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

Torra, Joaquim; Viela, Felipe; Megı́as, Diego; Sot, Begoña; Flors, Cristina

doi:10.1002/chem.202200026

Cited by 11 publications

(6 citation statements)

References 42 publications

(86 reference statements)

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“…For in vitro protein systems, Nile Red fluorescence switch-on and spectral blueshifts often occur at amyloid fibrils. ,, To examine this possibility, we utilized CRANAD-2 (Figure a), a far-red fluorogenic probe with high affinity and specificity to amyloid fibrils. , A recent study has demonstrated CRANAD-2 for the SMLM of α-synuclein fibrils, in which the stochastic amyloid binding of individual CRANAD-2 molecules gives bursts of single-molecule fluorescence.…”

Section: Resultsmentioning

confidence: 99%

Multidimensional Super-Resolution Microscopy Unveils Nanoscale Surface Aggregates in the Aging of FUS Condensates

He,

Wu,

et al. 2023

J. Am. Chem. Soc.

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The intracellular liquid−liquid phase separation (LLPS) of biomolecules gives rise to condensates that act as membrane-less organelles with vital functions. FUS, an RNAbinding protein, natively forms condensates through LLPS and further provides a model system for the often disease-linked liquidto-solid transition of biomolecular condensates during aging. However, the mechanism of such maturation processes, as well as the structural and physical properties of the system, remains unclear, partly attributable to difficulties in resolving the internal structures of the micrometer-sized condensates with diffractionlimited optical microscopy. Harnessing a set of multidimensional super-resolution microscopy tools that uniquely map out local physicochemical parameters through single-molecule spectroscopy, here, we uncover nanoscale heterogeneities in FUS condensates and elucidate their evolution over aging. Through spectrally resolved single-molecule localization microscopy (SR-SMLM) with a solvatochromic dye, we unveil distinct hydrophobic nanodomains at the condensate surface. Through SMLM with a fluorogenic amyloid probe, we identify these nanodomains as amyloid aggregates. Through single-molecule displacement/diffusivity mapping (SMdM), we show that such nanoaggregates drastically impede local diffusion. Notably, upon aging or mechanical shears, these nanoaggregates progressively expand on the condensate surface, thus leading to a growing low-diffusivity shell while leaving the condensate interior diffusion-permitting. Together, beyond uncovering fascinating structural arrangements and aging mechanisms in the single-component FUS condensates, the demonstrated synergy of multidimensional super-resolution approaches in this study opens new paths for understanding LLPS systems at the nanoscale.

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Section: Resultsmentioning

confidence: 99%

Multidimensional Super-Resolution Microscopy Unveils Nanoscale Surface Aggregates in the Aging of FUS Condensates

He,

Wu,

et al. 2023

J. Am. Chem. Soc.

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“…A further development is the re‐design of Nile Red, rendering it sensitive to polarity, which allowed long‐time live‐cell super‐resolution microscopy of endocytosis events [11] . Another target accessible with exchangeable small‐molecule labels are amyloid fibers, which are bound by Thioflavin T [12] or the curcumin derivative CRANAD‐2 [13] …”

Section: Types Of Low‐affinity Labels Underlying Molecular Interactio...mentioning

confidence: 99%

“…[11] Another target accessible with exchangeable small-molecule labels are amyloid fibers, which are bound by Thioflavin T [12] or the curcumin derivative CRANAD-2. [13] A second group are molecules that consist of a lowaffinity target-binding unit conjugated to a fluorophore. This group comprises, among others, conjugates of the DNA minor groove binder Hoechst with fluorophores, which were used for both PAINT [14] and STED microscopy; [2] fluorophore-peptide labels, such as the Lifeact peptide that targets cellular actin [15] and fluorophore-aptamer conjugates.…”

Section: Types Of Low-affinity Labels Underlying Molecular Interactio...mentioning

confidence: 99%

When Weak Is Strong: A Plea for Low‐Affinity Binders for Optical Microscopy

Albertazzi

Heilemann

2023

Angew Chem Int Ed

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The exploitation of low‐affinity molecular interactions in protein labeling is an emerging topic in optical microscopy. Such non‐covalent and low‐affinity interactions can be realized with various concepts from chemistry and for different molecule classes, and lead to a constant renewal of fluorescence signals at target sites. Further benefits are a versatile use across microscopy methods, in 3D, live and many‐target applications. In recent years, several classes of low‐affinity labels were developed and a variety of powerful applications demonstrated. Still, this research field is underdeveloped, while the potential is huge.

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“…We and others − have recently showed that super-resolution and AFM can be performed correlatively to provide contextual information via topographical mapping to target structures imaged under super-resolution. Various super-resolution techniques have been combined with AFM, including structured illumination microscopy (SIM) and stimulated emission depletion microscopy (STED) .…”

Section: Introductionmentioning

confidence: 99%

Live-Cell SOFI Correlation with SMLM and AFM Imaging

Hargreaves

Duwé

Rozario

et al. 2023

ACS Bio Med Chem Au

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Standard optical imaging is diffraction-limited and lacks the resolving power to visualize many of the organelles and proteins found within the cell. The advent of super-resolution techniques overcame this barrier, enabling observation of subcellular structures down to tens of nanometers in size; however these techniques require or are typically applied to fixed samples. This raises the question of how well a fixed-cell image represents the system prior to fixation. Here we present the addition of live-cell Super-Resolution Optical Fluctuation Imaging (SOFI) to a previously reported correlative process using Single Molecule Localization Microscopy (SMLM) and Atomic Force Microscopy (AFM). SOFI was used with fluorescent proteins and low laser power to observe cellular ultrastructure in live COS-7 cells. SOFI-SMLM-AFM of microtubules showed minimal changes to the microtubule network in the 20 min between live-cell SOFI and fixation. Microtubule diameters were also analyzed through all microscopies; SOFI found diameters of 249 ± 68 nm and SMLM was 71 ± 33 nm. AFM height measurements found microtubules to protrude 26 ± 13 nm above the surrounding cellular material. The correlation of SMLM and AFM was extended to two-color SMLM to image both microtubules and actin. Two target SOFI was performed with various fluorescent protein combinations. rsGreen1-rsKAME, rsGreen1-Dronpa, and ffDronpaF-rsKAME fluorescent protein combinations were determined to be suitable for two target SOFI imaging. This correlative application of super-resolution live-cell and fixed-cell imaging revealed minimal artifacts created for the imaged target structures through the sample preparation procedure and emphasizes the power of correlative microscopy.

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

Cited by 11 publications

References 42 publications

Multidimensional Super-Resolution Microscopy Unveils Nanoscale Surface Aggregates in the Aging of FUS Condensates

Multidimensional Super-Resolution Microscopy Unveils Nanoscale Surface Aggregates in the Aging of FUS Condensates

When Weak Is Strong: A Plea for Low‐Affinity Binders for Optical Microscopy

Live-Cell SOFI Correlation with SMLM and AFM Imaging

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