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

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.


Synthesis of pure silica nanoparticles (Me_series). Synthesis of pure silica
nanoparticles using L-arginine as a base catalyst was conducted as described in the literature [1]. In a typical experiment, 91 mg (0.52 mmol) L-arginine was dissolved in 69 mL of water. After addition of 4.5 mL of cyclohexane, the biphasic mixture was heated to 60 °C under stirring. Particle formation was induced by addition of 5.5 mL (28.1 mmol) TEOS and further heating for 20 h. Then the organic cyclohexane phase was separated from the aqueous particle suspensions.

Synthesis of pure silica nanoparticles by the Stöber method (Stoe15 and
Stoe60). Undoped silica nanoparticles were synthesised by the Stöber method [2].
To obtain small silica nanoparticles with a mean particle size of 15 nm, 0.41 mL of water, 23.57 mL of ethanol and 0.50 mL of ammonia (25%) were mixed. In a next step 0.554 mL (2.5 mmol) of TEOS was added and the reaction mixture was stirred for 2 d at room temperature. For larger silica particles with a mean diameter of 60 nm, the precursor ratio was changed to 0.41 mL of water, 22.55 mL of ethanol, 1.04 mL of ammonia and 1.05 mL (4.7 mmol) of TEOS.
Synthesis of pure silica nanoparticles using the C-dot method (CD). Pure C-dots were synthesised following the method described by Herz et al. [3]. 0.28 mL (1.3 mmol) of TEOS was added to a mixture of 21.75 mL ethanol, 0.39 mL of water, 2.5 mL of ammonia (2.0 M in ethanol). Then the reaction mixture was stirred for 12 h at room temperature to obtain the particle cores. Addition of further TEOS aliquots to the core solution and stirring of the reaction mixture for additional 8 h formed a pure S3 silica shell around the cores. To avoid secondary nucleation 0.4 mL (1.8 mmol) of TEOS was added stepwise over a maximum period of 300 min (20 µL / 15 min).

Synthesis of dyed silica nanoparticles
Synthesis of 25 nm large, dyed Star635 silica nanoparticles (FD25_Star635). To determine the influence of the dye concentration on the particle growth, Star635          Size and dispersity were similar for Dy-647 and Dy-648 dyes which carry one or two sulphonate groups, respectively. Smaller particles were obtained by introduction of Dy649 dye. A first regrowth step of (b) yielded particles with a mean diameter of d SEM = 60 ± 8 nm (13%) (d) that could be regrown to 80 ± 8 nm (10%) large particles (e). The graph (f) shows the mean particle diameter and size deviation obtained from SEM image analysis and logNormal fitting of the histograms. All scale bars are 300 nm.       The dye leaching was determined by comparing the relative fluorescence intensity before and after centrifugation of the particle suspension through two different membranes with molecular weight cut off (MWCO) of 30 kDa and 100 kDa. The measurements revealed that there was nearly no dye leaching in the particle suspensions.    Figure S22: STED imaging of A549 cells incubated with 10 µg/mL FD25_Star635 (A) or FD25_Atto647N (D) for 24 h. The actin cytoskeleton is depicted in green, the cis-Golgi network in cyan and silica nanoparticles in magenta. Particles were imaged in STED mode (B and E), cellular structures in confocal mode and recorded z-stacks were deconvolved. C and F show an intensity plot through a sample nanoparticle (dotted line) within the cell. The full width of half maximum (FWHM) was 88 or 85 nm, which is below the classical optical resolution limit. Brightness and contrast of images was adjusted for better visualization using ImageJ.