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)

Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion

The tethered particle motion (TPM) allows the direct detection of activity of a variety of biomolecules at the single molecule level. First pioneered for RNA polymerase, it has recently been applied also to other enzymes. In this work we employ TPM for a systematic investigation of the kinetics of DNA looping by wild-type Lac repressor (wt-LacI) and by hinge mutants Q60G and Q60 + 1. We implement a novel method for TPM data analysis to reliably measure the kinetics of loop formation and disruption and to quantify the effects of the protein hinge flexibility and of DNA loop strain on such kinetics. We demonstrate that the flexibility of the protein hinge has a profound effect on the lifetime of the looped state. Our measurements also show that the DNA bending energy plays a minor role on loop disruption kinetics, while a strong effect is seen on the kinetics of loop formation. These observations substantiate the growing number of theoretical studies aimed at characterizing the effects of DNA flexibility, tension and torsion on the kinetics of protein binding and dissociation, strengthening the idea that these mechanical factors in vivo may play an important role in the modulation of gene expression regulation

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

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca(2+) increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site

Volume/thrust optimal shape criteria for arches under static vertical loads

Arches are widely used when large spans are necessary, e.g. to overpass large rivers, and further possess unquestioned aesthetics advantages. Their structural efficiency depends primarily on optimal material exploitation, i.e. minimization of internal stress eccentricity, and on minimization of structural material volume. An efficient structure, under these terms, further requires simpler and lighter scaffolding, contributing in minimizing construction costs.Although arches have millenary use and many researches dealing with this typology are available in literature, there is still scope for design optimization. The proposed study is framed within this context. Investigation is limited to statically determinate plane arches under vertical load. The problem of finding the profile of an equal strength catenary subjected to its self-weight is spread out to the case of an inverted catenary of equal strength under its self-weight and an external constant load. In the first optimization step, constant normal stress is imposed at all sections, to maximize material exploitation, and the resulting arch centerline shape is computed in closed form. In the second step, the ensemble of foundations and arch is considered and optimized, taking the linear combination of arch weight and thrust as objective function. The linear combination is dependent on a single variable, and minima of the objective function (i.e. optimal geometric shape parameters) are computed and charted to be simply used in the design process. Keywords: Plane arch, Vertical loads, Optimal shape, Volume/thrust objective functions, Analytical solutio

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

Whole-brain functional imaging to highlight differences between the diurnal and nocturnal neuronal activity in zebrafish larvae

Most living organisms show highly conserved physiological changes following a 24-hour cycle which goes by the name of circadian rhythm. Among experimental models, the effects of light-dark cycle have been recently investigated in the larval zebrafish. Owing to its small size and transparency, this vertebrate enables optical access to the entire brain. Indeed, the combination of this organism with light-sheet imaging grants high spatio-temporal resolution volumetric recording of neuronal activity. This imaging technique, in its multiphoton variant, allows functional investigations without unwanted visual stimulation. Here, we employed a custom two-photon light-sheet microscope to study whole-brain differences in neuronal activity between diurnal and nocturnal periods in larval zebrafish. We describe for the first time an activity increase in the low frequency domain of the pretectum and a frequency-localised activity decrease of the anterior rhombencephalic turning region during the nocturnal period. Moreover, our data confirm a nocturnal reduction in habenular activity. Furthermore, whole-brain detrended fluctuation analysis revealed a nocturnal decrease in the self-affinity of the neuronal signals in parts of the dorsal thalamus and the medulla oblongata. Our data show that whole-brain nonlinear light-sheet imaging represents a useful tool to investigate circadian rhythm effects on neuronal activity.Comment: 18 pages, 6 figure