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.

Peptide-Membrane Interactions Monitored by Fluorescence Lifetime Imaging: A Study Case of Transportan 10.

The interest on detailed analysis of peptide-membrane interactions is of great interest in both fundamental and applied sciences as these may relate to both functional and pathogenic events. Such interactions are highly dynamic and spatially heterogeneous, making the investigation of the associated phenomena highly complex. The specific properties of membranes and peptide structural details, together with environmental conditions, may determine different events at the membrane interface, which will drive the fate of the peptide-membrane system. Here, we use an experimental approach based on the combination of spectroscopy and fluorescence microscopy methods to characterize the interactions of the multifunctional amphiphilic peptide transportan 10 with model membranes. Our approach, based on the use of suitable fluorescence reporters, exploits the advantages of phasor plot analysis of fluorescence lifetime imaging microscopy measurements to highlight the molecular details of occurring membrane alterations in terms of rigidity and hydration. Simultaneously, it allows following dynamic events in real time without sample manipulation distinguishing, with high spatial resolution, whether the peptide is adsorbed to or inserted in the membrane.

Improved Photocatalytic Activity of Polysiloxane TiO2 Composites by Thermally Induced Nanoparticle Bulk Clustering and Dye Adsorption.

Fine control of nanoparticle clustering within polymeric matrices can be tuned to enhance the physicochemical properties of the resulting composites, which are governed by the interplay of nanoparticle surface segregation and bulk clustering. To this aim, out-of-equilibrium strategies can be leveraged to program the multiscale organization of such systems. Here, we present experimental results indicating that bulk assembly of highly photoactive clusters of titanium dioxide nanoparticles within an in situ synthesized polysiloxane matrix can be thermally tuned. Remarkably, the controlled nanoparticle clustering results in improved degradation photocatalytic performances of the material under 1 sun toward methylene blue. The resulting coatings, in particular the 35 wt % TiO2-loaded composites, show a photocatalytic degradation of about 80%, which was comparable to the equivalent amount of bare TiO2 and two-fold higher with respect to the corresponding composites not subjected to thermal treatment. These findings highlight the role of thermally induced bulk clustering in enhancing photoactive nanoparticle/polymer composite properties.

Deciphering amyloid fibril molecular maturation through FLIM-phasor analysis of thioflavin T.

The investigation of amyloid fibril formation is paramount for advancing our understanding of neurodegenerative diseases and for exploring potential correlated therapeutic strategies. Moreover, the self-assembling properties of amyloid fibrils show promise for the development of advanced protein-based biomaterials. Among the methods employed to monitor protein aggregation processes, fluorescence has emerged as a powerful tool. Its exceptional sensitivity enables the detection of early-stage aggregation events that are otherwise challenging to observe. This research underscores the pivotal role of fluorescence analysis, particularly in investigating the aggregation processes of hen egg white lysozyme, a model protein extensively studied for insights into amyloid fibril formation. By combining classical spectroscopies with fluorescence microscopy and by exploiting the fluorescence properties (intensity and lifetime) of the thioflavin T, we were able to noninvasively monitor key and complex molecular aspects of the process. Intriguingly, the fluorescence lifetime imaging-phasor analysis of thioflavin T fluorescence lifetime on structures at different stages of aggregation allowed to decipher the complex fluorescence decay behavior, highlighting that their changes rise from the combination of specific binding to amyloid typical cross-ß structures and of the rigidity of the molecular environment.

Surface-catalyzed liquid-liquid phase separation and amyloid-like assembly in microscale compartments.

Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid-liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process.

Expansion and Light-Sheet Microscopy for Nanoscale 3D Imaging.

Expansion Microscopy (ExM) and Light-Sheet Fluorescence Microscopy (LSFM) are forefront imaging techniques that enable high-resolution visualization of biological specimens. ExM enhances nanoscale investigation using conventional fluorescence microscopes, while LSFM offers rapid, minimally invasive imaging over large volumes. This review explores the joint advancements of ExM and LSFM, focusing on the excellent performance of the integrated modality obtained from the combination of the two, which is refer to as ExLSFM. In doing so, the chemical processes required for ExM, the tailored optical setup of LSFM for examining expanded samples, and the adjustments in sample preparation for accurate data collection are emphasized. It is delve into various specimen types studied using this integrated method and assess its potential for future applications. The goal of this literature review is to enrich the comprehension of ExM and LSFM, encouraging their wider use and ongoing development, looking forward to the upcoming challenges, and anticipating innovations in these imaging techniques.

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.

Transportan 10 Induces Perturbation and Pores Formation in Giant Plasma Membrane Vesicles Derived from Cancer Liver Cells.

Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime.

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.

Sustainable soy protein microsponges for efficient removal of lead (II) from aqueous environments.

Protein-based materials recently emerged as good candidates for water cleaning applications, due to the large availability of the constituent material, their biocompatibility and the ease of preparation. In this work, new adsorbent biomaterials were created from Soy Protein Isolate (SPI) in aqueous solution using a simple environmentally friendly procedure. Protein microsponge-like structures were produced and characterized by means of spectroscopy and fluorescence microscopy methods. The efficiency of these structures in removing Pb2+ ions from aqueous solutions was evaluated by investigating the adsorption mechanisms. The molecular structure and, consequently, the physico-chemical properties of these aggregates can be readily tuned by selecting the pH of the solution during production. In particular, the presence of ß-structures typical of amyloids as well as an environment characterized by a lower dielectric constant seem to enhance metal binding affinity revealing that hydrophobicity and water accessibility of the material are key features affecting the adsorption efficiency. Presented results provide new knowledge on how raw plant proteins can be valorised for the production of new biomaterials. This may offer extraordinary opportunities towards the design and production of new tailorable biosorbents which can also be exploited for several cycles of purification with minimal reduction in performance. SYNOPSIS: Innovative, sustainable plant-protein biomaterials with tunable properties are presented as green solution for water purification from lead(II) and the structure-function relationship is discussed.

α-casein micelles-membranes interaction: Flower-like lipid protein coaggregates formation.

BACKGROUND: Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects. METHODS: By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes. RESULTS: The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part. GENERAL SIGNIFICANCE: These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins.

Insight into mechanisms of creatinine optical sensing using fluorescein-gold complex.

Creatinine level in biological fluids is a clinically relevant parameter to monitor vital functions and it is well assessed that measuring creatinine levels in the human body can be of great utility to evaluate renal, muscular, or thyroid dysfunctions. The accurate detection of creatinine levels may have a critical role in providing information on health status and represents a tool for the early diagnosis of severe pathologies. Among different methods for creatinine detection that have been introduced and that are evolving with increasing speed, fluorescence-based and colorimetric sensors represent one of the best alternatives, thanks to their affordability, sensitivity and easy readability. In this work, we demonstrate that the fluorescein-Au3+complex provides a rapid, selective, and sensitive tool for the quantification of creatinine concentrations in ranges typical of sweat and urine. UV-visible absorption, diffuse reflectance spectroscopy, steady state and time resolved fluorescence spectroscopy were used to shed light on the molecular mechanisms involved in the changes of optical properties, which underlie the multiplexed sensor analytical reply. Interestingly, sensing can be performed in solution or on solid nylon support accessing different physiological concentrations from micromolar to millimolar range. As a proof-of-concept, the nylon-based platform was used to demonstrate its effectiveness in creatinine detection on a solid and flexible substrate, showing its analytical colorimetric properties as an easy and disposable creatinine point-of-care test.

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.

Lead(II) ions adsorption onto amyloid particulates: An in depth study.

The production of new cost-effective biocompatible sorbent sustainable materials, with natural origins, able to remove heavy metals from water resources is nowadays highly desirable in order to reduce pollution and increase clean water availability. In this context, self-assembled protein materials with amyloid structures seem to have a great potential as natural platform for a broader development of highly-tunable structures. In this work we show how protein particulates, a generic form of protein aggregates, with spherical micro sized shape can be used as adsorbents of Pb2+ ions from aqueous solution. The effect of pH, ionic medium, ionic strength and temperature of the metal ion solution on the adsorption ability and affinity has been evaluated revealing the complexity of adsorption mechanisms which are the result of the balance of specific interactions with functional groups in protein structure and not specific ones common to all polypeptide chains, and possibly related to amyloid state and to modification of particulates hydration layer.

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).

Power-effective scanning with AODs for 3D optogenetic 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.

3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy.

The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH-H2O2-antigen Retrieval-TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ.

Identification of microplastics using 4-dimethylamino-4'-nitrostilbene solvatochromic fluorescence.

In this work, we introduce the use of 4-dimethylamino-4'-nitrostilbene (DANS) fluorescent dye for applications in the detection and analysis of microplastics, an impendent source of pollution made of synthetic organic polymers with a size varying from less than 5 mm to nanometer scale. The use of this dye revealed itself as a versatile, fast and sensitive tool for readily discriminate microplastics in water environment. The experimental evidences herein presented demonstrate that DANS efficiently absorbs into a variety of polymers constituting microplastics, and its solvatochromic properties lead to a positive shift of the fluorescence emission spectrum according to the polarity of the polymers. Therefore, under UV illumination, microplastics glow a specific emission spectrum from blue to red that allows for a straightforward polymer identification. In addition, we show that DANS staining gives access to different detection and analysis strategies based on fluorescence microscopy, from simple epifluorescence fragments visualization, to confocal microscopy and phasor approach for plastic components quantification.

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 brain-extracted myelin were produced. The MyVes have an average diameter of 100-150 â€‹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.

Nile Red lifetime reveals microplastic identity.

Microplastic pollution is recognized as a worldwide environmental problem. The increasing daily use and release of plastics into the environment have led to the accumulation of fragmented microplastics, with potentially awful consequences for the environment, and animal and human health. The detection and identification of microplastics are of utmost importance, but available methods are still limited. In this work, a new approach is presented for the analysis of microplastics based on hydrophobic fluorescence staining with Nile Red, using spectrally resolved confocal fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). Significant differences were observed in the emission spectra and fluorescence lifetimes of the analyzed microplastics. Nile Red fluorescence shows determinable behavior based on the polymer matrix and provides a fingerprint for the identification of fragments from different types of plastics. Lifetime imaging coupled with phasor analysis constitutes a fast, robust, and straightforward method for mapping and identifying different microplastics within the same sample in an aquatic environment.