Understanding alpha-synuclein aggregation propensity in animals and humans.

Alpha-synuclein (α-syn) aggregation plays a critical role in the pathogenicity of Parkinson's Disease (PD). This study aims to evaluate the aggregation propensity of α-syn fragment peptides designed using the variability found in humans and animals. Thioflavin T (ThT) and transmission electron microscopy (TEM) were used to validate the formation of fibrils to identify important amino acid residues. Human α-syn fragments 51-75, 37-61, 62-86, 76-100, and 116-140 demonstrate a significantly higher tendency to aggregate compared to fragments 1-25, 26-50, and 91-115. All species analyzed of the α-syn 37-61 and 62-86 regions were shown to form fibrils on both ThT and TEM. The α-syn 37-61 and 62-86 fragment regions exhibited a high susceptibility to aggregation, with fibril formation observed in all species. The A53T mutation in several α-syn 37-61 fragments may enhance their propensity for aggregation, suggesting a correlation between this mutation and the capacity for fibril formation. Furthermore, the presence of the non-amyloid-ß component (NAC) region, specifically in α-syn 62-86, was consistently observed in several fragments that displayed fibril formation, indicating a potential correlation between the NAC region and the process of fibril formation in α-syn. Finally, the combination of a high quantity of valine and a low quantity of acidic amino acids in these fragments may serve as indicators of α-syn fibril formation.

Potential thioflavin T false positives in lipomembranous changes in adipocytes during systemic amyloidosis diagnosis.

The diagnosis of systemic amyloidosis relies on the detection of amyloid deposition in the tissue, often utilizing biopsy specimens from the abdominal skin owing to their minimal invasiveness. Several amyloid staining methods, including Congo Red, Direct Fast Scarlet (DFS), and Thioflavin T (ThT), have been employed for visualization. Lipomembranous fat necrosis (LFN) is a non-specific reaction pattern of adipose tissue to injury, typically derived from blood insufficiency across a wide range of clinical conditions or diseases. It is characterized by the presence of eosinophilic, crenulated, and/or serpiginous membranes in fat lobules. We encountered a patient in whom ThT yielded suspiciously positive results in amyloidosis screening tests. Furthermore, our retrospective observations suggested that ThT staining was positive for LFN, whereas DFS and Congo red staining yielded negative results. The awareness that LFN can result in false-positive ThT staining during amyloid screening is crucial to avoiding the misdiagnosis of systemic amyloidosis. Furthermore, skin samples should not be collected from areas prone to developing lipomembranous changes. The use of more than two different stains for skin biopsy specimens is recommended.

Comparison of Double-Stranded DNA at the 5' and 3' Ends of the G-Triplex and Its Application in the Detection of Hg(II).

Leveraging the fluorescence enhancement effect of the G-triplex (G3)/thioflavin T (ThT) catalyzed by the adjacent double-stranded DNA positioned at the 5' terminus of the G3, the G3-specific oligonucleotide (G3MB6) was utilized to facilitate the rapid detection of mercury (Hg(II)) through thymine-Hg(II)-thymine (T-Hg(II)-T) interactions. G3MB6 adopted a hairpin structure in which partially complementary strands could be disrupted with the presence of Hg(II). It prompted the formation of double-stranded DNA by T-Hg(II)-T, inducing the unbound single strand of G3MB6 to spontaneously form a parallel G3 structure, producing a solid fluorescence signal by ThT. Conversely, fluorescence was absent without Hg(II), since no double strand and formation of G3 occurred. The fluorescence intensity of G3MB6 exhibited a positive correlation with Hg(II) concentrations from 17.72 to 300 nM (R2 = 0.9954), boasting a notably low quality of limitation (LOQ) of 17.72 nM. Additionally, it demonstrated remarkable selectivity for detecting Hg(II). Upon application to detect Hg(II) in milk samples, the recovery rates went from 100.3% to 103.2%.

Dissecting Henipavirus W proteins conformational and fibrillation properties: contribution of their N- and C-terminal constituent domains.

The Nipah and Hendra viruses are severe human pathogens. In addition to the P protein, their P gene also encodes the V and W proteins that share with P their N-terminal intrinsically disordered domain (NTD) and possess distinct C-terminal domains (CTDs). The W protein is a key player in the evasion of the host innate immune response. We previously showed that the W proteins are intrinsically disordered and can form amyloid-like fibrils. However, structural information on W CTD (CTDW) and its potential contribution to the fibrillation process is lacking. In this study, we demonstrate that CTDWS are disordered and able to form dimers mediated by disulfide bridges. We also show that the NTD and the CTDW interact with each other and that this interaction triggers both a gain of secondary structure and a chain compaction within the NTD. Finally, despite the lack of intrinsic fibrillogenic properties, we show that the CTDW favors the formation of fibrils by the NTD both in cis and in trans. Altogether, the results herein presented shed light on the molecular mechanisms underlying Henipavirus pathogenesis and may thus contribute to the development of targeted therapies.

Exploring the amyloid degradation potential of nanoformulated carrageenan-bridging in vitro and in vivo perspectives.

Amyloids, with their ß-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17-20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vitro. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

Key charged residues influence the amyloidogenic propensity of the helix-1 region of serum amyloid A.

Increased plasma levels of serum amyloid A (SAA), an acute-phase protein that is secreted in response to inflammation, may lead to the accumulation of amyloid in various organs thereby obstructing their functions. Severe cases can lead to a systemic disorder called AA amyloidosis. Previous studies suggest that the N-terminal helix is the most amyloidogenic region of SAA. Moreover, computational studies implicated a significant role for Arg-1 and the residue-specific interactions formed during the fibrillization process. With a focus on the N-terminal region of helix-1, SAA1-13, mutational analysis was employed to interrogate the roles of the amino acid residues, Arg-1, Ser-5, Glu-9, and Asp-12. The truncated SAA1-13 fragment was systematically modified by substituting the key residues with alanine or uncharged but structurally similar amino acids. We monitored the changes in the amyloidogenic propensities, associated conformational markers, and morphology of the amyloids resulting from the mutation of SAA1-13. Mutating out Arg-1 resulted in much reduced aggregation propensity and a lack of detectable ß-structures alluding to the importance of salt-bridge interactions involving Arg-1. Our data revealed that by systematically mutating the key amino acid residues, we can modulate the amyloidogenic propensity and alter the time-dependent conformational variation of the peptide. When the behaviors of each mutant peptide were analyzed, they provided evidence consistent with the aggregation pathway predicted by MD simulation studies. Here, we detail the important temporal molecular interactions formed by Arg-1 with Ser-5, Glu-9, and Asp-12 and discuss its mechanistic implications on the self-assembly of the helix-1 region of SAA.

Binding Interaction of Coumarin Derivative Daphnetin with Ovalbumin: Molecular Insights into the Complexation Process and Effects of Metal Ions and pH in the Binding and Antifibrillation Studies.

This study investigates the interaction between daphnetin and ovalbumin (OVA) as well as its potential to inhibit OVA fibrillation using both spectroscopic and computational analysis. A moderate binding affinity of 1 × 104 M-1 was observed between OVA and daphnetin, with a static quenched mechanism identified during the fluorescence quenching processes. Metal ions' (Cu2+ and Zn2+) presence led to an increase in the binding affinities of daphnetin toward OVA, mirroring a similar trend observed with the pH variation. Synchronous and 3D fluorescence studies indicated an increase in the polarity of the microenvironment surrounding the Trp residues during binding. Interestingly, circular dichroism and Fourier transform infrared studies showed a significant change in the secondary structure of OVA upon binding with daphnetin. The efficacy of daphnetin in inhibiting protein fibrillation was confirmed through thioflavin T and Congo Red binding assays along with fluorescence microscopic imaging analysis. The thermodynamic assessment showed positive ΔH° [+(29.34 ± 1.526) kJ mol-1] and ΔS° [+(181.726 ± 5.465) J mol-1] values, indicating the presence of the hydrophobic forces, while negative ΔG° signifies spontaneous binding interactions. These experimental findings were further correlated with computational analysis, revealing daphnetin dynamics within the binding site of OVA.

Effects of medium pH on the yeast plasma membrane potential.

The effects of different pH incubation values and K+ on yeast plasma membrane potential (PMP) were studied both by the fluorescence changes and the accumulation of thioflavin T (ThT), a method that has been shown most adequate for both procedures. By the changes in fluorescence of ThT, the qualitative observation of PMP at the 3 evaluated pHs indicated that cells at pH 4.0 maintain a PMP lower, but close to the observed at pH 6.0 and 7.0. By measuring the accumulation of ThT and applying the Nernst equation on the different concentrations in and out, the values of PMP could also be estimated at the different pHs, resulting in values in mV, in agreement with our observations by following the fluorescence. Yeast cells at their native niches, or during fermentations must cope with low pHs, so the importance to maintain a robust PMP to survive. The contribution of bicarbonate, derived from the fermentation to the establishment of the PMP is also described. The experiments showed once more the efficacy of the methods used with this dye.

Ferroptosis induces nucleolar stress as revealed by live-cell imaging using thioflavin T.

Nucleolar stress induced by stressors like hypoxia, UV irradiation, and heat shock downregulates ribosomal RNA transcription, thereby impairing protein synthesis capacity and potentially contributing to cell senescence and various human diseases such as neurodegenerative disorders and cancer. Live-cell imaging of the nucleolus may be a feasible strategy for investigating nucleolar stress, but currently available nucleolar stains are limited for this application. In this study using mouse hippocampal HT22 cells, we demonstrate that thioflavin T (ThT), a benzothiazole dye that binds RNA with high affinity, is useful for nucleolar imaging in cells where RNAs predominate over protein aggregates. Nucleoli were stained with high intensity simply by adding ThT to the cell culture medium, making it suitable for use even in damaged cells. Further, ThT staining overlapped with specific nucleolar stains in both live and fixed cells, but did not overlap with markers for mitochondria, lysosomes, endoplasmic reticulum, and double-stranded DNA. Ferroptosis, an iron-dependent nonapoptotic cell death pathway characterized by lipid peroxide accumulation, reduced the number of ThT-positive puncta while endoplasmic reticulum stress did not. These findings suggest that ferroptosis is associated with oxidative damage to nucleolar RNA molecules and ensuing loss of nucleolar function.

A Label-Free Fluorescent Amplification Strategy for High-Sensitive Detection of Pseudomonas aeruginosa based on Protective-EXPAR (p-EXPAR) and Catalytic Hairpin Assembly.

This study presents a fluorescent mechanism for two-step amplification by combining two widely used techniques, exponential amplification reaction (EXPAR) and catalytic hairpin assembly (CHA). Pseudomonas aeruginosa (P. aeruginosa) engaged in competition with the complementary DNA in order to attach to the aptamer that had been fixed on the magnetic beads. The unbound complementary strand in the liquid above was utilized as a trigger sequence to initiate the protective-EXPAR (p-EXPAR) process, resulting in the generation of a substantial quantity of short single-stranded DNA (ssDNA). The amplified ssDNA can initiate the second CHA amplification process, resulting in the generation of many double-stranded DNA (dsDNA) products. The CHA reaction was initiated by the target/trigger DNA, resulting in the release of G-quadruplex sequences. These sequences have the ability to bond with the fluorescent amyloid dye thioflavin T (ThT), generating fluorescence signals. The method employed in this study demonstrated a detection limit of 16 CFU/ml and exhibited a strong linear correlation within the concentration range of 50 CFU/ml to 105 CFU/ml. This method of signal amplification has been effectively utilized to create a fluorescent sensing platform without the need for labels, enabling the detection of P. aeruginosa with high sensitivity.

Crystal Violet Selectively Detects Aß Oligomers but Not Fibrils In Vitro and in Alzheimer's Disease Brain Tissue.

Deposition of extracellular Amyloid Beta (Aß) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer's Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aß and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aß42 oligomers (AßOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans.

Redesign of a thioflavin-T-binding protein with a flat ß-sheet to evaluate a thioflavin-T-derived photocatalyst with enhanced affinity.

Amyloids, proteinous aggregates with ß-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat ß-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model. In this study, we further explored the residue-specific properties of ThT-binding to the flat ß-sheet by introducing systematic mutations. We found that site-specific mutations at the ThT-binding channel enhanced affinity. We also evaluated the binding of a ThT-based photocatalyst, which showed the photooxygenation activity on the amyloid fibril upon light radiation. Upon binding of the photocatalyst to the PSAM variant, singlet oxygen-generating activity was observed. The results of this study expand our understanding of the detailed binding mechanism of amyloid-specific molecules.

A G-quadruplex/thioflavin T-based label-free biosensor to detect ClO- in stress-induced hypertension.

Hypochlorous acid (HClO), as an essential reactive oxygen species (ROS) in biological systems, plays a pivotal role in processes of physiology and pathology. Abnormal fluctuations in HClO concentration can lead to various diseases, such as inflammation, cardiovascular diseases, and neurodegeneration. Therefore, developing an approach to rapidly and sensitively quantify ClO- content is vital to biomedicine development and bioassays. Herein, we fabricated a novel "turn-on" label-free fluorescence DNA probe to specifically detect hypochlorite ion (ClO-) based on G-quadruplex formation. To this end, we designed a G-rich signal DNA sequence (S-DNA) and a block DNA sequence (B-DNA), followed by the introduction of ClO--responsive phosphorothioate (PS) into B-DNA. In the absence of ClO-, B-DNA hybridized with S-DNA, preventing G-quadruplex formation from S-DNA; this resulted in the relatively low fluorescence intensity of ThT. Once ClO- was added, the hydrolysis between PS and ClO- split the B-DNA into two fragments, resulting in B-DNA breaking away from S-DNA, allowing G-quadruplex formation from S-DNA and increasing the fluorescence intensity of ThT. Using this method, we can detect ClO- without the interference of additional reactive oxygen species. The detection limit of ClO- was as low as 10 nM. Furthermore, this method facilitates the detection of ClO- within the tissues of rats with stress-induced hypertension.

Engineering of a DNAzyme-Based dimeric G-quadruplex rolling circle amplification for robust analysis of lead ion.

Detecting heavy metal pollution, particularly lead ion (Pb2⁺) contamination, is imperative for safeguarding public health. In this study, we introduced an innovative approach by integrating DNAzyme with rolling circle amplification (RCA) to propose an amplification sensing method termed DNAzyme-based dimeric-G-quadruplex (dimer-G4) RCA. This sensing approach allows for precise and high-fidelity Pb2⁺ detection. Strategically, in the presence of Pb2⁺, the DNAzyme undergoes substrate strand (S-DNA) cleavage, liberating its enzyme strand (E-DNA) to prime isothermal amplification. This initiates the RCA process, producing numerous dimer-G-Quadruplexes (dimer-G4) as the signal reporting transducers. Compared to conventional strategies using monomeric G-quadruplex (mono-G4) as the reporting transducers, these dimer-G4 structures exhibit significantly enhanced fluorescence when bound with Thioflavin T (ThT), offering superior target signaling ability for even detection of Pb2⁺ at low concentration. Conversely, in the absence of Pb2⁺, the DNAzyme structure remains intact so that no primers can be produced to cause the RCA initiation. This nucleic acid amplification-based Pb2⁺ detection method combing with the high specificity of DNAzymes for Pb2⁺ recognition ensures highly sensitive detection of Pb2+ with a detection limit of 0.058 nM, providing a robust tool for food safety analysis and environmental monitoring.

Lantern-type G-quadruplex fluorescent sensors for detecting divalent metal ions.

Three thioflavin T (ThT) derivatives, namely ThT/ethylenediaminetetraacetic acid conjugates (E1T, E2T, and E1T1P), were designed and synthesized as sensing components for divalent metal ion detection. Furthermore, these ThT derivatives were used to design lantern-type G-quadruplex (G4) fluorescent sensors. The fluorescence intensities of the ThT derivatives decreased by 1.2- to 5.6-folds in the presence of Ni2+ and Cu2+, respectively, regardless of the topology of the utilized G4. Conversely, when Mn2+ and Zn2+ coexisted in antiparallel G4, the fluorescence intensities of E2T increased to approximately 3.3- and 2.3-folds, respectively, depending on the concentration of the divalent metal ion, allowing for quantitative analyses. The Job plot analysis revealed that the binding ratio of G4 and E2T changed from 2:1 to 1:2 with the increasing concentration of the divalent metal ions. These results indicated that the basic principle of such a lantern-type G4 sensor can be applied to the detection of divalent metal ions and other types of targets, such as proteins, and small molecules via ThT derivatization.

Comparative negation of amphiphile production using nutrition factors: Amyloids versus biosurfactants.

Microbial amphiphiles play an important role in environmental activities such as microbial signaling, bioremediation, and biofilm formation. Microorganisms rely on their unique characteristics of interfaces to carry out critical biological functions, which are helped by amphipathic biomolecules known as amphiphiles. Bacillus amyloids aid in cell adhesion and biofilm formation. Pseudomonas sp. are essential in biofilm development and are a vital survival strategy for many bacteria. Furthermore, Pseudomonas and Bacillus are well-known for their ability to produce biosurfactants with a range of applications, including bioremediation and removing biological pollutants from different environments. The study employed 31 different media types and a range of analytical techniques to assess the presence of amyloid proteins and the absence of biosurfactants in Bacillus licheniformis K125 (GQ850525.1) and Pseudomonas fluorescens CHA0. The presence of amyloid proteins was confirmed through Congo red and thioflavin T staining. The carefully constructed medium also efficiently inhibited the synthesis of biosurfactants by these bacteria. Additionally, surface tension measurements, emulsification index, thin-layer chromatography, and high-performance thin-layer chromatography analyses indicated the absence of biosurfactants in the tested media.

Fibrillization of lentil proteins is impacted by the protein extraction conditions and co-extracted phenolics.

Legume proteins can be induced to form amyloid-like fibrils upon heating at low pH, with the exact conditions greatly impacting the fibril characteristics. The protein extraction method may also impact the resulting fibrils, although this effect has not been carefully examined. Here, the fibrillization of lentil protein prepared using various extraction methods and the corresponding fibril morphology were characterized. It was found that an acidic, rather than alkaline, protein extraction method was better suited for producing homogeneous, long, and straight fibrils from lentil proteins. During alkaline extraction, co-extracted phenolic compounds bound proteins through covalent and non-covalent interactions, contributing to the formation of heterogeneous, curly, and tangled fibrils. Recombination of isolated phenolics and proteins (from acidic extracts) at alkaline pH resulted in a distinct morphology, implicating a role for polyphenol oxidase also in modifying proteins during alkaline extraction. These results help disentangle the complex factors affecting legume protein fibrillization.

Polyallylamine Binds to Aß Amyloid and Inhibits Antibody Recognition.

Protein amyloids have attracted attention for their application as functional amyloid materials because of their strong properties, such as high resistance to chemical or biological degradation, despite their medical issues. Amyloids can be used for various applications by modifying the amyloid surface with functional materials, such as proteins and polymers. In this study, we investigated the effect of polyallylamine (PAA), a functional cationic polymer as a candidate for amyloid modification, on the amyloids formed from amyloid ß (Aß) peptide. It was demonstrated for the first time that PAA can bind to Aß amyloids through fluorescence observations and the quenched emission from the tyrosine at site 10 near the fibrillogenic core. These results suggest that PAA could be used to develop new functional amyloids. However, notably, coating Aß amyloid with PAA could affect conventional amyloid detection assays such as thioflavin T assay and detection using antibodies. Thus, our results also indicate that consideration would be necessary for the analysis of functional amyloids coated with various polymers.

Microfluidic chip immunoassay based on rolling circle amplification and G-quadruplex/Thioflavin T for multiplex detection of CTX I.

A label-free immunoassay based on rolling circle amplification (RCA) and G-quadruplex/Thioflavin T (G4/ThT) is proposed to realize the sensitive detection of carboxy-terminal cross-linked fragment of type I collagen (CTX I) for bone loss. Under the optimal conditions, as low as 38.02 pg/mL of CTX I can be detected. To improve the detecting throughput and simplify the operation, a microfluidic chip was designed, fabricated, and used for CTX I detection based on the proposed assay. The detection can be completed with only a single on-chip magnetic separation step, which was easy to operate, less time-consuming, and has only low reagent consumption. The limit of detection was 131.83 pg/mL by observing with fluorescence microscope. With further improvement of detection equipment, the sensitivity of on-chip detection can be improved. It can be expected that the proposed RCA/G4/ThT immunoassay for sensitive and high-throughput automated detection of CTX I might be chosen as a potential analytical tool for clinical osteoporosis diagnosis and in-orbit bone loss detection.

Use of the Fluorescent Dye Thioflavin T to Track Amyloid Structures in the Pathogenic Yeast Candida albicans.

The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm. Key features • Study of amyloid properties of fungal proteins. • Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy. • Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation. • Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.