Bright fluorescent silica-nanoparticle probes for high-resolution STED and confocal microscopy

Abstract

In recent years, fluorescent nanomaterials have gained high relevance in biological applications as probes for various fluorescence-based spectroscopy and imaging techniques. Among these materials, dye-doped silica nanoparticles have demonstrated a high potential to overcome the limitations presented by conventional organic dyes such as high photobleaching, low stability and limited fluorescence intensity. In the present work we describe an effective approach for the preparation of fluorescent silica nanoparticles in the size range between 15 and 80 nm based on L-arginine-controlled hydrolysis of tetraethoxysilane in a biphasic cyclohexane–water system. Commercially available far-red fluorescent dyes (Atto647N, Abberior STAR 635, Dy-647, Dy-648 and Dy-649) were embedded covalently into the particle matrix, which was achieved by aminosilane coupling. The physical particle attributes (particle size, dispersion, degree of agglomeration and stability) and the fluorescence properties of the obtained particles were compared to particles from commonly known synthesis methods. As a result, the spectroscopic characteristics of the presented monodisperse dye-doped silica nanoparticles were similar to those of the free uncoupled dyes, but indicate a much higher photostability and brightness. As revealed by dynamic light scattering and ζ-potential measurements, all particle suspensions were stable in water and cell culture medium. In addition, uptake studies on A549 cells were performed, using confocal and stimulated emission depletion (STED) microscopy. Our approach allows for a step-by-step formation of dye-doped silica nanoparticles in the form of dye-incorporated spheres, which can be used as versatile fluorescent probes in confocal and STED imaging.

Advances in highly doped upconversion nanoparticles

“…This doping process requires a rational design that offers optimal interactions of a network of the sensitizer and activator ions, and the upconversion efficiency is highly dependent on the separating distance between the dopants. Therefore, the proper management of the doping concentration in a given nanoparticle will be the deciding factor in leveraging the energy transfer process and ultimately the luminescence performance of the nanoparticle 9 – 11 .…”

Silica Nanoparticles for Intracellular Protein Delivery: a Novel Synthesis Approach Using Green Fluorescent Protein

“…This study aims at functionalising silica nanoparticles with GFP under suitable conditions that maintain the biochemical characteristics and functionality of the protein. In previous work, we synthesised near IR dye-doped monodisperse fluorescent silica nanoparticles in the size range between 15 and 80 nm, using a ʟ-arginine controlled hydrolysis of tetraethoxysilane (TEOS) in a biphasic cyclohexane/water system [ 26 ]. Here, we have adopted this synthesis procedure to embed GFP, as a model protein, into the silica matrix.…”

A simple empirical algorithm for optimising depletion power and resolution for dye and system specific STED imaging

“…In general, anything that alters absorption or scattering in the sample (or, like bead index, changes the lifetime of the probe) may affect P STED . Although there are many sites which give helpful information about the ‘best’ STED fluorophores (https://nanobiophotonics.mpibpc.mpg.de/dyes/, https://www.leica-microsystems.com/science-lab/quick-guide-to-sted-sample-preparation/, https://www.abberior-instruments.com/site/assets/files/2190/recommended_fluorescent_markers_for_sted_by_abberior_instruments-1.pdf) and new STED probes are being developed (Hense et al ., ; Maksim et al ., ; Rosales et al ., ; Butkevich et al ., ; Byrne et al ., ; Laporte & Psaltis, ; Butkevich et al ., ; Erdmann et al ., ; Tavernaro et al ., ), it is important to know how the dye is working locally on a given nanoscope.…”