Characterization via Two-Color STED Microscopy of Nanostructured Materials Synthesized by Colloid Electrospinning

Abstract

A model system for multicompartment nanofibers was fabricated by colloid electrospinning. The obtained nanostructured material consisted of fluorescent polymer nanoparticles that were synthesized in a miniemulsion and then embedded in fluorescently labeled polymer nanofibers. Because of the absence of contrast between both polymers, the immobilized nanoparticles cannot be reliably identified in the nanofibers via electron microscopy or other techniques. Here, we describe investigations on the hybrid material with two-color STED microscopy to localize the nanoparticles and to quantify their distribution along nanofibers with particle and fiber radii down to 50 nm.

Lens-based fluorescence nanoscopy

“…Blom et al 2011;Kellner et al 2007;Kittel et al 2006;Lau et al 2012;Muller et al 2012;Opazo et al 2012;Persson et al 2011;Schmidt et al 2008Schmidt et al , 2009Sieber et al 2007;Wagner et al 2012), making a STED microscope a uniquely helpful tool nowadays in cell-biological laboratories (Clausen et al 2013). Furthermore, STED has important applications outside biology, ranging from nanoscale imaging of assemblies of colloidal particles and polymeric structures (Friedemann et al 2011;Harke et al 2008b;Ullal et al 2009Ullal et al , 2011 to solid-state physics . With its capabilities and simplifications steadily growing, as outlined further on, and commercial instrumentation improving (Clausen et al 2013), STED nanoscopy may become a workhorse of imaging facilities, greatly extending the resolving power of confocal microscopes.…”

“…Therefore, initial two-color STED images were recorded sequentially. This limitation has been solved by straightforward optimizations of the choices of labels and wavelengths: (1) the combination of two labels with overlapping emission spectra and with a long (Stokes) shift between the excitation and emission spectrum of one of the labels allows the recording of two-color STED images with two excitation lasers but only one STED laser (serving both labels) (Clausen et al 2013;Dean et al 2012;Friedemann et al 2011;Pellett et al 2011;Schmidt et al 2008). (Quasi-) simultaneous recording of both colors is possible in either a line-by-line or a pulse-interleaved excitation scheme, in both cases rapidly switching between the two excitation lasers.…”

“…CW-STED imaging was first realized with strong Argon-Krypton lasers or Ti:Sa lasers running in CW mode (Ding et al 2009;Harke, 2008), but since then it has been shown that it is possible to utilize compact fibre lasers Moneron & Hell, 2009;Moneron et al 2010), diode-pumped solid-state (DPSS) lasers Mueller et al 2012) or amplified diode lasers (Honigmann et al 2013a) at wavelengths between 560 and 765 nm, also on commercial systems (Clausen et al 2013;Friedemann et al 2011). However, CW-STED comes with two drawbacks compared to the pulsed STED modality.…”

Pathways to optical STED microscopy

“…Initially realized with strong Argon-Krypton lasers [49] or Ti:Sa lasers running in CW mode [50,51], CW-STED microscopy has now been demonstrated with compact fibre lasers [52][53][54] or diode-pumped solid-state (DPSS) lasers [55,56], on a commercial system [57]. However, the performance of CW-STED nanoscopy lacks behind that using the pulsed STED modality.…”

“…This usually requires the use of moleculespecific tags with different fluorescence emission properties such as well-separated emission wavelength ranges or colors. A preferred approach for multi-color STED imaging is the use of two labels with overlapping emission spectra but with a long (Stokes) shift between the excitation and emission spectrum of one of the labels allowing the recording of two-color images with two excitation but only one STED laser (serving both labels) [57,80,82,83]. This avoids the supply of a multitude of additional lasers (strictly speaking one STED laser for each label) and the cross-photobleaching of the more red-emitting dyes by the blue-shifted STED lasers, previously only allowing a onetime subsequent recording of two colors [84,85].…”