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The authors present the application of a retarding field between the electron objective lens and sample in an integrated fluorescence and electron microscope. The retarding field enhances signal collection and signal strength in the electron microscope. This is beneficial for samples prepared for integrated fluorescence and electron microscopy as the amount of staining material added to enhance electron microscopy signal is typically lower compared to conventional samples in order to preserve fluorescence. We demonstrate signal enhancement through the applied retarding field for both 80-nm post-embedding immunolabeled sections and 100-nm in-resin preserved fluorescence sections. Moreover, we show that tuning the electron landing energy particularly improves imaging conditions for ultra-thin (50 nm) sections, where optimization of both retarding field and interaction volume contribute to the signal improvement. Finally, we show that our integrated retarding field setup allows landing energies down to a few electron volts with 0.3 eV dispersion, which opens new prospects for assessing electron beam induced damage by in situ quantification of the observed bleaching of the fluorescence following irradiation.
We describe a method for patterning substrates with colloidal particles in any designed two-dimensional structure. By using optical tweezers particles are brought from a reservoir to a surface that carries a surface charge opposite to that of the particles. Using this technique large, two-dimensional patterns can be created, where the pattern can be manipulated on a single particle level. We show that these structures can be dried using critical point drying thus preventing distortions due to surface tension forces. After drying patterned surfaces can be used for further processing, which includes repeating the procedure of patterning. We show some first results of three-dimensional structures created using this layer-by-layer method. The method is generally applicable and has been demonstrated for a variety of (core-shell) colloidal particles including particles that are interesting for photonic applications like high-refractive index (ZnS)-core – silica shell particles, metallodielectric (gold)-core – silica-shell particles, fluorescently labeled particles and small (several nanometers large) gold particles. Particle sizes used range from a few nanometers to several micrometers.
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