Plaques of amyloid fibrils composed of neuronal protein α-synuclein are one of the hallmarks of Parkinson's disease, and their selective imaging is crucial to study the mechanism of its pathogenesis However, the existing fluorescent probes for amyloids are efficient only in solution and tissue systems, and they are not selective enough for the visualization of amyloid fibrils in living cells In this study, we present two molecular rotor-based probes RB1 and RB2 These thiazolium probes show affinity to α-synuclein fibrils and turn-on fluorescence response upon interactions Because of its extended π-conjugation and high rotational degree of freedom, RB1 exhibits a 76 nm red-shift of absorption maxima and 112-fold fluorescence enhancement upon binding to amyloid fibrils Owing to its strong binding affinity to α-synuclein fibrils, RB1 can selectively stain them in the cytoplasm of living HeLa and SH-SY5Y cells with high optical contrast RB1 is a cell-permeable and noncytotoxic probe Taken together, we have demonstrated that RB1 is an amyloid probe with an outstanding absorption red-shift that can be used for intracellular imaging of α-synuclein fibrils

Fluorescent Probe for Selective Imaging of α-Synuclein Fibrils in Living Cells.

The binding mechanism of thioflavin T (ThT) to DNA was studied using polarized light spectroscopy and fluorescence-based techniques in solutions and in solid films. Linear dichroism measurements showed that ThT binds to DNA duplex by intercalation. Time-resolved fluorescence studies revealed a second binding mode which is the external binding to the DNA phosphate groups. Both binding modes represent the nonspecific type of interactions. The studies were complemented with the analysis of short oligonucleotides having DNA cavities. The results indicate that the interplay between three binding modes-intercalation, external binding, and binding inside DNA cavities-determines the effective fluorescence quantum yield of the dye in the DNA structures. External binding was found to be responsible for fluorescence quenching because of energy transfer between intercalated and externally bound molecules. Finally, amplified spontaneous emission (ASE) was successfully generated in the ThT-stained films and used for detecting different DNA structures. ASE measurements show that ThT-stained DNA structures can be used for designing bioderived microlasers.

Identification of Thioflavin T Binding Modes to DNA: A Structure-Specific Molecular Probe for Lasing Applications.

There is currently no definitive test for early detection of neurodegeneration which is linked with protein aggregation. Finding methods capable of detecting intermediate states of protein aggregates, named oligomers, is critical for the early stage diagnosis of over 30 neurodegenerative diseases including Alzheimer's or Parkinson's. Currently, fluorescence-based imaging using Thioflavin T (ThT) dye is the gold standard for detecting protein aggregation. It is used to detect aggregation in vitro and in various tissues, including the cerebrospinal fluid (CSF), whereby the disease-related protein recombinant is seeded with the patient's fluid. The major drawback of ThT is its lack of sensitivity to oligomeric forms of protein aggregates. Here, we overcome this limitation by transferring a ThT-oligomer mixture into solid state thin films and detecting fluorescence of ThT amplified in the process of stimulated emission. By monitoring the amplified spontaneous emission (ASE) we achieved a remarkable recognition sensitivity to prefibrillar oligomeric forms of insulin and lysozyme aggregates in vitro, to Aβ42 oligomers in the human protein recombinants seeded with CSF and to Aβ42 oligomers doped into brain tissue. Seeding with Alzheimer patient's CSF containing Aβ42 and Tau aggregates revealed that only Aβ42 oligomers allowed generating ASE. Thus, we demonstrated that, in contrast to the current state-of-the-art, ASE of ThT, a commonly used histological dye, can be used to detect and differentiate amyloid oligomers and evaluate the risk levels of neurodegenerative diseases to potential patients before the clinical symptoms occur.

Laser Emission of Thioflavin T Uncovers Protein Aggregation in Amyloid Nucleation Phase.

Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a ‘reservoir’ of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix pointspread function, the tetrapod point-spread function and singlemolecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

resPAINT: Accelerating Volumetric Super‐Resolution Localisation Microscopy by Active Control of Probe Emission

A review of the current research on in vivo and in vitro detection for alpha-synuclein: a biomarker of Parkinson’s disease

The benzothiazolium salt, Thioflavin T (ThT), has been widely adopted as the "gold-standard" fluorescent reporter of amyloid in vitro. Its properties as a molecular rotor result in a large-scale (∼1000-fold) fluorescence turn-on upon binding to β-sheets in amyloidogenic proteins. However, the complex photophysics of ThT combined with the intricate and varied nature of the amyloid binding motif means these interactions are poorly understood. To study this important class of fluorophores, we present a detailed photophysical characterization and comparison of a novel library of 12 ThT-inspired fluorescent probes for amyloid protein (PAPs), where both the charge and donor capacity of the heterocyclic and aminobenzene components have been interrogated, respectively. This enables direct photophysical juxtaposition of two structural groups: the neutral "PAP" (class 1) and the charged "mPAP" fluorophores (class 2). We quantify binding and optical properties at both the bulk and single-aggregate levels with some derivatives showing higher aggregate affinity and brightness than ThT. Finally, we demonstrate their abilities to perform super-resolution imaging of α-synuclein fibrils with localization precisions of ∼16 nm. The properties of the derivatives provide new insights into the relationship between chemical structure and function of benzothiazole probes.

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Felix Gorka

Papers

A Comparative Photophysical Study of Structural Modifications of Thioflavin T-Inspired Fluorophores