High spatial and temporal resolution using upconversion nanoparticles and femtosecond pulsed laser in single particle tracking

Lee, Jinmin; Lee, Hyeryeong; Kang, Minchae; Baday, Murat; Lee, Sang Hak

doi:10.1016/j.cap.2022.09.002

Cited by 6 publications

(4 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Tracking time ranges from tens of minutes up to hours with localization accuracies of a few nm [ 304 ]. A recent study exploited UCNPs to perform single-particle tracking on molecular motors in live cells, achieving 2.4 nm localization accuracy with 2 ms time resolution [ 305 ]. However, several challenges still limit the use of these powerful probes, mainly due to large sizes (UCNPs have sizes of 40–50 nm) and complex surface chemistries.…”

Section: Fluorescent Probes For Single Molecule Microscopymentioning

confidence: 99%

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Spagnolo

Luin

2022

IJMS

View full text Add to dashboard Cite

Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the various issues in fluorophore choice, and discuss how this can become more effective and decisive for increasingly demanding experiments in single- and multiple-color applications.

show abstract

Section: Fluorescent Probes For Single Molecule Microscopymentioning

confidence: 99%

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Spagnolo

Luin

2022

IJMS

View full text Add to dashboard Cite

show abstract

“…68,69 On the instrumental side, other excitation strategies could further maximize the upconversion efficiency, resulting in the collection of more photons and thus allowing for better localization accuracy and/or higher frame rates. 70…”

Section: Discussionmentioning

confidence: 99%

“…Further improvements could also be anticipated by replacing the bulky antibodies (150–180 kDa, ∼15 nm) with much smaller and flexible aptamers (6–30 kDa, ∼2 nm), nanobodies (∼15 kDa, ∼3 nm), or other artificial small protein binders which were successfully conjugated to nanoparticules . Also the large streptavidin–biotin link (60 kDa) could be replaced by an irreversible conjugation using genetically encoded protein tags like the SpyTag/SpyCatcher system (12 kDa) or even a smaller link via click chemistry. , On the instrumental side, other excitation strategies could further maximize the upconversion efficiency, resulting in the collection of more photons and thus allowing for better localization accuracy and/or higher frame rates …”

Section: Discussionmentioning

confidence: 99%

Targeted Single Particle Tracking with Upconverting Nanoparticles

Dukhno,

Ghosh,

Greiner

et al. 2024

ACS Appl. Mater. Interfaces

View full text Add to dashboard Cite

Single particle tracking (SPT) is a powerful technique for real-time microscopic visualization of the movement of individual biomolecules within or on the surface of living cells. However, SPT often suffers from the suboptimal performance of the photon-emitting labels used to tag the biomolecules of interest. For example, fluorescent dyes have poor photostability, while quantum dots suffer from blinking that hampers track acquisition and interpretation. Upconverting nanoparticles (UCNPs) have recently emerged as a promising anti-Stokes luminescent label for SPT. In this work, we demonstrated targeted SPT using UCNPs. For this, we synthesized 30 nm diameter doped UCNPs and coated them with amphiphilic polymers decorated with polyethylene glycol chains to make them waterdispersible and minimize their nonspecific interactions with cells. Coated UCNPs highly homogeneous in brightness (as confirmed by a single particle investigation) were functionalized by immunoglobulin E (IgE) using a biotin−streptavidin strategy. Using these IgE-UCNP SPT labels, we tracked high-affinity IgE receptors (FcεRI) on the membrane of living RBL-2H3 mast cells at 37 °C in the presence and absence of antigen and obtained good agreement with the literature. Moreover, we used the FcεRI-IgE receptorantibody system to directly compare the performance of UCNP-based SPT labels to organic dyes (AlexaFluor647) and quantum dots (QD655). Due to their photostability as well as their backgroundless and continuous luminescence, SPT trajectories obtained with UCNP labels are no longer limited by the photophysics of the label but only by the dynamics of the system and, in particular, the movement of the label out of the field of view and/or focal plane.

show abstract

“…11 On the instrumental side, other excitation strategies could further maximize the upconversion e ciency, resulting in the collection of more photons and thus allowing better localization accuracy and/or higher frame rates. 40…”

Section: Discussionmentioning

confidence: 99%

Targeted single-particle tracking with upconverting nanoparticles

Dukhno

Ghosh

Greiner

et al. 2023

Preprint

View full text Add to dashboard Cite

Single-particle tracking (SPT) is a powerful technique to elucidate the behavior of biomolecules inside or on the surface of living cells in real time. SPT is based on labeling the component of interest with a phosphor or a light-scattering gold nanoparticle, and visualizing its motion by real time microscopy. However, SPT generally suffers from suboptimal performance of phosphors. For example, fluorescent dyes have poor photostability, while quantum dots suffer from sporadic blinking that hampers track acquisition and interpretation. Light-scattering gold nanoparticles require custom experimental setups and relatively large particle size for tracking purposes. In recent years, upconverting nanoparticles (UCNPs) have emerged as a promising phosphor that enables backgroundless anti-Stokes luminescence imaging. They are also attractive for SPT applications due to their extreme photostability and non-blinking luminescence. Previously, SPT has been used to monitor UCNP endocytosis in cells. However, targeted SPT with UCNPs that specifically bind to biomolecules of interest has not yet been achieved. In this work, we provided a first proof-of-concept experiment of targeted SPT with UCNPs and showed the superior performance of UCNPs compared to organic dyes and quantum dots. As a benchmark system to assess the performance of UCNPs, we tracked FcεRI receptors on the surface of RBL-2H3 cell membranes using UCNP-IgE conjugates and obtained good agreement with data from the literature. Importantly, the versatility of the biotin-streptavidin architecture of our system allows UCNPs to be used to track a wide range of targets. Overall, this report lays the foundation for UCNP applications in targeted SPT.

show abstract

High spatial and temporal resolution using upconversion nanoparticles and femtosecond pulsed laser in single particle tracking

Cited by 6 publications

References 28 publications

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Choosing the Probe for Single-Molecule Fluorescence Microscopy

Targeted Single Particle Tracking with Upconverting Nanoparticles

Targeted single-particle tracking with upconverting nanoparticles

Contact Info

Product

Resources

About