The feasibility of cryo in-SEM Raman microspectroscopy.
J Hazekamp1, M G Reed1, C V Howard2, A van Apeldoorn3 & C Otto4.
1. Unilever R&D Vlaardingen, The Netherlands
2. School for Bioimaging, University of Ulster, U.K.
3. Dept. developmental bioengineering, MIRA institute for biomedical technology and technical medicine,
University of Twente, the Netherlands
4. MIRA Institute for biomedical technology and technical medicine, University of Twente, The
Keywords: Raman, Cryo-SEM, Microspectroscopy
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 [1, 2, 3, 4]. 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 [5, 6, 7,
8]. Therefore, a complete cryo transfer flange was designed and built to ad 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.
For cryo In-SEM Raman measurements, a 2.3 M sucrose specimen was prepared by placing a
small drop on a Leica specimen stub and was immersed in cold Nitrogen gas for vitrification. The
glass state of the sample was inspected under the optical system of the cryo ultramicrotome (Leica
Ultracut UCT + EM FCS, (Leica Microsystems GmbH, Austria) used for cryo planing the sample
block face. After cryo transfer into the SEM, the collected frost at the sample surface was
extensively sublimed at -85° for 20 minutes to ensure that all frost was removed. Raman spectra
were collected at -150m °C using the EMRAM MCS-A1 and a focused laser spot at 10 seconds
integration time. A small volume of polystyrene beads (diameter 10 µm), for cryogenic experiments,
was dried on a sample stub and cooled during transfer inside the cold stage of the microscope.
Raman spectra were collected at -150m °C using the EMRAM MCS-A1 and a focused laser spot at
10 seconds integration time. The spectra were baseline subtracted using Wire spectral software
(Renishaw plc, United Kingdom).
Figure 1a shows a single polystyrene bead dispersed over a TEM finder grid and imaged under
low temperature conditions (-150 °C). Figure 1d shows a vitrified 2.3 M sucrose solution covering a
relocation grid kept at -150 °C. The spectra depicted in figure 1b & c for polystyrene and figures 1 e
& f for sucrose show the resulting Raman signals collected both at ambient conditions and cooled to
-150 °C. In the fingerprint region the characteristic spectrum of polystyrene was collected both at
ambient and under cryogenic conditions. In the 700 – 400 cm-1 region a series of well resolved
bands can be observed at cryogenic conditions (1c, arrows). The bands are due to wagging modes,
rocking modes and out of plane vibrations, which appear as broader and weaker features at
elevated (room) temperature, due to dynamic broadening effects as a result of the large amount of
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 (figure 1). A pronounced sharpening of the bands however occurred in the 800–400
cm−1 region, a result of the reduction of intermolecular interactions (figure 1 c & f). This enhanced
visibility of the lower frequency modes may offer interesting potential for more detailed interpretation
of Raman spectra. In general, cryo preservation results in an undisturbed chemical and structural
preservation which, when combined with the aforementioned higher specificity of the Raman signal, Download full-text
results in a more accurate correlation of microstructure and molecular composition.
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 A A van Apeldoorn et al, Journal of the Royal Society Interface 2 (2005) p. 39–45.
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 P Echlin, Low-temperature microscopy and analysis. Plenum Press, New York.
Figure 1. In-SEM Raman analysis of polystyrene beads (a,b,c) and of 2.3M sucrose (d, e, f) using a 685 nm
laser in the EMRAM In-SEM Raman instrument. The unprocessed Raman spectra collected at ambient (b/c &
e/f, red curves) and cryogenic (−150◦C, blue curves) conditions. Under low-temperature conditions, the peaks
in the 800–400 cm−1 regions sharpen (c & f, arrows) as a result of the reduction of dynamic interaction.