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

Isabella Tavernaro,Henrike Peuschel,Christian Cavelius,Annette Kraegeloh

doi:10.3762/bjnano.8.130

Isabella Tavernaro, Henrike Peuschel + Show 2 more

Open Access

https://doi.org/10.3762/bjnano.8.130

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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.

Highlights

With the emergence of nanotechnology, numerous applications have been developed using the size-related properties of nanoparticles [1,2]
Our main goal was the synthesis of monodisperse silica nanoparticles in the size range of 15 to 80 nm and their modification with fluorescent NIR dyes to enable stimulated emission depletion (STED) and confocal imaging
We adopted a literature-known synthesis of silica nanoparticles using an L-arginine-catalysed hydrolysis of tetraethoxysilane (TEOS) in a biphasic water–cyclohexane system [49] and incorporated different fluorescent NIR dyes into the silica particle matrix

Summary

Introduction

With the emergence of nanotechnology, numerous applications have been developed using the size-related properties of nanoparticles [1,2]. We present the improved syntheses of the covalently incorporated (fully dyed) fluorescent silica nanoparticles in more detail by describing the syntheses of the dye–APTES conjugates, the different synthesis steps, a comparison with commonly used methods and variations of the used dyes. The shape of the 15 nm large silica nanoparticles, obtained by the Stöber synthesis and the C-dots method, are more irregular and less spherical than FD15_Atto647N (Figure S15, Supporting Information File 1).

Results

Conclusion