Phasor-FLIM analysis of cellulose paper ageing mechanism with carbotrace 680 dye

Ageing of paper is a complex process of great relevance for application purposes because of its widespread use as support for information storage in books and documents, and as common low-cost and green packaging material, to name a few. A key factor in paper ageing is the oxidation of cellulose, a macromolecule of natural origin that constitutes the main chemical component of paper. Such a complex process results in changes in the cellulose polymeric chains in chemical and structural properties. The scope of this work is to explore the effects of oxidation of cellulose as one of the principal mechanisms of ageing of paper using a fluorescence-based approach. To this aim, fluorescence-lifetime imaging microscopy (FLIM) measurements on pure cellulose samples stained using Carbotrace 680 dye were performed, and data were analyzed by phasor approach. The comparison with results from conventional techniques allowed to map paper microstructure as a function of the sample oxidation degree correlating the fluorescence-lifetime changes to cellulose oxidation. A two-step oxidation kinetics that produced specific modification in paper organization was highlighted indicating that FLIM measurements using Carbotrace 680 dye may provide a simple tool to obtain information on the oxidation process also adding spatial information at sub-micrometric scale

Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

A Guide to Perform 3D Histology of Biological Tissues with Fluorescence Microscopy

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 nuclear and eosin Y staining that enabled us to obtain hematoxylin and eosin pseudo-coloring comparable with the gold standard H&E analysis. The computational reconstructions obtained with 3D optical imaging can be analyzed by a pathologist without any specific training in volumetric microscopy, paving the way for new biomedical applications in clinical pathology

Fast multi-directional DSLM for confocal detection without striping artifacts

In recent years light-sheet fluorescence microscopy (LSFM) has become a cornerstone technology for neuroscience, improving the quality and capabilities of 3D imaging. By selectively illuminating a single plane, it provides intrinsic optical sectioning and fast image recording, while minimizing out of focus fluorescence background, sample photo-damage and photo-bleaching. However, images acquired with LSFM are often affected by light absorption or scattering effects, leading to un-even illumination and striping artifacts. In this work we present an optical solution to this problem, via fast multi-directional illumination of the sample, based on an acousto-optical deflector (AOD). We demonstrate that this pivoting system is compatible with confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) by using a pivoted elliptical-Gaussian beam. We tested its performance by acquiring signals emitted by specific fluorophores in several mouse brain areas, comparing the pivoting beam illumination and a traditional static one, measuring the point spread function response and quantifying the striping reduction. We observed real-time shadow suppression, while preserving the advantages of confocal detection for image contrast

Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

Engineered Ferritin with Eu3+ as a Bright Nanovector: A Photoluminescence Study

Ferritin nanoparticles play many important roles in theranostic and bioengineering applications and have been successfully used as nanovectors for the targeted delivery of drugs due to their ability to specifically bind the transferrin receptor (TfR1, or CD71). They can be either genetically or chemically modified for encapsulating therapeutics or probes in their inner cavity. Here, we analyzed a new engineered ferritin nanoparticle, made of the H chain mouse ferritin (HFt) fused with a specific lanthanide binding tag (LBT). The HFt-LBT has one high affinity lanthanide binding site per each of the 24 subunits and a tryptophane residue within the tag that acts as an antenna able to transfer the energy to the lanthanide ions via a LRET process. In this study, among lanthanides, we selected europium for its red emission that allows to reduce overlap with tissue auto-fluorescence. Steady state emission measurements and time-resolved emission spectroscopy have been employed to investigate the interaction between the HFt-LBT and the Eu3+ ions. This allowed us to identify the Eu3+ energy states involved in the process and to pave the way for the future use of HFt-LBT Eu3+ complex in theranostics

Power-effective scanning with AODs for 3D optogenic applications

Two-photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub-micrometric-resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto-optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD-based scheme for 2P 3D scanning that improves the power delivery between different illumination planes. We applied this approach to photostimulate an optogenetic actuator in zebrafish larvae, demonstrating the method efficiency observing increased activity responses and uniform activation probabilities from neuronal clusters addressed in the volume. This novel driving scheme can open to new AOD applications in neuroscience, allowing more effective 3D interrogation in large neuronal networks

Nano-structured myelin: new nanovesicles for targeted delivery to white matter and microglia, from brain-to-brain

Neurodegenerative diseases affect millions of people worldwide and the presence of various physiological barriers limits the accessibility to the brain and reduces the efficacy of various therapies. Moreover, new carriers having targeting properties to specific brain regions and cells are needed in order to improve therapies for the brain disorder treatment. In this study, for the first time, Myelin nanoVesicles (hereafter defined MyVes) from brainextracted myelin were produced. The MyVes have an average diameter of 100-150 nm, negative zeta potential, spheroidal morphology, and contain lipids and the key proteins of the myelin sheath. Furthermore, they exhibit good cytocompatibility. The MyVes were able to target the white matter and interact mainly with the microglia cells. The preliminary results here presented allow us to suppose the employment of MyVes as potential carrier to target the white matter and microglia in order to counteract white matter microglia-related diseases