dissecting-mechanoenzymatic-properties-processive-myosins-with

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

National Institute of Optics, National Research Council

LENS, European Laboratory for Non-Linear Spectroscopy

Physics Department, University of Florence

The distinct organization of the brain's vascular network ensures that it is adequately supplied with oxygen and nutrients. However, despite this fundamental role, a detailed reconstruction of the brain-wide vasculature at the capillary level remains elusive, due to insufficient image quality using the best available techniques. Here, we demonstrate a novel approach that improves vascular demarcation by combining CLARITY with a vascular staining approach that can fill the entire blood vessel lumen and imaging with light-sheet fluorescence microscopy. This method significantly improves image contrast, particularly in depth, thereby allowing reliable application of automatic segmentation algorithms, which play an increasingly important role in high-throughput imaging of the terabyte-sized datasets now routinely produced. Furthermore, our novel method is compatible with endogenous fluorescence, thus allowing simultaneous investigations of vasculature and genetically targeted neurons. We believe our new method will be valuable for future brain-wide investigations of the capillary network.

Whole-Brain Vasculature Reconstruction at the Single Capillary Level.

Optogenetics is an emerging method that uses light to manipulate electrical activity in excitable cells exploiting the interaction between light and light-sensitive depolarizing ion channels, such as channelrhodopsin-2 (ChR2). Initially used in the neuroscience, it has been adopted in cardiac research where the expression of ChR2 in cardiac preparations allows optical pacing, resynchronization and defibrillation. Recently, optogenetics has been leveraged to manipulate cardiac electrical activity in the intact heart in real-time. This new approach was applied to simulate a re-entrant circuit across the ventricle. In this technical note, we describe the development and the implementation of a new software package for real-time optogenetic intervention. The package consists of a single LabVIEW program that simultaneously captures images at very high frame rates and delivers precisely timed optogenetic stimuli based on the content of the images. The software implementation guarantees closed-loop optical manipulation at high temporal resolution by processing the raw data in workstation memory. We demonstrate that this strategy allows the simulation of a ventricular tachycardia with high stability and with a negligible loss of data with a temporal resolution of up to 1 ms.

A Software Architecture to Mimic a Ventricular Tachycardia in Intact Murine Hearts by Means of an All-Optical Platform.

Recent improvements in optical tools that can perturb brain activity and simultaneously reveal the elicited alterations in the associated regions offer an exceptional means to understand and map the connectivity of the brain. In this work, we exploit a combination of recently developed optical tools to monitor neural population at the meso-scale level and to mould the cortical patterns of targeted neuronal population. Our goal was to investigate the propagation of neuronal activity over the mouse cortex that is triggered by optogenetic stimulation in the contralateral hemisphere. Towards this aim, we developed a wide-field fluorescence microscope that is characterized by a double illumination path allowing for the optogenetic stimulation of the transfected area in the left hemisphere and the simultaneous recording of cortical activity in the right hemisphere. The microscope was further implemented with a custom shutter in order to split the LED illumination path, resulting in a half-obscured field of view. By avoiding the spectral crosstalk between GCaMP6f and channelrhodopsin 2 (ChR2), this system offered the possibility of simultaneous "pumping and probing" of inter-hemispheric functional connectivity on Thy1-GCaMP6f mice.

Large Scale Double-Path Illumination System with Split Field of View for the All-Optical Study of Inter-and Intra-Hemispheric Functional Connectivity on Mice.

Single Photon Avalanche Diode (SPAD) arrays are increasingly exploited and have demonstrated potential in biochemical and biomedical research, both for imaging and single-point spectroscopy applications. In this study, we explore the application of SPADs together with fiber-optic-based delivery and collection geometry to realize fast and simultaneous single-point time-, spectral-, and depth-resolved fluorescence measurements at 375 nm excitation light. Spectral information is encoded across the columns of the array through grating-based dispersion, while depth information is encoded across the rows thanks to a linear arrangement of probe collecting fibers. The initial characterization and validation were realized against layered fluorescent agarose-based phantoms. To verify the practicality and feasibility of this approach in biological specimens, we measured the fluorescence signature of formalin-fixed rabbit aorta samples derived from an animal model of atherosclerosis. The initial results demonstrate that this detection configuration can report fluorescence spectral and lifetime contrast originating at different depths within the specimens. We believe that our optical scheme, based on SPAD array detectors and fiber-optic probes, constitute a powerful and versatile approach for the deployment of multidimensional fluorescence spectroscopy in clinical applications where information from deeper tissue layers is important for diagnosis.

Multispectral Depth-Resolved Fluorescence Lifetime Spectroscopy Using SPAD Array Detectors and Fiber Probes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Article	Year
Whole-Brain Vasculature Reconstruction at the Single Capillary Level. Scientific reports\| PubMed ID: 30135559	2018
A Software Architecture to Mimic a Ventricular Tachycardia in Intact Murine Hearts by Means of an All-Optical Platform. Methods and protocols\| PubMed ID: 31164591	2019
Large Scale Double-Path Illumination System with Split Field of View for the All-Optical Study of Inter-and Intra-Hemispheric Functional Connectivity on Mice. Methods and protocols\| PubMed ID: 31164593	2019
Multispectral Depth-Resolved Fluorescence Lifetime Spectroscopy Using SPAD Array Detectors and Fiber Probes. Sensors (Basel, Switzerland)\| PubMed ID: 31200569	2019
Publisher Correction: Whole-Brain Vasculature Reconstruction at the Single Capillary Level. Scientific reports\| PubMed ID: 31201354	2019

F. S. Pavone

Biography

JoVE Journal Publications

Other Publications