Environmental Engineering Reference
In-Depth Information
proper samples can directly address the problem of the origin of a pathogenic
pollution.
10.2 The Technique
It is particularly useful to have a technique that preserves the sample and allows the
investigation to be repeatable, but it is mandatory that the technique or the preser-
vation process do not add laboratory pollution.
The pieces of equipment we use to observe our specimens are environmental
scanning electron microscopes (ESEMQuanta 200 or FEG ESEMQuanta 250, both
by FEI Company, The Netherlands). The choice is due to their possibility to work
not in low vacuum the way SEMs do, but, thanks to a particular air trap placed in the
column, at environmental conditions, so that no preparation of the sample (dehy-
dration, Carbon or Gold/Palladium coating to make the sample electroconductive)
is necessary. This is a measure to prevent the formation of artifacts.
An EDS microprobe (Energy Dispersion System by EDAX, USA) equipping the
ESEM collects the X-rays generated by the sample
s surface when hit by the
electron beam, sorts and plots them by energy, and allows to identify the elements,
each of which produces peaks characteristic to that particular element in this energy
distribution. Suitable software identifies the elemental composition of the materials
imaged of all elements with an atomic number greater than Boron
'
s. Most elements
'
can be detected at concentrations of the order of 0.1 %.
For the morphological observations and for the chemical analyses the sample is
placed on an Aluminum stub which supports an adhesive Carbon substrate whose
contribution in terms of Carbon and Oxygen does not affect the analytical result,
since the particles to be studied are inorganic.
The samples can be observed in different modalities: in low vacuum, from 20 to
30 kV, in secondary and in backscattered electron mode, with spots from 3 to 5. The
most common mode of detection is by secondary electrons emitted by the atoms of
the sample excited by the electron beam. That way the surface of the specimen is
shown. Backscattered electrons are electrons emitted by the beam that are reflected
from the sample. The intensity of that signal is strongly related to the atomic
number of the element. The backscattered mode allows to see materials with a
higher atomic density than the biological matrix that contains them.
The analyses on biological specimens we usually carry out are either on fresh or
on preserved materials. Common fresh samples are fluids like blood, sperm, and
sweat, while preserved ones, generally in paraffin blocks, are tissues coming from
postmortem or biopsic takings. When the sample relates to an infectious disease, a
fixation in formalin is performed beforehand. Unlike fresh samples that need no
special preparation, those stored in paraffin blocks must be cut into 5-10-micron
sections with a microtome and freed from paraffin with xylol. For the various
preparation steps, please see the topic Nanopathology [
1
].
