A Comparative Study of High-Contrast Fluorescence Lifetime Probes for Imaging Amyloid in Tissue.

Optical imaging of protein aggregates in living and post-mortem tissue can often be impeded by unwanted fluorescence, prompting the need for novel methods to extract meaningful signal in complex biological environments. Historically, benzothiazolium derivatives, prominently Thioflavin T, have been the state-of-the-art fluorescent probes for amyloid aggregates, but their optical, structural, and binding properties typically limit them to in vitro applications. This study compares the use of novel uncharged derivative, PAP_1, with parent Thioflavin T as a fluorescence lifetime imaging probe. This is applied specifically to imaging recombinant α-synuclein aggregates doped into brain tissue. Despite the 100-fold lower brightness of PAP_1 compared to that of Thioflavin T, PAP_1 binds to α-synuclein aggregates with an affinity several orders of magnitude greater than Thioflavin T; thus, we observe a specific decrease in the fluorescence lifetime of PAP_1 bound to α-synuclein aggregates, resulting in a separation of >1.4 standard deviations between PAP_1-stained brain tissue background and α-synuclein aggregates that is not observed with Thioflavin T. This enables contrast between highly fluorescent background tissue and amyloid fibrils that is attributed to the greater affinity of PAP_1 for α-synuclein aggregates, avoiding the substantial off-target staining observed with Thioflavin T.

From sieve to microscope: An efficient technique for sample transfer in the process of microplastics' quantification.

In the field of microplastics' quantification, efficient and reproducible methodology is still needed. Procedures of sample fractionation and transfer are often insufficiently reported, although fractionating a sample in similarly sized particles is a crucial prerequisite for the subsequent detection and identification process. At the same time, fractionation is error-prone as particles can be lost during transfer between different vessels. This article presents a four-step technique of sample preparation and microscopic examination, suited for different kind of environmental samples (e.g., water, sediment, soil): The sample is size-fractionated in a sieve cascade (I), rinsed from the sieve and vacuum-filtrated onto a filter (II), rinsed from the filter into a glass petri dish with a low amount of water (III), and examined under the microscope in wet or dry condition (IV). The technique manages on standard laboratory equipment and is reliable for fragments > 300 µm: In a validation experiment with polypropylene, the average recovery was 94 ± 13.5% (arithmetic mean ± standard deviation) and 100% (median), respectively.•Reliable sample transfer after wet-sieving.•Concentration of the pretreated sample in a very small amount of water.•Usage of transmitted light in microscopy.

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

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 ß-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.