Journal of Applied Microbiology, June 2014, Vol.116(6), pp.1521-1530
To purchase or authenticate to the full-text of this article, please visit this link: http://onlinelibrary.wiley.com/doi/10.1111/jam.12492/abstract Byline: V. Lausch, P. Hermann, M. Laue, N. Bannert Keywords: detection; electron microscopy; tip-enhanced Raman spectroscopy; rapid methods Abstract Aims Successive application of negative staining transmission electron microscopy (TEM) and tip-enhanced Raman spectroscopy (TERS) is a new correlative approach that could be used to rapidly and specifically detect and identify single pathogens including bioterrorism-relevant viruses in complex samples. Our objective is to evaluate the TERS-compatibility of commonly used electron microscopy (EM) grids (sample supports), chemicals and negative staining techniques and, if required, to devise appropriate alternatives. Methods and Results While phosphortungstic acid (PTA) is suitable as a heavy metal stain, uranyl acetate, paraformaldehyde in HEPES buffer and alcian blue are unsuitable due to their relatively high Raman scattering. Moreover, the low thermal stability of the carbon-coated pioloform film on copper grids (pioloform grids) negates their utilization. The silicon in the cantilever of the silver-coated atomic force microscope tip used to record TERS spectra suggested that Si-based grids might be employed as alternatives. From all evaluated Si-based TEM grids, the silicon nitride (SiN) grid was found to be best suited, with almost no background Raman signals in the relevant spectral range, a low surface roughness and good particle adhesion properties that could be further improved by glow discharge. Conclusions Charged SiN grids have excellent particle adhesion properties. The use of these grids in combination with PTA for contrast in the TEM is suitable for subsequent analysis by TERS. Significance and Impact of the Study The study reports fundamental modifications and optimizations of the negative staining EM method that allows a combination with near-field Raman spectroscopy to acquire a spectroscopic signature from nanoscale biological structures. This should facilitate a more precise diagnosis of single viral particles and other micro-organisms previously localized and visualized in the TEM.
Detection ; Electron Microscopy ; Tip‐Enhanced Raman Spectroscopy ; Rapid Methods