https://pure.mpg.de/pubman/item/item_1795834_7/component/file_1795841/1795834.pdf

Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution

Water-soluble organic fluorophores are widely used as labels in biological systems. However, in many cases these fluorophores can interact strongly with lipid bilayers, influencing the interaction of the target with the bilayer and/or leading to misleading fluorescent signals. Here, we quantify the interaction of 32 common water-soluble dyes with model lipid bilayers to serve as an additional criterion when selecting a dye label.

/pdf/choose-your-label-wisely-water-soluble-fluorophores-often-4ylmpfxn8y.pdf

Choose your label wisely: water-soluble fluorophores often interact with lipid bilayers.

Fluorescence bioimaging is a powerful, versatile, method for investigating, both in vivo and in vitro, the complex structures and functions of living organisms in real time and space, also using super-resolution techniques. Being poorly invasive, fluorescence bioimaging is suitable for long-term observation of biological processes. Long-term detection is partially prevented by photobleaching of organic fluorescent probes. Semiconductor quantum dots, in contrast, are ultrastable, fluorescent contrast agents detectable even at the single nanoparticle level. Emission color of quantum dots is size dependent and nanoprobes emitting in the near infrared (NIR) region are ideal for low back-ground in vivo imaging. Biocompatibility of nanoparticles, containing toxic elements, is debated. Recent safety concerns enforced the search for alternative ultrastable luminescent nanoprobes. Most recent results demonstrated that optimized silicon quantum dots (Si QDs) and fluorescent nanodiamonds (FNDs) show almost no photobleaching in a physiological environment. Moreover in vitro and in vivo toxicity studies demonstrated their unique biocompatibility. Si QDs and FNDs are hence ideal diagnostic tools and promising non-toxic vectors for the delivery of therapeutic cargos. Most relevant examples of applications of Si QDs and FNDs to long-term bioimaging are discussed in this review comparing the toxicity and the stability of different nanoprobes.

Nanodiamonds and silicon quantum dots: ultrastable and biocompatible luminescent nanoprobes for long-term bioimaging

We review the use of luminescent nanoparticles in super-resolution imaging and single-molecule tracking, and showcase novel approaches to super-resolution imaging that leverage the brightness, stability, and unique optical-switching properties of these nanoparticles. We also discuss the challenges associated with their use in biological systems, including intracellular delivery and molecular targeting. In doing so, we hope to provide practical guidance for biologists and continue to bridge the fields of super-resolution imaging and nanoparticle engineering to support their mutual advancement.

Nanoparticles for super-resolution microscopy and single-molecule tracking.

Despite its short history, diffraction-unlimited fluorescence microscopy techniques have already made a substantial imprint in the biological sciences. In this review, we describe how stimulated emission depletion (STED) imaging originally evolved, how it compares to other optical super-resolution imaging techniques, and what advantages it provides compared to previous golden-standards for biological microscopy, such as diffraction-limited optical microscopy and electron microscopy. We outline the prerequisites for successful STED imaging experiments, emphasizing the equally critical roles of instrumentation, sample preparation, and photophysics, and describe major evolving strategies for how to push the borders of STED imaging even further in life science. Finally, we provide examples of how STED nanoscopy can be applied, within three different fields with particular potential for STED imaging experiments: neuroscience, plasma membrane biophysics, and subcellular clinical diagnostics. In these areas, and...

Stimulated Emission Depletion Microscopy

Exploring the maximum spatial resolution achievable in far-field optical imaging, we show that applying solid immersion lenses (SIL) in stimulated emission depletion (STED) microscopy addresses single spins with a resolution down to 2.4 ± 0.3 nm and with a localization precision of 0.09 nm.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/adma.201203033

Solid immersion facilitates fluorescence microscopy with nanometer resolution and sub-ångström emitter localization.

In optical microscopy, most red-emitting dyes provide only moderate performance due to unspecific binding, poor labeling efficiency, and insufficient brightness. Here we report on four novel red fluororescent dyes, including the first phosphorylated dye, created by combining a rigidized rhodamine backbone with various polar groups. They exhibit large fluorescence quantum yields and improved NHS ester stability. While these fluorophores are highly suitable for fluorescence microscopy in general, they excel in stimulated emission depletion (STED) microscopy, providing < 25 nm spatial resolution in raw images of cells.

/pdf/novel-red-fluorophores-with-superior-performance-in-sted-4c90mso40p.pdf

Heiko Schill

Papers

Solid immersion facilitates fluorescence microscopy with nanometer resolution and sub-ångström emitter localization.

Novel red fluorophores with superior performance in STED microscopy