Spontaneous Functional Recovery after Focal Damage in Neuronal Cultures.

Damage in biological neuronal networks triggers a complex functional reorganization whose mechanisms are still poorly understood. To delineate this reorganization process, here we investigate the functional alterations of in vitro rat cortical circuits following localized laser ablation. The analysis of the functional network configuration before and after ablation allowed us to quantify the extent of functional alterations and the characteristic spatial and temporal scales along recovery. We observed that damage precipitated a fast rerouting of information flow that restored network's communicability in about 15 min. Functional restoration was led by the immediate neighbors around trauma but was orchestrated by the entire network. Our in vitro setup exposes the ability of neuronal circuits to articulate fast responses to acute damage, and may serve as a proxy to devise recovery strategies in actual brain circuits. Moreover, this biological setup can become a benchmark to empirically test network theories about the spontaneous recovery in dynamical networks.

Enhanced Light Sheet Elastic Scattering Microscopy by Using a Supercontinuum Laser.

Light sheet fluorescence microscopy techniques have revolutionized biological microscopy enabling low-phototoxic long-term 3D imaging of living samples. Although there exist many light sheet microscopy (LSM) implementations relying on fluorescence, just a few works have paid attention to the laser elastic scattering source of contrast available in every light sheet microscope. Interestingly, elastic scattering can potentially disclose valuable information from the structure and composition of the sample at different spatial scales. However, when coherent scattered light is detected with a camera sensor, a speckled intensity is generated on top of the native imaged features, compromising their visibility. In this work, we propose a novel light sheet based optical setup which implements three strategies for dealing with speckles of elastic scattering images: (i) polarization filtering; (ii) reducing the temporal coherence of the excitation laser light; and, (iii) reducing the spatial coherence of the light sheet. Finally, we show how these strategies enable pristine light-sheet elastic-scattering imaging of structural features in challenging biological samples avoiding the deleterious effects of speckle, and without relying on, but complementing, fluorescent labelling.

Multiphoton imaging with blue-diode-pumped SESAM-modelocked Ti:sapphire oscillator generating 5 nJ 82 fs pulses.

We report a directly blue diode pumped Ti:Sapphire oscillator that generates 5 nJ pulses. This is five times higher pulse energy than previously reported for a directly diode pumped Ti:sapphire laser. With 460 mW of average power at 92 MHz and 82 fs pulses, its peak power reaches 61 kW, also several times higher the value than previously published. Direct diode pumping significantly reduces the complexity and therefore the footprint and the cost of the laser, while SESAM modelocking ensures reliable selfstarting and robust operation. Such a laser is ideally suited for biomedical imaging and nanostructuring applications. As a demonstration of sufficient peak power for microscopy applications, we perform different modalities of nonlinear microscopy of biological samples.

Imaging tissue-mimic with light sheet microscopy: A comparative guideline.

Tissue mimics (TMs) on the scale of several hundred microns provide a beneficial cell culture configuration for in vitro engineered tissue and are currently under the spotlight in tissue engineering and regenerative medicine. Due to the cell density and size, TMs are fairly inaccessible to optical observation and imaging within these samples remains challenging. Light Sheet Fluorescence Microscopy (LSFM)- an emerging and attractive technique for 3D optical sectioning of large samples- appears to be a particularly well-suited approach to deal with them. In this work, we compared the effectiveness of different light sheet illumination modalities reported in the literature to improve resolution and/or light exposure for complex 3D samples. In order to provide an acute and fair comparative assessment, we also developed a systematic, computerized benchmarking method. The outcomes of our experiment provide meaningful information for valid comparisons and arises the main differences between the modalities when imaging different types of TMs.

Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters.

We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C-H (2800-3100cm(-1)) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman·3D-imaging on biological samples.

A transcriptome-proteome integrated network identifies endoplasmic reticulum thiol oxidoreductase (ERp57) as a hub that mediates bone metastasis.

Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared with parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein-protein interaction network and differential expression. The protein-protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub that retained four down-regulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E, and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.

Femtosecond laser axotomy in Caenorhabditis elegans and collateral damage assessment using a combination of linear and nonlinear imaging techniques.

In this work highly localized femtosecond laser ablation is used to dissect single axons within a living Caenorhabditis elegans (C. elegans). We present a multimodal imaging methodology for the assessment of the collateral damage induced by the laser. This relies on the observation of the tissues surrounding the targeted region using a combination of different high resolution microscopy modalities. We present the use of Second Harmonic Generation (SHG) and Polarization Sensitive SHG (PSHG) to determine damage in the neighbor muscle cells. All the above is done using a single instrument: multimodal microscopy setup that allows simultaneous imaging in the linear and non-linear regimes and femtosecond-laser ablation.

Image formation by linear and nonlinear digital scanned light-sheet fluorescence microscopy with Gaussian and Bessel beam profiles.

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivoCaenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

Measurement and correction of in vivo sample aberrations employing a nonlinear guide-star in two-photon excited fluorescence microscopy.

We demonstrate that sample induced aberrations can be measured in a nonlinear microscope. This uses the fact that two-photon excited fluorescence naturally produces a localized point source inside the sample: the nonlinear guide-star (NL-GS). The wavefront emitted from the NL-GS can then be recorded using a Shack-Hartmann sensor. Compensation of the recorded sample aberrations is performed by the deformable mirror in a single-step. This technique is applied to fixed and in vivo biological samples, showing, in some cases, more than one order of magnitude improvement in the total collected signal intensity.