The aggregation process of amyloidogenic peptides can be characterized by advanced correlative techniques to understand the molecular mechanisms of misfolded protein diseases. We performed correlative AFM-STED measurements to study amyloid aggregates formation starting from monomeric peptides. A previous study, performed by our group on insulin and β-amyloid peptides (MW∼ 4kDa) suggested that the presence of fluorophores not only decreased the kinetics of the aggregation, a very well-known effect, but also favored the coexistence of labeled and unlabeled fibrils. The results suggested the coexistence of different aggregation pathways. Labeled molecules can follow just some of these pathways. However, clear structural diversity between fluorescent, or dark fibrils was not detected. Particularly, we investigated the in vitro aggregation of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease (PD), which is significantly larger (MW∼ 14kDa) with respect to insulin, previously investigated. The fluorophore was covalently bind at the C-terminal group of the peptide, an area of the polypeptide chain not or scarcely involved in fibrillation. We employed ATTO488 with a functional maleimide group that covalently bind the free cysteine at the C-terminal domain. We used dye-to-protein ratio 1:20 and 1:10. We demonstrated that the fibrillary aggregates of α-syn are all fluorescent, or at least just very minor portions of the sample are not labeled. These results show that labeling artifacts can be avoided, but also that the labeling strategies must be carefully designed to the molecular system under investigation. This work provides evidence of a fundamental drawback in fluorescence microscopy, generating a warning for all the fluorescence users: a deeper control for possible artifacts induced by the fluorophore is needed. The use of a correlative microscopy technique would play a key role in the control of the setting of these conditions.

Hypericin (Hyp), a pigment extracted from Hypericum perforatum, is a naturally occurring photosensitizer (PS), i.e. a compound that sensitizes the production of singlet oxygen (1O2) upon visible photo-excitation. 1O2 is an oxidizing molecule capable of damaging biomolecules such as lipids, nucleic acids and amino acids. The combination of a PS, visible light and molecular oxygen is at the basis of photodynamic therapy (PDT), a clinically approved treatment against cancer cells, but also effective against pathogenic bacteria, fungi and viruses. Hyp represents a very interesting PS, because it shows high singlet-oxygen quantum yield (φΔ=0.33) and high fluorescence quantum yield (φF=0.35) in DMSO. Due to the hydrophobic character of the molecule, these properties are lost in aqueous solution, where Hyp tends to form photo-inactive aggregates. The photo-physical and photo-dynamic properties are recovered when Hyp is bound to apolar systems, such as phospholipidic membranes or protein hydrophobic pockets. These features are useful for localization of the PS by fluorescence microscopy. In particular, Hyp was found to effectively inactivate viruses with a phospholipidic envelope, like SARS-CoV-2 upon light exposure, and sometimes also in dark conditions.We used fluorescence spectroscopy and microscopy to explore the interaction between SARS-CoV-2 and Hyp, and correlative AFM-fluorescence microscopy on a bilayer model membrane, to study the morphological changes induced by the presence of the PS. The aim of this research project is to assess the binding and photosensitizing properties of Hyp with SARS-CoV-2 and a model membrane, in order to provide more data for a future therapeutic implementation of PDT with this antiviral agent against SARS-CoV-2 and, more generally, all enveloped viruses.

The toxicity of α-synuclein (α-syn), the amyloidogenic protein responsible for Parkinson's disease, is likely related to its interaction with the asymmetric neuronal membrane. α-Syn exists as cytoplasmatic and as extracellular protein as well. To shed light on the different interactions occurring at the different α-syn localizations, we have here modelled the external and internal membrane leaflets of the neuronal membrane with two complex lipid mixtures, characterized by phase coexistence and with negative charge confined to either the ordered or the disordered phase, respectively. To this purpose, we selected a five-component (DOPC/SM/DOPE/DOPS/chol) and a four-component (DOPC/SM/GM1/chol) lipid mixtures, which contained the main membrane lipid constituents and exhibited a phase separation with formation of ordered domains. We have compared the action of α-syn in monomeric form and at different concentrations (1 nM, 40 nM, and 200 nM) with respect to lipid systems with different composition and shape by AFM, QCM-D, and vesicle leakage experiments. The experiments coherently showed a higher stability of the membranes composed by the internal leaflet mixture to the interaction with α-syn. Damage to membranes made of the external leaflet mixture was detected in a concentration-dependent manner. Interestingly, the membrane damage was related to the fluidity of the lipid domains and not to the presence of negatively charged lipids.

SLBs are a well-established model of the plasma membrane of eukaryotic cells. It is known that α-synuclein (α-syn), a peptide involved in Parkinson's disease, is present in both the inner (cytosol) and extracellular space. Both the endogenous and exogenous components seem to have a role in the neurodegenerative process typical of the disorder. It is also demonstrated that the physiological membrane has an asymmetric structure, i.e., the lipid compositions of the inner and outer leaflet are different. As a consequence of this asymmetry, also the organization of the lipid phase in the two leaflets are different. α-syn interacts with supported lipid bilayers (SLBs) with two different compositions, mimicking the inner and the outer leaflet of the cell membrane respectively, to reveal the possible destabilization induced by the pathological peptides. It is known that the presence of the rigid substrate, that support the membrane, reduces the diffusion of the single lipid molecules within the membrane and induce a mechanical stabilization of the membrane, that increase the resistance of the bilayer toward the action of an external agent. We proposed a new approach, making the peptides interact with lipid vescicles in solution, creating a planar bilayer after this interaction. in this way, α-syn interacts with a tridimensional membrane, that is better resembling the properties of the cell membrane. Before and after the interaction with α-syn the membrane were characterized by atomic force microscopy, quartz micor-balance, and fluorescence techniques.

Atomic force microscopy (AFM) is a nano-mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction between the tip and the sample, a mechanism of contrast capable of measuring different information, including surface topography, mechanical, and electrical properties. However, the lack of specificity highlights the need to integrate AFM data with other techniques providing compositional hints. In particular, optical microscopes based on fluorescence as a mechanism of contrast can access the local distribution of specific molecular species. The coupling between AFM and super-resolved fluorescence microscopy solves the resolution mismatch between AFM and conventional fluorescence optical microscopy. Recent advances showed that also the inherently label-free imaging capabilities of the AFM are fundamental to complement the fluorescence images. In this review, we have presented a brief historical view on correlative microscopy, and, finally, we have summarized the progress of correlative AFM-super-resolution microscopy in biological research.

Atomic force microscopy (AFM) is a nano-mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction between the tip and the sample, a mechanism of contrast capable of measuring different information, including surface topography, mechanical, and electrical properties. However, the lack of specificity highlights the need to integrate AFM data with other techniques providing compositional hints. In particular, optical microscopes based on fluorescence as a mechanism of contrast can access the local distribution of specific molecular species. The coupling between AFM and super-resolved fluorescence microscopy solves the resolution mismatch between AFM and conventional fluorescence optical microscopy. Recent advances showed that also the inherently label-free imaging capabilities of the AFM are fundamental to complement the fluorescence images. In this review, we have presented a brief historical view on correlative microscopy, and, finally, we have summarized the progress of correlative AFM-super-resolution microscopy in biological research.