Search results for " Fluorescence microscopy"
showing 10 items of 35 documents
Shedding Light on the Formation and Structure of Kombucha Biofilm Using Two-Photon Fluorescence Microscopy
2021
Kombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in thede novoformation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected t…
Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures
2019
The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (alpha-La). alpha-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize alpha-La-membrane mechanisms of interaction and alpha-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imag…
Flexible multi-beam light-sheet fluorescence microscope for live imaging without striping artifacts
2018
The development of light-sheet fluorescence microscopy (LSFM) has greatly expanded the experimental capabilities in many biological and biomedical research fields, enabling for example live studies of murine and zebrafish neural activity or of cell growth and division. The key feature of the method is the selective illumination of a sample single plane, providing an intrinsic optical sectioning and allowing direct 2D image recording. On the other hand, this excitation scheme is more affected by absorption or scattering artifacts in comparison to point scanning methods, leading to un-even illumination. We present here an easily implementable method, based on acousto-optical deflectors (AOD),…
4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)
2015
AbstractIn the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of…
A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue
2016
AbstractFor 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular mat…
Fast Inertia-Free Volumetric Light-Sheet Microscope
2017
Fast noninvasive three-dimensional (3D) imag-ing is crucial for quantitatively studying highly dynamic events ranging from flow cytometry to developmental biology. Light-sheet microscopy has emerged as the tool-of-choice for 3D characterization of rapidly evolving systems. However, to obtain a 3D image, either the sample or parts of the microscope are moved, limiting the acquisition speed. Here, we propose a novel inertia-free light-sheet-based scheme for volumetric imaging at high temporal resolution. Our approach comprises a novel combination of an acousto-optic scanner to produce tailored illumination and an acoustic-optofluidic lens, placed in the detection path to provide extended dept…
Swift light sheet volumetric charting of large human brain portions
2020
Using a custom light sheet fluorescence microscope, we image large stained human brain portions, labelled for NeuN and GAD67 neuronal markers, discerning the inhibitory population via neural-network based image analysis and exposing the brain connectivity.
A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy
2023
The analysis of histological alterations in all types of tissue is of primary importance in pathology for highly accurate and robust diagnosis. Recent advances in tissue clearing and fluorescence microscopy made the study of the anatomy of biological tissue possible in three dimensions. The combination of these techniques with classical hematoxylin and eosin (H&E) staining has led to the birth of three-dimensional (3D) histology. Here, we present an overview of the state-of-the-art methods, highlighting the optimal combinations of different clearing methods and advanced fluorescence microscopy techniques for the investigation of all types of biological tissues. We employed fluorescence …
Advanced fluorescence microscopy for in vivo imaging of neuronal activity
2019
Brain function emerges from the coordinated activity, over time, of large neuronal populations placed in different brain regions. Understanding the relationships of these specific areas and disentangling the contributions of individual neurons to overall function remain central goals for neuroscience. In this scenario, fluorescence microscopy has been proved as the tool of choice for in vivo recording of brain activity. Optical advances combined with genetically encoded indicators allow a large flexibility in terms of spatiotemporal resolution and field of view while keeping invasiveness in living animals to a minimum. Here we describe the latest advancements in the field of linear and nonl…
Optical sectioning by two-pinhole confocal fluorescence microscopy.
2003
A two-pinhole axially superresolving confocal fluorescence imaging system is presented. Based on the concept of subtractive incoherent imaging, the system described here is equipped with a zero-focus complex-transmittance pupil filter in one of the collector paths. The optical sectioning capacity of the system is 25% superior to that of a free-pupil one-pinhole instrument.