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

Abstract

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 largescale (~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 fluorophore, we present a detailed photophysical characterisation and comparison of a novel library of twelve 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; Class 1 the neutral 'PAP' and Class 2 the charged 'mPAP' fluorophores. We quantify binding and optical properties at both the bulk and singleaggregate levels with some derivatives showing higher aggregate affinity and brightness than ThT. Finally, we demonstrate their abilities to perform super-resolution imaging of αsynuclein fibrils with localisation precisions of ~16 nm. The properties of the derivatives provide new insights into the relationship between chemical structure and function of benzothiazole probes.

Versatile Near‐Infrared Super‐Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD‐2

“…For SMLM, stacks of 6,000 images with 50 ms exposure per frame were recorded. Super‐resolution images were reconstructed in ImageJ using the PeakFit package (GDSC SMLM, University of Sussex) as described elsewhere [19,20] with minor changes, using a Gaussian spot filter, signal strength=20, minimum photon count=50, precision=100. The resolution of the super‐resolved images was determined using the Fourier ring correlation GDSC SMLM package with a defined threshold of 1/7 [40] …”

“…Alternatively, recent super‐resolution imaging reports have exploited the ability of most amyloid probes to transiently bind to amyloid structures and fluoresce strongly only when bound. The fluorogenic properties and the reversible binding favors both the rapid exchange of photobleached labels enabling prolonged STED imaging of amyloids [14,15] as well as the generation of blinking events that can be analyzed for SMLM [16–20] . However, the fluorophores explored so far have limited application for in vivo studies, as they suffer from either low specificity and/or affinity to amyloid aggregates, poor penetration of the blood‐brain barrier (BBB), or display short emission wavelengths.…”

“…The fluorogenic properties and the reversible binding favors both the rapid exchange of photobleached labels enabling prolonged STED imaging of amyloids [14,15] as well as the generation of blinking events that can be analyzed for SMLM. [16][17][18][19][20] However, the fluorophores explored so far have limited application for in vivo studies, as they suffer from either low specificity and/or affinity to amyloid aggregates, poor penetration of the blood-brain barrier (BBB), or display short emission wavelengths. In this regard, near-infrared (NIR) fluorophores are advantageous given the minimal absorption and scattering of light between 650 and 900 nm by tissue, resulting in low autofluorescence and deeper light penetration.…”

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

“…anti-hCD45-Fab off rate imaging and calculation: T cells were prepared and adhered to a coverslip using PLL as for apical surface imaging before incubation with 200 nM of anti-hCD45-Fab-SiR (Gap8.3-Fab-SiR) for 15 minutes. Imaging was performed on a bespoke microscope as in previous work 66 using a 641 nm excitation laser (Obis, Coherent). The beam was filtered with an appropriate excitation bandpass filter (FF01-640/14-25, Semrock) and circularly polarised using a wavelength specific quarterwave plate.…”

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

“…Small fluorescent dye Thioflavin T (ThT) drastically increases its fluorescence intensity upon binding to amyloid fibrils and is the gold-standard tool for fibril detection and investigation of the fibrillization kinetics in solutions. , Several ThT analogues with improved optical properties − as well as other solvatochromic probes , were recently developed and can be efficiently used in solution and tissue systems. However, their application in living cells is limited by too strong background signal that resulted from off-target binding.…”