Journal of Microscopy, Vol. 188, Pt 1, October 1997, pp. 7278.

Received 13 June 1996; accepted 1 April 1997

Backscattered electron imaging of cultured cells:

application to electron probe X-ray microanalysis
using a scanning electron microscope
NDEZ-SEGURA,* F. J. CAN
IZARES,* M. A. CUBERO,* F. REVELLES* & A. CAMPOS*
E. FERNA
*Department of Cell Biology, School of Medicine, University of Granada, E-18071 Granada, Spain
Institute of Neurosciences, University of Granada, E-18071 Granada, Spain

Key words. Backscattered electron imaging, cell culture, scanning electron

microscopy, X-ray microanalysis.

Summary
We report a simple method to study the elemental content
in cultured human adherent cells by electron probe X-ray
microanalysis with scanning electron microscopy. Cells
were adapted to grow on polycarbonate tissue culture cell
inserts, washed with distilled water, plunge-frozen with
liquid nitrogen and freeze-dried. Unstained, freeze-dried
cultured cells were visualized in the secondary and backscattered electron imaging modes of scanning electron
microscopy. With backscattered electron imaging it was
possible to identify unequivocally major subcellular compartments, i.e. the nucleus, nucleoli and cytoplasm. X-ray
microanalysis was used simultaneously to determine the
elemental content in cultured cells at the cellular level. In
addition, we propose some improvements to optimize backscattered electron and X-ray signal collection. Our findings
demonstrate that backscattered electron imaging offers a
powerful method to examine whole, freeze-dried cultured
cells for scanning electron probe X-ray microanalysis.

Introduction
Cultured cells provide a model to study the composition,
distribution and transport of ions in defined physiological
states by electron probe X-ray microanalysis with the
electron microscope. Different investigators have used this
technique to determine the elemental composition of
primary cultures of vascular smooth muscle cells ( JamesKracke et al., 1980), chondrocytes (Wroblewski et al.,
1983), fibroblasts (Abraham et al., 1985), cardiac myocytes
(Buja et al., 1985), renal epithelial cells (Larsson et al.,
1986), hepatocytes (Zierold & Schafer, 1988), endothelial
cells (Hall et al., 1992), airway smooth muscle cells (Warley
Correspondence to: Eduardo Fernandez-Segura, Tel: 34 58 243415; fax: 34 58
244034; email: efernandez@histolii.ugr.es

72

et al., 1994) and glandular epithelial cells (Hongpaisan et

al., 1996). In addition, several studies have investigated the
element content of established cell lines such as glioma C6
cells (Zierold, 1981), HeLa S3 cells (Warley et al., 1983),
colon carcinoma cell lines (von Euler & Roomans, 1992;
von Euler et al., 1993) and A6 epithelial cells (Borgmann et
al., 1994). These cells were adapted to grow on solid
substrates (plastic and glass coverslips, graphite discs) or
thin films (Pioloform-coated gold and titanium grids) to
determine the elemental content at the cellular level. In
contrast, high-resolution X-ray microanalysis at the subcellular level requires ultrathin cryosections of cultured
cells (James-Kracke et al., 1980; Warley, 1994).
Although a variety of studies have documented the
usefulness of X-ray microanalysis of whole cultured cells
using SEM (Zierold, 1981; Abraham et al., 1985; Larsson et
al., 1986; Hall et al., 1992; Borgmann et al., 1994; Hall et
al., 1994), this method has some methodological problems.
One is the need to remove the culture medium which may
overlie the surface of the cells after freezing and freezedrying (Wroblewski & Roomans, 1984). In addition, difficulties can arise with absolute quantitative analyses of
elemental content because the substrate may contribute to
the background, decreasing the peak-to-background (P/B)
ratio (Roomans, 1981). Moreover, because of its inferior
spatial resolution, SEM X-ray microanalysis does not permit
the precise identification of subcellular structures or the
measurement of elements in different compartments of
cultured cells (Zierold, 1981).
To overcome these problems and enhance lateral resolution, Zierold (1981) adapted freeze-etching methods for
X-ray microanalysis of cultured cells with SEM. This
method made it possible to recognize the major intracellular
compartments, i.e. the nucleus, cytoplasm and mitochondria. Another potential way to localize subcellular structures with SEM is to use backscattered electrons (BSE),
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which produces an image with z contrast which provides

useful structural information from a significant depth
within the sample (Richards & ap Gwynn, 1995). Different
studies have reported the utility of this method to examine
the subcellular structures in cultured myocardial cells
(LeFurgey et al., 1983) and in bulk, hydrated human tissue
(Oates et al., 1984). Echlin & Taylor (1986) used simultaneous BSE imaging and X-ray microanalysis to examine
and measure element concentrations in bulk frozen hydrated
vacuolar plant tissue with a combined BSE/X-ray detector.
In addition, backscattered electron signals in combination
with quantitative electron probe X-ray microanalysis has
been used to measure the dry mass fractions of biological
specimens at the subcellular level (Stols et al., 1986).
Recently, different investigators have used BSE and X-ray
microanalysis to obtain simultaneous structural and compositional information on mineralized tissues (Kaufman et
al., 1990; Kodaka et al., 1995; Roschger et al., 1995;
Tjaderhane et al., 1995). However, this combined method
has not been used to study the elemental content of whole,
freeze-dried cultured cells with SEM.
We present here a simple method with X-ray microanalysis and scanning electron microscopy with BSE imaging to study the elemental composition at the cellular level
in unstained, freeze-dried cultured cells. In addition, we
propose some improvements in specimen preparation and
instrumentation for BSE and SEM X-ray microanalysis of
whole cultured cells.

Material and methods

Materials
Dulbeccos modified Eagles essential medium (DMEM) and
fetal bovine serum (FBS) were obtained from Sigma (St
Louis, MO, U.S.A.). Tissue culture flasks (75 cm2) and 24well tissue culture plates were from Nunc (Roskilde, Denmark). Sterile 0.4-mm pore size polycarbonate (Millicell-PCF)
tissue culture plate cell inserts were obtained from Millipore
(Bedford, MA, U.S.A).

Cell culture
The MCF-7 (human breast cancer) and the LLC-PK1 (pig
kidney) cell lines were used throughout this study. The
cultures were maintained in DMEM supplemented with 10%
FBS at 310 K in a humid atmosphere containing 5% CO2.
Cells were subcultured every 7 days in 75-cm2 tissue culture
flasks, and the culture medium was changed every 48 h.

Sample preparation for X-ray microanalysis

For X-ray microanalysis, MCF-7 and LLC-PK1 cells were
prepared by a modified method of Wroblewski & Wroblewski
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73

(1993). Briefly, MCF-7 and LLC-PK1 cells were subcultured

using trypsin-EDTA at a density of 2.5 104 cells mL 1 in
polycarbonate tissue culture plate well inserts placed into
24-well tissue culture plates. Cells were grown in DMEM
supplemented with 10% FBS at 310 K in a humid atmosphere containing 5% CO2. After 48 h, cultured cells were in
the logarithmic growth phase and cell viability was >95%
as determined by trypan blue exclusion. At this time the
cells were washed with ice-cold distilled water for 5 s to
remove the extracellular medium. This washing solution
was more effective than 0.15 M ammonium acetate or 0.3 M
sucrose in removing the culture medium and preserving
the morphology of cultured cells (not shown). After
washing, excess fluid was drained from the surface and
the polycarbonate membrane filter was immediately plungefrozen in liquid nitrogen (Abraham et al., 1985; Warley et
al., 1994). After cryofixation the polycarbonate membrane
filter was placed in a precooled aluminium specimen
holder at liquid nitrogen temperature and freeze-dried at
213 K for 20 h in an E5300 Polaron freeze-drier apparatus
equipped with a vacuum rotatory pump system (Polaron,
Watford, U.K.). The samples were left in the chamber until
they had reached room temperature. Freeze-dried membrane filters were fixed to adhesive graphite lamina in SEM
holders, carbon-coated in a high-vacuum coating system
(Hitachi, Tokyo, Japan) and stored in a desiccator cabinet.

Instrumentation and analytical conditions

Electron probe X-ray microanalysis of the specimens was
performed using a Philips XL30 scanning electron microscope (Philips, Eindhoven, The Netherlands) equipped with
a conical objective lens, an ultrathin window (UTW) energydispersive detector (EDAX International) and a solid-state
backscattered electron detector (KE Development, Cambridge, U.K.) positioned directly under the conical lens. The
geometric arrangement of this configuration makes it
possible simultaneously to detect high-deflected BSE,
which provide z contrast, and to perform X-ray microanalysis with a high take-off angle. The samples were
examined with SEM using a combination of secondary
electron (SE) and BSE imaging modes. For BSE imaging, the
microscope was operated at different accelerating voltages
to optimize the collection of images, with a tilt angle of 358
and a free working distance of 10 mm. The BSE images were
observed and photographed with reverse signal polarity.
Micrographs were made using Ilford FP4 125 film.
For X-ray microanalysis, the analytical conditions were:
tilt angle 358, take-off angle 608 and working distance
10 mm. All spectra were collected in the spot mode at
10 000 for 200 s live time, and the number of counts per
second by the detector was about 500. To determine total
ion content, we used the peak-to-local-background (P/B)
ratio method (Statham & Pawley, 1978; Roomans, 1981;

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Boekestein et al., 1984) with reference to standards composed of 20% dextran containing known amounts of
inorganic salts (Warley, 1990). Students t-test was used
to evaluate the statistical significance of the differences
between means; a value of P < 0.05 was considered
statistically significant.

Results and discussion

BSE imaging of whole, freeze-dried cells
Figure 1a shows a scanning electron micrograph of whole
freeze-dried MCF-7 cells as seen with SE. MCF-7 cells were
flattened and polygonal in shape; some spindle-shaped and
rounded cells were also seen. The LLC-PK1 cells displayed
similar cell shapes (not shown). Because the SE imaging
mode does not allow the accurate identification of any
subcellular structures in whole, freeze-dried cells, we used
BSE which provides structural information within samples.
When unstained freeze-dried cultured cells were observed
with BSE, the major subcellular compartments, i.e. nucleus,
nucleoli and cytoplasm, were readily identifiable (Fig. 2b,c).
To optimize BSE signal collection, the emission current
was increased to induce a higher production of BSE, which
improved contrast (Richards & ap Gwynn, 1995). In addition, we compared the images obtained at different accelerating voltages; this allowed us to assess the depth in the
specimen from which the BSE emerged and thus obtain
information from different depths in the whole, freeze-dried
cells (Fig. 2). Figure 2a shows a backscattered electron
micrograph of whole, freeze-dried LLC-PK1 cells at 7 keV. At
this accelerating voltage, no subcellular structures were
noted. In contrast, when unstained, cultured cells where
examined with BSE at 913 keV, it was possible to identify
the nucleus, nucleoli, and cytoplasm (Fig. 2bd). However,
as accelerating voltage increased (1517 keV), the edges of
the cytoplasm of freeze-dried cells faded, revealing the
polycarbonate membrane filter (Fig. 2e,f). These findings
suggest that moderate accelerating voltages (911 keV) can
provide structural information within cells when the BSE
imaging mode is used, because of the different densities of
the subcellular compartments in unstained, freeze-dried
cultured cells. However, we caution that these conditions
will need to be tested and confirmed for each particular type
of cell under investigation.
To cultivate MCF-7 and LLC-PK1 cells we used polycarbonate tissue culture plate cell inserts. This thick growth
substrate does not interfere with the elements of the cellular
spectra, provides stability of the element content and cell
morphology during culture, and makes it possible to detect
changes in these features during pharmacological assays
(Wroblewski & Roomans, 1984; Hall et al, 1994). In
addition, it does not absorb significant numbers of BSE,
thanks to its low z value. Therefore, polycarbonate

Fig. 1. Scanning electron micrograph of MCF-7 cells cultured on

polycarbonate tissue culture plate well inserts, as seen with secondary (a) and backscattered electrons (b,c). Cells were washed with
distilled water, plunge-frozen in liquid nitrogen, freeze-dried and
evaporated with carbon. (a) Secondary electron micrograph of a
cluster of MCF-7 cells. Scale bar: 50 mm. (b) Backscattered electron
micrograph with reverse signal polarity of the same cells. Note
that the major intracellular compartments can be recognized. (c)
Higher power backscattered electron micrograph with reverse signal polarity of an MCF-7 cell. Note the outline of the nucleus (n),
nucleolus (nu) and cytoplasm (c). Scale bar: 10 mm.

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Fig. 2. Backscattered electron micrographs of LLC-PK1 cells cultured on polycarbonate tissue culture plate well inserts at increasing accelerating voltages. (a) 7-keV accelerating voltage image. Scale bar: 10 mm. Note that the major intracellular compartments cannot be recognized. (b) 9-keV and (c) 11-keV accelerating voltage images. Note that the subcellular compartments, i.e. nucleus, nucleoli and
cytoplasm, are recognizable. (d) 13-keV accelerating voltage image. At this voltage subcellular structures can be recognized, but fine details
of the cytoplasm are lost. (e) 15-keV and (f) 17-keV accelerating voltage images. Note that the image of cytoplasm has disappeared, revealing
the microporous polycarbonate membrane filter below. Some details of the nucleus are also lost. Micrographs were taken with reverse signal
polarity. Samples were processed as indicated in Fig. 1.

membrane filters are an excellent, growth-compatible, low-z

matrix that make it possible to obtain information on the
structural elements of unstained cells, with good image
contrast when the cells are viewed with BSE.
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X-ray microanalysis of whole freeze-dried cells

The application of the BSE imaging to freeze-dried cultured
cells plated on polycarbonate tissue culture plate well inserts
allowed us to investigate the elemental composition at the

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Fig. 3. Spectra from whole, freeze-dried MCF-7 cells cultured on

polycarbonate tissue culture plate well inserts and washed with distilled water (a), and from a washed polycarbonate membrane filter
(b). Note the absence of elements from the culture medium on the
filter. Samples were processed as indicated in Fig. 1.

cellular level, i.e. the nucleus and cytoplasm together, by

SEM X-ray microanalysis. However, penetration of the
electron probe is a problem in SEM X-ray microanalysis of
cells cultured on a thick substrate (von Euler & Roomans
1992). The dependence between beam penetration and
accelerating voltage has been documented in several SEM
X-ray microanalysis studies of bulk, hydrated specimens
(Echlin & Taylor, 1986) and freeze-dried specimens (Abraham
et al., 1985; Hall et al., 1992; Borgmann et al., 1994). These
studies demonstrate that the use of higher accelerating
voltages to analyse cells on a thick support induces overpenetration into the solid support, leading to a decrease in
the detection of cellular elements.
To test for the possible overpenetration in whole, freezedried cultured cells, we calculated the theorical maximum
penetration depth of the electron probe into the specimen at
10 and 15 keV accelerating voltages based on the Bethe
.
range equation: RB 70 (E1 66/d), where E incident
energy in keV and d specimen density (kg cm 3 ). These
accelerating voltages provide structural information from
the depth of cells with BSE imaging, and are sufficient to
achieve critical excitation energy for Cl and K. To compare
maximum penetration depths, we also calculated the
thickness of the LLC-PK1 cell line. Transmission electron
micrographs of LLC-PK1 cells showed that the mean thickness was 8.95 mm (not shown). Wroblewski & Wroblewski
(1993) reported that the thickness of cultured monolayers is
about 5 mm, rarely exceeding 10 mm. If we assume that the
density of freeze-dried cytoplasm is <0.5 kg cm 3 (Echlin &
Taylor, 1986), the theoretical maximum overpenetration in

whole, freeze-dried cells is 12.5 mm at 15 keV and 6.3 mm

at 10 keV. Taken together, these data indicate that an
accelerating voltage of 10 keV does not induce overpenetration of the electron probe in X-ray microanalysis of whole,
freeze-dried adherent cells on polycarbonate tissue culture
plate well inserts.
We also tested the elemental content in whole, freezedried MCF-7 and LLC-PK1 cells at 10 and 15 keV. To analyse
cultured cells, the static spot probe was positioned above the
nucleus imaged with BSE. Figure 3 shows X-ray spectra
from whole, freeze-dried cultured cells. A spectrum from a
polycarbonate membrane filter washed with distilled water
is also shown. Our results demonstrate that in MCF-7 and
LLC-PK1 cells washed with distilled water, Na, Mg, P, S, Cl
and K can be detected. However, Ca was detected only in
damaged cultured cells, which were also characterized by
their high Na and Cl contents. Our results show that MCF-7
and LLC-PK1 cells were characterized by high K/Na ratios
when analysed at 10 and 15 keV. MCF-7 cells had K/Na
ratios of 11 6 1 and 10 6 1, and LLC-PK1 yielded K/
Na ratios of 13 6 1 and 12 6 1, respectively. These
analytical results, together with the good morphological
appearance of MCF-7 and LLC-PK1 cells, suggest that our
method of preparing cultured cells for electron probe X-ray
microanalysis yields satisfactory results.
Table 1 compares the elemental composition in MCF-7
and LLC-PK1 cells analysed at 10 and 15 keV. Use of an
accelerating voltage of 10 keV in freeze-dried cells causes a
significant increase in the intracellular concentrations of
Na, P, Cl and K. The fact that these elemental contents did
not increase at 15 keV suggests that the electron probe
penetrated the thick support, which thus contributed to the
X-ray spectra. In contrast, at 10 keV the X-ray signals
detected were generated exclusively from the cells.
In addition, elemental concentrations were related to the
P content as an indicator of dry mass of the cell analysed
(Abraham et al., 1985; Larsson et al., 1986). Figure 4 shows

Fig. 4. Elemental contents (Na/P, Cl/P and K/P ratios) in whole,

freeze-dried LLC-PK1 cells analysed at 10 (open bars) and 15 keV
(filled bars). The values are the means 6 SE from 25 cells.
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Table 1. Elemental composition of whole, freeze-dried cultured cells at accelerating voltages of 10 and 15 keV.

Cell type

Na

Mg

Cl

MCF-7 (clone BUS )

10 keV
25
15 keV
25

38 6 2
39 6 2

33 6 1*
37 6 1

285 6 5**
260 6 5

78 6 2
78 6 2

101 6 3**
87 6 3

395 6 8*
367 6 7

LLC-PK1
10 keV
15 keV

32 6 2*
28 6 2

26 6 1*
30 6 5

239 6 4**
202 6 5

76 6 3
77 6 2

84 6 5**
63 6 3

383 6 9**
296 6 9

20
20

Data given in mmol kg1 dry weight 6 SE; n, no. of cells analysed; * P < 0.05, ** P < 0.01.

the content ratios for Na/P, Cl/P and K/P in LLC-PK1 cells
analysed at 10 and 15 keV. Our results demonstrated that
the concentration ratios of K/P obtained at 10 keV were
significantly higher (P < 0.01) than the corresponding ratio
obtained at 15 keV. Taken together, these results are
compatible with previous reports that accelerating voltages
of about 1011 keV should be used to analyse the elemental
content in whole, freeze-dried cells plated on thick supports
(Abraham et al., 1985; Hall et al., 1992; Borgmann et al.,
1994). In addition, we demonstrate that this range of
accelerating voltages was high enough to provide structural
information from a depth of several micrometres within
unstained whole, freeze-dried cells viewed with BSE.

Conclusions
We describe a method to measure the elemental content in
whole cultured adherent cells by X-ray microanalysis with
SEM, using backscattered electron imaging. This imaging
mode provides useful structural information from subcellular compartments, i.e. the nucleoli, nucleus and cytoplasm, and makes it possible to analyse the elemental
composition of cultured cells at the cellular level. We believe
that backscattered electron imaging will prove useful as a
complementary technique to analyse whole, freeze-dried
cultured cells with scanning electron probe X-ray microanalysis.

Acknowledgments
We thank Dr M. Ruiz de Almodovar (Department of Radiology, Granada, Spain) for providing the MCF-7 cell line, Dr
R. Garcia del Moral (Department of Pathology, Granada,
Spain) for providing the LLC-PK1 cell line, M. A. Robles for
her expert technical assistance and K. Shashok for translating parts of the original manuscript into English.

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