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Jun. 01, 2008

FIB/SEM for Biological Data Mining

Cell Focused Ion Beam (FIB) Sectioning for X-ray Analysis - Part
I+II
PART I
Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron
dissection of organs, tissues, cells and subcellular structures of prepared
(i.e. chemically fixed) samples. In combination with a Scanning Electron
Microscope (SEM), FIB makes electron and ion microscopy available at the
same time, for complementary morphological information, that moreover
can be completed by EDS (Energy Dispersive X-ray Spectroscopy). The case
of metal granules in P. scaber hepatopancreas is presented. Part I will be
devoted to FIB sectioning and SEM imaging of the sample. Part II will be
devoted to EDS analysis.
Introduction
Electron microscopy has immensely contributed to the field of biology. Electron
microscopes are getting more and more used not only for imaging surfaces at high
magnification, but also for problem solving in different disciplines. Electron
microscopy has crossed disciplines to a degree that not one single discipline can
claim ownership of this tool. Anatomy, biochemistry, botany, cell biology, forensic
medicine, microbiology, pathology, physiology, toxicology are biological and
biomedical fields in the life sciences area that heavily rely on the electron
microscope. To some degree, all biologically related journals publish electron
micrographs [1, 2]. In electron microscopy, a specimen, when exposed to a high
(0.2-30 KeV) energy electron beam, reacts and simultaneously generates a number
of different signals, although only a few of them are usually recorded at a time.
Secondary Electron Imaging (SEI) is the main method to understand structural
characteristics of specimens (with limitation to depths ranging from 5 to 50 nm
depending on the energy of primary electrons). Emitted backscattered electrons
(electrons having high energy, as those from primary beam, generated by incident
electron collisions with atoms in the specimen and backwards scattered at very low
angles with respect to the incident beam) are used to discriminate sample volumes

with different mean atomic number (high atomic numbered elements give off more
backscattered electrons and therefore appear brighter than lower numbered
elements).

By this method (BEI, Backscattered Electron Imaging) an image is generated that is

more keen to atomic distribution (sample composition) than to morphology.
Elemental characteristic X-rays, associated to the chemical composition of the
rastered area, are captured for sample composition analytical studies (EDS). As
important as the development of the electron microscope itself are various
specialized techniques that are associated with electron microscopy and that have
been developed to prepare and evaluate tissue. The preparation of samples for
biological SEM lagged considerably behind Transmission Electron Microscopy
(TEM) techniques. This is probably due to the fact that SEMs were not
commercially available until 1965, while TEM microscopes entered the market
already in 1939. Major advances in biological knowledge often follow the
introduction of new analytical techniques and this has certainly been the case with
metals in biological systems. The interest to study metals in terms of notion,
pollution or as biocides (insecticide) is constantly present; here electron microscopy
and X-ray microanalysis have a considerable role. Aim of the present paper is to
prove that FIB [3] milling and sectioning is a reliable tool in exposing surfaces
(even polygonal ones) of biological samples [4], as well as it happens since years in
the semiconductor and materials science fields. These sections are of good quality
[5] to allow reliable SEI, BEI and EDS; we therefore hope to facilitate application of
advanced scanning electron microscopy methods in research of metals in biological
systems in a normal physiological state or in an altered one as a result of pollution.
A Case History: TheP. scaber in Structural Research of Cells and Tissues
We investigated digestive glands (hepatopancreas) of a terrestrial isopod
crustacean Porcellio scaber (Isopoda, Crustacea). Hepatopancreas consists of a

single layer epithelium, whose cells are covered by microvilli and face the gland
lumen. It is composed of B- and S-cells. A characteristic of S-cells is to store metals
in granules of homogenous material, which are containing primarily copper and
sulphur. Copper plays an important role in many biochemical processes. These
granules are insoluble in water, alcohol and acetic acid. Animals from
uncontaminated field locations contain 80% of the total copper in the
hepatopancreas. In B-cells, granules of more loosely-bound deposits of flocculent
material in which iron was detected, are present [6]. In studies of metal metabolism
it is necessary to conduct, apart molecular and physiological studies, also structural
analysis of the investigated organ, thus information can be obtained about the
distribution pattern, size, shape and possibly structure of granules. When
concentrations of metals in the food are elevated, the metals are stored in the
hepatopancreas in high amounts. There is an extensive literature on metal
metabolism and storage in the hepatopancreas of isopods, and a lot of interest is
posed on metal metabolism and storage in animals from metal polluted
environments. The most applied methods beside biochemical are TEM, SEM and
light microscopy [6].
Sample Preparation [7, 8] and FIB Sectioning
Four digestive gland tubes (fig. 1) of a terrestrial isopod P. scaber were isolated
and fixed in 0.1% glutaraldehyde and 0.4% paraformaldehyde in 0.1 M sodium
cacodylate buffer (pH 7.2) for 2.5 hours at room temperature. The chemically fixed
samples were dehydrated in a graded series of ethanol, dried at the critical point
(Balzers Critical Point Dryer 030) and carbon coated (Sputter coater SCD 050, BALTEC, Germany). The samples were glued on aluminium stubs with silver paint (High
purity silver paint, SPI ) and then mounted on the sample holder into the specimen
chamber of a Dual Beam system for FIB / SEM operation (FEI Strata DB 235 M and
FEI Quanta 3D). In order to expose the cell region of interest in the gland, a cut
was performed by rough milling along a selected plane, with ion currents ranging
from 5 to 7 nA, at 30 kV. Lower beam currents of 100 to 300 pA were used to polish
the cross section, i.e. to get rid of most of the morphological artifacts associated to
milling operations and at the same time of the majority of redeposited material and
of implanted Gallium. Spot size in the case of rough milling was approximately 150100 nm of diameter, and for polishing it ranged from 20 nm to 35 nm of diameter.
Dwell time for milling was 1 s and the overlap was 50%. Secondary electron
detectors used to track the section status and quality were: Everhardt Thornley
Detector (ETD), Continuous Dynode Electron Multiplier (CDEM) and BackScattered Electron; CDEM was used as secondary ion detector as well. The SEM
imaging was performed by means of a FEG electron column, or by a tungsten

column (examples are seen in figs.1 c,d). The system operated with column

-4

pressures in the 10-5 Pa range and the specimen chamber pressure between 10 -10
-3

Pa.

Imaging is a common step to recognize sample morphology and structure and can
be performed by:
* Secondary Electron Imaging (SEI) to characterize the morphology of the sample
features;
* Back Scattered Electron Imaging (BEI) to enhance the chemical differences in the
area of interest;
* X ray Analysis (EDS) to check the chemical composition.
In this paper we show that all these techniques can rely on preliminary FIB
operations. The X-ray analysis discussion will be postponed to a forthcoming paper.
E-beam Imaging: SE (Secondary Electrons) versus BE (Backscattered
Electrons)
SE are low energy (with less than 50 eV) electrons emitted from the sample.
Because of their low energy, they can only escape from the sample if they are
produced very close to the sample surface with typical escape depth ranging from 5
nm (for a conductor) to 50 nm (for an insulator). Proper collection of secondary
electrons is the basis of SEI.
BE are formed through the interaction (elastic collision) of an electron beam with
the Coulomb field of the atomic nuclei. They will be redirected without energy
losses, and after many of these elastic interactions, they will eventually leave the
sample. The larger the Coulomb field, i.e. the higher the average atomic number,
the more likely a beam electron will interact with it and produce a backscattered
electron. Mapping the abundance of backscattered electrons, BEI will provide
information about the composition of the sample and may identify areas occupied
by domains made of elements with different weight without any chemical analyses.
BEI has lower resolution than SEI because of the depth from which signal can be
produced.
BEI clearly distinguishes the regions on the FIB exposed cell surface that are
composed of elements with high atomic number from the surrounding (fig. 2 a, b).
This is in accordance with X-ray analyses on TEM samples and with histochemical
investigations of isopod hepatopancreas [6]. Since sample was prepared without
metal postfixation or conductive staining, one could expect charging effects due to
sample low conductivity and porous structure. The presence of Ga is likely, because
Ga is the constituent element of the beam used for milling. This will be discussed in
more detail in Part II. Our results indicate that charging was not a problem (fig. 2

b). We therefore observe that primary fixation alone allows investigation of samples
where high amounts of metals are expected, although it is necessary to mention
that such a fixation is not suitable to preserve cell ultrastructure and that material
is somewhat dissolved. When the elemental composition of these granules is of
interest, EDS is a method of choice (see Part II).
References - PART I:
[1] Bozzola J. J., Russell L. D.: Electron Microscopy: Principles and Techniques for
Biologists. Jones and Bartlett Publishers (1998)
[2] Hayat M. A.: Principles and Techniques of Electron Microscopy, Cambridge
University Press (2000)
[3] Milani M., et al.: An atlas of FIB/SEM applications in soft materials and life
sciences, (A05, 16), Aracne (2006)
[4] Ballerini M., et al., Eur J Histochem 4: 89-90 (1997)
[5] Milani M., Imaging & Microscopy 8 (3) 38-40 (2006)
[6] Hopkin S. P. Ecophysiology of Metals in Terrestrial, Elsevier (1989)
[7] Drobne D., et al., J. Biomed. Opt. 9: 1238-1243 (2004)
[8] Drobne D., et al., J. Microsc. 219: 29-35 (2005)

PART II
Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron
dissection of organs, tissues, cells and subcellular structures. In
combination with a Scanning Electron Microscope (SEM), FIB makes
electron and ion microscopy available at the same time, for complementary
morphological information, that moreover can be completed by EDS
(Energy Dispersive X-ray Spectroscopy). In a previous paper [1] we have
shown that Secondary Electron Imaging (SEI) and Back Scattered Electron
Imaging (BEI) allow to recognize sample morphology; these techniques can
rely on preliminary FIB operations (such as FIB sectioning, smoothing and
lamellae formation) that provide specific manipulation of the sample in
order to enhance the out-coming results. Here we discuss X ray analysis
(EDS) to check the chemical composition of FIB exposed digestive gland
cells of a terrestrial isopod Porcellio scaber.
When high energy beam electrons interact with the shell electrons of the specimen
atoms, displacement of inner shell electron results in an electrically unbalanced
atom, leading to shifts of outer-orbital electrons to fill inner shells. As these shifts
take place, energy is often released in the form of characteristic X-rays. The output

of an EDS analysis is an EDX spectrum. The more the element is concentrated in

the specimen, the higher characteristic peaks are in a spectrum, and elements are
identified in an EDX spectrum on the basis of the energy content in the X-rays
emitted by their electrons. A qualitative X-ray analysis is the usual procedure to
search for those elements that are present in a particular location of any kind of
sample. Different operation modes are available; among them spot and map
analysis. As the name suggests, in spot mode the primary beam does not scan over
the area but stays in a single point (spot) for the whole spectrum acquisition time.
Spot mode analysis is used mainly to check the chemical composition of small
features on sample surface, for example inclusions. The area targeted by the
primary beam has the same size as the beam spot (in the sub micron range, down to
a few nanometers). The region that is the source of the X-Ray signal is much larger
than the spot size and it mainly depends on material density and beam energy,
having in the worst case linear sizes up to 10 m both in diameter and depth. This
must be taken into account when interpreting data of a Spot Mode Analysis,
especially when the size of the feature of interest is of the order of a micron or less;
the same consideration applies to X-ray maps as well. Figure 1 shows the X-ray
energy distribution in a signal collected at 15 KV from the section created via FIB
milling (see Fig. 4a of Ref. 1).
X-ray Map
X-ray map is an image showing the spatial distribution of a chosen element in the
field of view. While scanning a frame, corresponding to a matrix of pixels, the beam
stays at each pixel for a certain (dwell) time and produces a certain amount of
characteristic X-rays. When X-rays typical of the selected element are identified, a
bright signal is generated on the screen at the corresponding position whose
intensity is proportional to the intensity of the signal generated. At the end of the
frame the result is a pattern of more or less bright dots corresponding to the
presence of the selected elements (up to 16-32 at once).
Analysis of Bulk Biological Samples
SEM mode operations make it also possible to examine bulk samples (thick slices,
intact tissue, pieces of fractured specimens, fractured surface of the deep-frozen
tissue samples); this however creates some difficulties, and methods are less well
developed than for thin sections. The irregular surface of the specimen is one of the
problems related to X-ray analysis of bulk biological samples; the weight of such a
problem is strongly reduced, as can be seen from maps reported in this paper,
where object of EDS are internal cell sections, whose surface is smooth, having

been obtained by FIB milling.

Elemental analysis has been performed to extract a X-ray spectrum from a gland
cell section created by FIB milling to identify the most abundant elements,
especially metals; actually our aim is to analyze metal granules, identify the
component elements, evaluate their spatial distribution and when possible spatial
correlations among the relevant elements.
The Nova NanoSEM is an Hi Res FE SEM. The electron column has the objective
lens that can be used in "immersion" mode to reduce the lens aberrations and
increase the resolution power. In this mode the sample is fully immersed in the
middle of the generated magnetic field. It can also increase, in EDS analysis, the
emission current (and the beam current). This produces an higher number of
emitted X-rays making the analysis faster and more reliable. The machine reaches a
resolution of 1 nm at 15 kV and is equipped with a 6 inch stage motorized on 5 axis
where X, Y and Rot have piezo-motors for very high accuracy movement.
EDS was performed by EDAX Genesis System whose spatial resolution is dictated
by the X-ray emission law above mentioned.
As a first step we report the spot spectrum (Fig. 1) from one area of Fig.2a rich in
white spots.
Metals are present and significant contributions to the spectrum come also from
Phosphorus and Sulphur.
As a second step, we proceed to a more detailed analysis on the basis of X-ray
mapping and we report an example of operation on a section to get information
about the Ag presence in the sample. As seen in Ref.1, Ag is introduced in the
sample when gluing the sample to the stub by silver paste. It can be seen from Figs.
2 a,b,c that the Ag signal decreases non monotonically from the surface of the
gland towards the lumen; the signal is obviously strongly dependent on geometrical
factors such as beam alignment relative to the sample surface and from surface
rugosity; anyway as it is reasonably expected that the signal from Ag rapidly falls
off as one moves away from the surface towards the inner part of the sample.
A third step is the study of the white spots (as seen by BEI, see for instance Fig. 2
in ref.1) to check whether or not they are actually formed by metals, i.e. they are
metal granules, keeping in mind that Gallium from the ion beam can be an
undesired responsible for metal presence in the section (apart Ag introduced by
gluing).
Therefore in figure 3a we report BEI image of a FIB milled section obtained on the
very same animal object of the investigations in ref.1 and with the same procedure.
Then we compare this image (and the bright spots seen in it) with Gallium
distribution (Fig. 3b) in the same section by map analysis and subsequently we
proceed to the identification of Copper and Zinc presence and distribution (Figs. 4a
and b).

A significant presence of Gallium is seen, as expected, on the top of the gland

(external surface) where the ion beam starts the interaction with the gland and at
the bottom, where most likely Gallium ions are stopped after their many collisions
with the upper part of the gland that subtract the major amount of their energy.
The core of the section, where polishing was effective does not show significant
traces of Gallium thus indicating that implantation and redeposition processes were
counteracted by the selected operation protocols [2,3].
The fourth step in our analysis is devoted to the investigation of distribution of
other elements that are abundant in the spectrum of figure 1, Phosphorus and
Sulphur). We studied their distribution by mapping the surface of the section and as
it can be seen in figures 5a and b, their presence is maximally reported in regions
that overlap with the two major areas of important backscattered signal (white
spots in fig.3a), suggesting a link between P and S and metals of interest ( Cu and
Zn) (Fig.5c, d).
A subsequent step is to focus on the composition of granules; the sparse
distribution of Gallium in the central part of the section (Fig. 3b) is not associated
to any granule of this metal and therefore does not give rise to artefacts in the
interpretation of BEI (see Fig. 6).
A link can be established between the presence of metals such as Cu and Zn and
the white spots of BSE keeping in mind the correlations among the spectra of Cu,
Zn and Ga [4]. In figure 7 it appears that, although Zn and Cu are present in large
amounts in the same regions of the section, they do not concur in the formation of
the same granules, i.e. they are not components of the very same granule.
Of course this is a qualitative approach to the problem of metal granules
composition; for a more informative analysis one actually should consider that the
lines of interest referring to Ga, Zn and Cu have some width and they consequently
overlap [4,5] (this will be the issue in a forthcoming paper).The white spots can be
definitely associated to metal granules, confirming the conclusion driven from
figure 6.
Summary
Focused ion beam (FIB) milling has been proven to be a proper instrument to
section cells for BSE and qualitative X-ray analyses and FIB generated sections
provide good BEI and EDS in biological objects. Ga ions are not contaminating the
surface so much to seriously affect results and their interpretation.
On the basis of data reported in literature, we expected the presence of high
amounts of metal granules in S-cells, primarily made of copper, and of a certain
amount of iron in B-cells. The preliminary results here reported confirm the
presence of Cu in metal granules, significantly associated to Zn, while there is no

evidence of Fe content in B-cells. P and S are non homogeneously distributed in the

section and their concentration is related to the metal one where and when metals
are present.
A critical part of this analysis as it appears from the previous discussion is section
preparation, that is fundamental in determining the volume of interaction that
generates X- rays apart location selection and quality of the section.
Same order of magnitude of the granules size and depth of generation of X-rays
suggest the usefulness of a sample with a thickness lower than 1 micron. FIB role in
preparing quasi - lamellae in situ appears therefore promising (see fig. 8).
References - PART II:
[1] Milani M. et al., Imaging & Microscopy 9 (2) 51-53 (2007).
[2] Drobne D., et al.: Surface damage induced by FIB milling and imaging of
biological samples is controllable, MRT-Microscopy Research and Techniques, 2007
(submitted).
[3] Lehrer C. et al.: Defects and Gallium - Contamination During Focused Ion Beam
Machining, IEEE, 0-7803-6462-7/00, 695-698 (2000)
[4] http://ie.lbl.gov/xray/frame.html: Data collected by Eric Norman and Gregory
Rech.
[5] Statham, P.J., J. Res. Natl. Inst. Stand. Technol. 107, 531-546 (2002).

Authors
Prof. Marziale Milani (corresponding author), Damjana Drobne, Francesco Tatti

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