, CLEM enables one to investigate the same exact region of interest utilizing the two microscope platforms, and thereby virtually combine their capabilities. We describe a protocol based on immunolabeling of Tokuyasu cryosections that allows correlation of LM and EM images with excellent preservation of cellular ultrastructure. We will refer to this method as high-data-output CLEM (HDO-CLEM). The major benefits of HDO-CLEM are the possibility to (1) correlate several hundreds of events at the same time, (2) perform three-dimensional (3D) correlation, (3) immunolabel both endogenous and recombinantly tagged proteins at the same time, and (4) combine the high data analysis capability of FLM with the high precision of transmission EM in a CLEM hybrid morphometric analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D FLM reconstruction and set the basis for a hybrid light/EM morphometry approach

Excimer Laser-produced Biodegradable Photopolymer Scaffolds Do Not Induce Immune Rejection In Vivo

Following our previous works of in-vitro tests, the biocompatibility of photopolymer scaffolds was tested against immune responses in vivo. Neither relevant immune reactions nor the rejection of implanted scaffolds was detected, being an essential step for in vivo implantation of excimer laser-prepared scaffolds. The scaffolds were fabricated by UV excimer laser photocuring at 308 nm. After two weeks of transplantation neither inflammatory response nor reactive immune activation was detected based on the chemokine and cytokine profile. As a sign of biodegradability of the scaf-folds, we detected macrophage infiltration and phagocytosis of the biopolymer at the site of implan-tation. Our results suggest that poly(propylene fumarate) (PPF): diethyl fumarate (DEF) (7 : 3 w/w) scaffolds have appropriate properties for in vivo applications Excimer laser-produced biodegradable photopolymer scaffolds do not induce immune rejection in vivo - ResearchGate. Available from: http://www.researchgate.net/publication/268523447_Excimer_laser-produced_biodegradable_photopolymer_scaffolds_do_not_induce_immune_rejection_in_vivo [accessed Aug 17, 2015]