title: The feasibility of Cryo In-SEM Raman microspectroscopy.
abstract: The combination of noninvasive compositional analysis by Raman microspectrometry with high-resolution imaging in the scanning electron microscope greatly expands the analytical capabilities of the electron microscope. However, the chemical preparation of scanning electron microscope (SEM) specimens, although adequate for low-resolution imaging of superficial detail, is not the true representation of the chemistry and composition of the sample, as extraction and aggregation artefacts as a result of dehydrating and cross-linking agents are abundant. The original chemical composition and ultrastructure is only preserved using cryo preparation methods. Therefore, a complete cryo transfer flange was designed and built to add cryogenic control of specimens to the configuration of the EMRAM instrument, a combined Raman spectrometer and XL-30 ESEM instrument. The Raman spectra of two model specimen, polystyrene beads and 2.3M sucrose were studied at ambient and cryogenic temperatures as well as during a heating ramp. Comparing the fingerprint regions of polystyrene and sucrose, both measured at ambient and at cryogenic conditions, only small spectral differences were observed for the main peaks of both molecules. A pronounced sharpening of the bands occurred in the 800-400 cm(-1)  region, a result of the reduction of intermolecular interactions. The enhanced visibility of the lower frequency modes may offer interesting potential for more detailed interpretation of Raman spectra.
authors.affiliation: Unilever R&D Vlaardingen, The Netherlands. Johan.Hazekamp@unilever.com
authors.forename: J
authors.initials: J
authors.lastname: Hazekamp
authors.forename: M G
authors.initials: MG
authors.lastname: Reed
authors.forename: C V
authors.initials: CV
authors.lastname: Howard
authors.forename: A A
authors.initials: AA
authors.lastname: VAN Apeldoorn
authors.forename: C
authors.initials: C
authors.lastname: Otto
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D005615:Freezing; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*