Beyond DNA: new probes for PAINT super-resolution microscopy

Tholen, Marrit; Tas, Roderick P.; Wang, Yuyang; Albertazzi, Lorenzo

doi:10.1039/d3cc00757j

Cited by 9 publications

(7 citation statements)

References 71 publications

(94 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This enables miniaturization, because a low numerical aperture objective lens in combination with an affordable (uncooled) camera will still provide a sufficient signal-to-noise ratio. The modular design of this sensor allows adaptation to the detection of cytokines and other proteins using low-affinity probes such as aptamers, antibodies fragments, and peptides. , Competition assays are also easily implemented by employing detection probes that directly bind (with low affinity) to the capture probe, thereby competing with the analyte. SM interactions also provide access to molecular dynamics information allowing for kinetic fingerprinting, hence improved specificity.…”

Section: Discussionmentioning

confidence: 99%

Continuous Monitoring Biosensing Mediated by Single-Molecule Plasmon-Enhanced Fluorescence in Complex Matrices

Lamberti,

Dolci,

Zijlstra

2024

ACS Nano

View full text Add to dashboard Cite

Continuous detection of critical markers directly at the point of interest and in undiluted biological fluids represents the next fundamental step in biosensing. The goal of realizing such a platform is utterly challenging because it requires a reversible biosensor that enables the tracking of picoto nanomolar molecular concentrations over long time spans in a compact device. Here we describe a sensing method based on plasmon-enhanced fluorescence capable of single-molecule detection of unlabeled analyte by employing biofunctionalized gold nanoparticles. The very strong plasmon-enhanced fluorescence signals allow for single-molecule sensing in unaltered biological media, while the use of low-affinity interactions ensures the continuous tracking of increasing and decreasing analyte concentrations with picomolar sensitivity. We demonstrate the use of a sandwich assay for a DNA cancer marker with a limit of detection of picomolar and a time response of 10 min. The enhanced single-molecule signals will allow for miniaturization into a small and cheap platform with multiplexing capability for application in point-of-care diagnostics, monitoring of industrial processes, and safe keeping of the environment.

show abstract

Section: Discussionmentioning

confidence: 99%

Continuous Monitoring Biosensing Mediated by Single-Molecule Plasmon-Enhanced Fluorescence in Complex Matrices

Lamberti,

Dolci,

Zijlstra

2024

ACS Nano

View full text Add to dashboard Cite

show abstract

“…By repeatedly localizing "flashes" from individual fluorophores over time, SMLM has visualized the static and dynamic behavior of self-assembling fibrillar systems and their intermolecular interactions 17,18 . Beyond covalent labeling, bindingactivated blinking 19 of environment-sensitive amyloidophilic dyes can also be used for SMLM.…”

Section: Introductionmentioning

confidence: 99%

“…By repeatedly localizing “flashes” from individual fluorophores over time, SMLM has visualized the static and dynamic behavior of self-assembling fibrillar systems and their intermolecular interactions 17,18 . Beyond covalent labeling, binding-activated blinking 19 of environment-sensitive amyloidophilic dyes can also be used for SMLM. Termed transient amyloid binding (TAB) 20 , a variety of dyes, including p-TFAA 21 , Nile red 22–24 , thioflavin T 20 , thioflavin X 25 , SYPRO orange, and LDS772 26 , can visualize self-assembling fibrillar systems under physiological conditions for hours to days.…”

Section: Introductionmentioning

confidence: 99%

Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy

Zhou,

O’Neill,

Ding

et al. 2023

Preprint

View full text Add to dashboard Cite

Synthetic peptides that self-assemble into cross-β fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers, KFE8Land KFE8D, and the pathological amyloid-beta peptide Aβ42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and Aβ42, are consistent with a helical (bilayer) ribbon structure and convey the precise tilt of the fibrils' inner and outer backbones. SMOLM also finds polymorphic branched and curved morphologies of KFE8 whose backbones exhibit much more heterogeneity than those of more typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross β-rich fibrils.

show abstract

“…SMLM has enabled the imaging of subcellular structures beyond the diffraction limit. SMLM can be further divided into three main families depending on the mechanism of single-molecule fluorescent signal separation (Tholen et al 2023 ): (1) photo-activation/switching based microscopy, including PALM, STORM; (2) dynamic labelling and reversible binding based microscopy exemplified by point accumulation for imaging in nanoscale topography (PAINT) (Giannone et al 2010 ; Sharonov et al 2006 ); and (3) fluorescence lifetime separation based microscopy (Oleksiievets et al 2022 ). Among them, PAINT stands out due to straightforward implementation that does not require special experimental conditions for photoswitching, as long as probes possess the capability to diffuse and reach their target structures.…”

Section: Introductionmentioning

confidence: 99%

Deep learning-enabled fast DNA-PAINT imaging in cells

Zhu,

Zhang,

Jin

et al. 2023

swwlxb

View full text Add to dashboard Cite

DNA-based point accumulation in nanoscale topography (DNA-PAINT) is a well-established technique for single-molecule localization microscopy (SMLM), enabling resolution of up to a few nanometers. Traditionally, DNA-PAINT involves the utilization of tens of thousands of single-molecule fluorescent images to generate a single super-resolution image. This process can be time-consuming, which makes it unfeasible for many researchers. Here, we propose a simplified DNA-PAINT labeling method and a deep learning-enabled fast DNA-PAINT imaging strategy for subcellular structures, such as microtubules. By employing our method, super-resolution reconstruction can be achieved with only one-tenth of the raw data previously needed, along with the option of acquiring the widefield image. As a result, DNA-PAINT imaging is significantly accelerated, making it more accessible to a wider range of biological researchers.

show abstract

Beyond DNA: new probes for PAINT super-resolution microscopy

Abstract: In the last decade, point accumulation for imaging in nanoscale topography (PAINT) has emerged as a versatile tool for single-molecule localization microscopy (SMLM). Currently, DNA-PAINT is the most widely used,...

Cited by 9 publications

References 71 publications

Continuous Monitoring Biosensing Mediated by Single-Molecule Plasmon-Enhanced Fluorescence in Complex Matrices

Continuous Monitoring Biosensing Mediated by Single-Molecule Plasmon-Enhanced Fluorescence in Complex Matrices

Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy

Deep learning-enabled fast DNA-PAINT imaging in cells

Contact Info

Product

Resources

About