Protein conformation and molecular order probed by second-harmonic-generation microscopy

Second-harmonic-generation (SHG) microscopy has emerged as a powerful tool to image unstained living tissues and probe their molecular and supramolecular organization. In this article, we review the physical basis of SHG, highlighting how coherent summation of second-harmonic response leads to the sensitivity of polarized SHG to the three-dimensional distribution of emitters within the focal volume. Based on the physical description of the process, we examine experimental applications for probing the molecular organization within a tissue and its alterations in response to different biomedically relevant conditions. We also describe the approach for obtaining information on molecular conformation based on SHG polarization anisotropy measurements and its application to the study of myosin conformation in different physiological states of muscle. The capability of coupling the advantages of nonlinear microscopy (micrometer-scale resolution in deep tissue) with tools for probing molecular structure in vivo renders SHG microscopy an extremely powerful tool for the advancement of biomedical optics, with particular regard to novel technologies for molecular diagnostic in vivo. (C) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE)

Observation of an improved healing process in superficial skin wounds after irradiation with a blue-LED haemostatic device

The healing process of superficial skin wounds treated with a blue-LED haemostatic device is studied. Four mechanical abrasions are produced on the back of 10 Sprague Dawley rats: two are treated with the blue-LED device, while the other two are left to naturally recover. Visual observations, non-linear microscopic imaging, as well as histology and immunofluorescence analyses are performed 8 days after the treatment, demonstrating no adverse reactions neither thermal damages in both abraded areas and surrounding tissue. A faster healing process and a better-recovered skin morphology are observed: the treated wounds show a reduced inflammatory response and a higher collagen content. Blue LED induced photothermal effect on superficial abrasions

Second-harmonic generation sensitivity to transmembrane potential in normal and tumor cells.

Second-harmonic generation (SHG) is emerging as a powerful tool for the optical measurement of transmembrane potential in live cells with high sensitivity and temporal resolution. Using a patch clamp, we characterize the sensitivity of the SHG signal to transmembrane potential for the RH 237 dye in various normal and tumor cell types. SHG sensitivity shows a significant dependence on the type of cell, ranging from 10 to 17% per 100 mV. Furthermore, in the samples studied, tumor cell lines display a higher sensitivity compared to normal cells. In particular, the SHG sensitivity increases in the cell line Balb/c3T3 by the transformation induced with SV40 infection of the cells. We also demonstrate that fluorescent labeling of the membrane with RH 237 at the concentration used for SHG measurements does not induce any measurable alteration in the electrophysiological properties of the cells investigated. Therefore, SHG is suitable for the investigation of outstanding questions in electrophysiology and neurobiology

The Human Brain Project

The Human Brain Project aims at the simulation of the human brain and at the description of its physiology and computational capabilities. Consequently, a multidisciplinary approach will be developed, in which different disciplines such as physics or medicine will aim at the same objective.Lo Human Brain Project ambisce alla simulazione del cervello umano, e quindi alla comprensione dei suoi meccanismi che ne regolano il funzionamento. Per questo la ricerca tenderà a mettere insieme aspetti disciplinari molto diversi tra di loro, dalla fisica alla medicina, accomunati dal raggiungimento dello stesso obiettivo

Multimodal nonlinear microscopy: A powerful label-free method for supporting standard diagnostics on biological tissues

The large use of nonlinear laser scanning microscopy in the past decade paved the way for potential clinical application of this imaging technique. Modern nonlinear microscopy techniques offer promising label-free solutions to improve diagnostic performances on tissues. In particular, the combination of multiple nonlinear imaging techniques in the same microscope allows integrating morphological with functional information in a morpho-functional scheme. Such approach provides a high-resolution label-free alternative to both histological and immunohistochemical examination of tissues and is becoming increasingly popular among the clinical community. Nevertheless, several technical improvements, including automatic scanning and image analysis, are required before the technique represents a standard diagnostic method. In this review paper, we highlight the capabilities of multimodal nonlinear microscopy for tissue imaging, by providing various examples on colon, arterial and skin tissues. The comparison between images acquired using multimodal nonlinear microscopy and histology shows a good agreement between the two methods. The results demonstrate that multimodal nonlinear microscopy is a powerful label-free alternative to standard histopathological methods and has the potential to find a stable place in the clinical setting in the near future

Bessel beam illumination reduces random and systematic errors in quantitative functional studies using light-sheet microscopy

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a choice method to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20 fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams

Multi-Photon Nanosurgery in Live Brain

In the last few years two-photon microscopy has been used to perform in vivo high spatial resolution imaging of neurons, glial cells and vascular structures in the intact neocortex. Recently, in parallel to its applications in imaging, multi-photon absorption has been used as a tool for the selective disruption of neural processes and blood vessels in living animals. In this review we present some basic features of multi-photon nanosurgery and we illustrate the advantages offered by this novel methodology in neuroscience research. We show how the spatial localization of multi-photon excitation can be exploited to perform selective lesions on cortical neurons in living mice expressing fluorescent proteins. This methodology is applied to disrupt a single neuron without causing any visible collateral damage to the surrounding structures. The spatial precision of this method allows to dissect single processes as well as individual dendritic spines, preserving the structural integrity of the main neuronal arbor. The same approach can be used to breach the blood-brain barrier through a targeted photo-disruption of blood vessels walls. We show how the vascular system can be perturbed through laser ablation leading toward two different models of stroke: intravascular clot and extravasation. Following the temporal evolution of the injured system (either a neuron or a blood vessel) through time lapse in vivo imaging, the physiological response of the target structure and the rearrangement of the surrounding area can be characterized. Multi-photon nanosurgery in live brain represents a useful tool to produce different models of neurodegenerative disease

Towards a comprehensive understanding of brain machinery by correlative microscopy.

Unraveling the complexity of brain structure and function is the biggest challenge of contemporary science. Due to their flexibility, optical techniques are the key to exploring this intricate network. However, a single imaging technique can reveal only a small part of this machinery due to its inherent multilevel organization. To obtain a more comprehensive view of brain functionality, complementary approaches have been combined. For instance, brain activity was monitored simultaneously on different spatiotemporal scales with functional magnetic resonance imaging and calcium imaging. On the other hand, dynamic information on the structural plasticity of neuronal networks has been contextualized in a wider framework combining two-photon and light-sheet microscopy. Finally, synaptic features have been revealed on previously in vivo imaged samples by correlative light-electron microscopy. Although these approaches have revealed important features of brain machinery, they provided small bridges between specific spatiotemporal scales, lacking an omni-comprehensive view. In this perspective, we briefly review the state of the art of correlative techniques and propose a wider methodological framework fusing multiple levels of brain investigation