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Ultramicroscopy ] (]]]]) ]]]–]]]

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Ultramicroscopy
journal homepage: www.elsevier.com/locate/ultramic

A fluorescence scanning electron microscope

Takaaki Kanemaru a, Kazuho Hirata b,, Shin-ichi Takasu c, Shin-ichiro Isobe d, Keiji Mizuki e,
Shuntaro Mataka f, Kei-ichiro Nakamura g
a
Morphology and Core Unit, Kyushu University Hospital, Kyushu, Japan
b
Department of Anatomy and Cell Biology, Graduate School of Medical Sciences, Kyushu University, Higashi-ku, Maidashi 3-1-1, Fukuoka 812-8582, Japan
c
Advanced Technology Division, JEOL Ltd., Tokyo, Japan
d
Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kyushu Sangyo University, Fukuoka, Japan
e
Department of Nanoscience, Faculty of Engineering, Sojo University, Kumamoto, Japan
f
International Science Technology Co., Ltd, Kasuga Laboratory, Fukuoka, Japan
g
Department of Anatomy, Kurume University School of Medicine, Kurume, Japan

a r t i c l e in fo abstract

Article history: Fluorescence techniques are widely used in biological research to examine molecular localization, while
Received 12 August 2008 electron microscopy can provide unique ultrastructural information. To date, correlative images from
Received in revised form both fluorescence and electron microscopy have been obtained separately using two different
22 December 2008
instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current
Accepted 6 January 2009
study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence
digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In
Keywords: the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were
Correlative microscopy employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative
Scanning electron microscopy
fluorescence and electron microscopy.
Fluorescence microscopy
& 2009 Elsevier B.V. All rights reserved.
Organic EL fluorophore

1. Introduction localization. Fluorescent probes, which label an identical region of

Fluorescence microscopy has become an indispensable micro-
scopy technique for the examination of biological specimens,
because it allows selective and specific detection of molecules at
small concentrations with a good signal-to-background ratio [1,2].
It even allows one to work with intact samples, including living
cells, and to see samples with the naked eye; these advantages are
not available with other methods, such as electron microscopy [3].
Furthermore, recent developments in fluorescence imaging
techniques have enabled the well-known Abbe barrier of about
200 nm lateral resolution to be crossed, that is, the diffraction
limit for an optical microscope has approached the level of 100 nm
as with 3D structural illumination microscopy (3D-SIM) [4]
or even less than 100 nm, as with other techniques such as 4Pi,
simulated emission depletion (STED) and photoactivated localiza-
tion (PALM) [3,5].
In order to obtain a stable image with a higher resolution,
however, an electron microscope is still required. Corre-
lative study using both fluorescence and electron microscopy is
usually employed to obtain substantial information on molecular
 Corresponding author. Tel.: +81 92 642 6048; fax: +81 92 642 6050.
E-mail address: hirata@anat1.med.kyushu-u.ac.jp (K. Hirata).
a single specimen not only for fluorescence microscopy but also
for electron microscopy [6], such as FluoroNanogold [7], and more
recently ReAsH, which is used in the tetracystein–biarsenical
system [8] and small nanocrystals (Quantum dots; QDs) [9], have
all been being utilized; nevertheless, complications during the
specimen preparation process and during imaging with both types
of microscopy are unavoidable.
One of the ways in which the complex methods for correlative
microscopy could be improved would be to incorporate one
microscope into another. The technology of the scanning electron
microscope (SEM) is now well advanced and the image resolution
of the SEM approaches that of the transmission electron
microscope (TEM) [10]. It is clear that for the majority of biological
samples, it is easier and less time-consuming to prepare samples
for scanning electron microscopy than for transmission electron
microscopy. Furthermore, the structure of the SEM makes it easier
and more cost-effective to incorporate other devices into it. This
led us to attempt to make a hybrid ‘‘FL-SEM’’ instrument in which
an SEM is combined with an FM. In parallel with its set-up, some
fluorophores were tested for the labeling of biological samples
using the new instrument. We found that an organic EL
fluorophore named Fluolid (Pub. no. US 7015002) [11], which
has a high physical stability was suitable for this purpose.
The design of the FL-SEM is described and the first FL-SEM
images are presented.

0304-3991/$ - see front matter & 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ultramic.2009.01.002

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Some of our findings have been previously reported in abstract possible fluorescence damage which may be caused by the
form [12]. electron beam. Each of the FM and SEM images is separately
displayed on a single PC screen through the CCD camera and
the AD converter, respectively (Fig. 1C). Both images are then
2. Experimental manually merged using Adobe Photoshop CS.

2.1. Assembly of the FL-SEM

2.2. Animals

An SEM for wavelength dispersive spectrometry (WDS) (JEOL

JXA8600 M, JEOL, Tokyo Japan) was reconstructed to create a
dual-mode microscope, named the FL-SEM, which is capable of
obtaining both FM and SEM images without requiring to move the
specimen (Fig. 1A and B). All the parts of the built-in optical
microscope unit in the SEM for WDS, with the exception of a
mirror and the Cassegrain type (non-chromatic aberration type)
45  , NA 0.41 optical objective lens, both of which are placed
within the column and have a small hole for the electron beam to
pass through, were replaced with an FM fitted with a digital
camera unit that was assembled in our laboratory. The unit
consisted of a laser light source from an external unit (473 nm,
Showa Optronics, Tokyo, Japan) (a in Fig. 1A and B), an emission
filter (515 nm LP), an adaptor device for concentrating the laser
light (b in Fig. 1A and B), a unit consisting of mirrors and prisms (c
in Fig. 1A and B), an external CCD camera (Bitran Corporation,
Saitama, Japan) adapted via a C-mount adaptor (d in Fig. 1A and
B), and an eye piece (e in Fig. 1A and B). In the FL-SEM, an electron
beam passes through small holes at the center of a mirror and the
object lens in order to reach the specimen. The SEM image is then
sent to an AD converter (SemAfor, JEOL SA20) via a photomulti-
plier (PMT). On the other hand, the excitation beam from the
external unit reaches the specimen along the same passage as the
electron beam within the column. The fluorescence emission from
specimens is then directed to an external CCD camera. In practice,
the digital image from the FM is acquired first, with the image
from the SEM being sequentially acquired, in consideration of
Under deep anesthesia induced by diethyl ether, adult male
Wistar rats were perfused intracardially with PBS, followed
by a mixture of 2.8% paraformaldehyde, 0.2% picric acid and
0.06% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4.
The diaphragm and the kidney were removed and postfixed with
4% paraformaldehyde in 0.1 M PB.
These experiments were reviewed by the Committee on Ethics
for Animal Experiments of the Faculty of Medicine, Kyushu
University and were carried out according to the guidelines for
Animal Experiments of the University, and Law no. 105 and
Notification no. 6 of the Japanese Government.
2.3. Exploitation of a new probe for FL-SEM
In a preliminary study, we attempted to use some tissues
that had been labeled with GFP or FITC as FL-SEM samples.
These fluorophores were almost bleached out in the process of
dehydration. For this reason, we employed a new probe, Fluolid,
which is a small organic fluorophore originally synthesized as an
organic EL dye in our laboratory (Pub. no. US 7015002), [11]. The
Fluolid dye has a high physical stability and a large Stokes shift
and it shows strong fluorescence intensity in its solid state.
However, it has never been applied to the labeling of biological
samples. In order to test its viability, a kidney was immersed in
20% sucrose in PBS and 10-mm-thick frozen sections of the kidney
were made for peanut agglutinin (PNA) staining, in accordance

Fig. 1. (A). A view of the FL-SEM. (B). A schematic diagram of an inside view of the FL-SEM, which is made up of a combination of the SEM and the FM units. (C). A schematic
diagram depicting the flow of SEM and FM image signals to a PC display. (a): the laser light source of the external unit (473 nm), (b): an adaptor device for laser light, (c):
mirror and prism, (d): external CCD camera, and (e): eye piece. PMT, photomultiplier tube.

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with a report by Holthofer [13] regarding specific PNA binding
to the brush border of epithelial cells within the renal proximal
tubules. Fluolid-W-Orange was conjugated with streptavidin
utilizing the IST Fluolid-protein-labeling kit (CosmoBio, Japan)
before use. After being washed with PBS, the sections were
incubated with biotinylated PNA (Vector) (1:100) at room
temperature (RT) overnight and then with Fluolid-W-Orange-
conjugated streptavidin (1:10) at RT for 3 h. The sections were
then mounted with aqueous Vectashield (Vector). FITC-conjugated
streptavidin (Vector) (1:100) and Alexa-488-conjugated strepta-
vidin (Molecular Probe) (1:500) were used as controls. Under
conventional FM (Zeiss Axiophoto), it was revealed that the
Fluolid dye was able to successfully label the PNA binding site
of the apical region of the renal proximal tubule cells (Fig. 2A).
The fluorescent intensity of Fluolid became stronger in the same
section mounted with non-aqueous Histomount (Invitrogen) after
dehydration (Fig. 2B), whereas that of the FITC was attenuated
(Fig. 2C and D). Furthermore, Fluolid showed no change over 4
months, even under direct light (Fig. 2E and F), whereas the FITC
was completely bleached out (Fig. 2G and H). Alexa Fluor, which is
known to have a strong photostability [14], showed similar
findings to Fluolid (data not shown). Thus, Fluolid and Alexa
Fluor were confirmed to be suitable for specimen preparation for
the FL-SEM.

Fig. 2. Conventional fluorescent micrographs of the PNA staining of frozen sections of the rat kidney, which was labeled by Fluolid-W-Orange, an organic EL dye. (A, B, E,
and F). FITC (C, D, G, and H) was used as a control. The stability of the fluorophores to dehydration (A–D) and daylight (E–H) is shown. Scale bar: (A–D): 20 lm, (E–H):
100 lm.

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2.4. Preparation of FL-SEM samples
For FL-SEM specimens, the fixed diaphragm and kidney were
processed for Iba1 immunohistochemistry and PNA staining,
respectively. The immunohistochemical procedure used here has
been described elsewhere [15]. Briefly, after removing the
peritoneum, the diaphragm was preincubated with 1% bovine
serum albumin in PBS at RT, for 1 h, in order to block nonspecific
binding sites. The specimen was then incubated with a rabbit
polyclonal anti-Iba1-antibody (Wako) (1:100) as the primary
antibody at RT for 3 days and with an Alexa488-anti-rabbit
antibody (Molecular Probes) (1:200) as the secondary antibody at
RT for 1 day. The labeled specimen was then dehydrated with
acetone and coated with osmium (2.5 mm in thickness) in
an osmium-plasma coater (HPC-1S, Vacuum Device Inc., Ibaragi).
The fixed kidney was freeze-fractured using DMSO and was first
incubated with biotinylated PNA (Vector) (1:100) at RT for 3 days
and then with Fluolid-W-Orange-conjugated streptavidin (1:10) at
RT for 1 day. Some of the labeled specimens were thinly sectioned
after being embedded with Technovit 8100 resin (Heraeus Kulzer)
(cf. Fig. 4C and D, about 5 mm thick), due to the limitation of
the FM with respect to its depth of focus [1], compared to the
relatively large depth of focus of the SEM [10]. In order to
delineate the subcellular structure, an additional technique of ion-
etching [16,17] was employed. Very mild rapid etching was
conducted as follows: 12 mA for 8 min, employing an ion coater
above the sample bulk on silicon ware. The specimens were then
immersed in 100% acetone for 140 min and were processed for
osmium-plasma coating.
3. Results and discussion
The bulk of the Alexa Fluor-labeled diaphragm was first
observed by FL-SEM. The image at the maximum resolution of
the FM (650  ) was recorded through the FM unit (Fig. 3A).
Fig. 3. FL-SEM imaging of Iba1-immunostained rat diaphragm. FM (A) and SEM (B) images and the merged image (C) of an identical region. (D) An enlarged SEM image of
the white box in (C). Alexa 488-labeled Iba1-positive macrophages (green) can be seen among the skeletal muscles. Scale bar: (A–C): 10 lm, (D): 1 lm.
Please cite this article as: T. Kanemaru, et al., Ultramicroscopy (2009), doi:10.1016/j.ultramic.2009.01.002
The identical region was also recorded through the SEM unit
without moving the specimen (Fig. 3B). Both images were finally
merged on the PC display (Fig. 3C). In the merged image, the Iba1-
positive macrophages distributed among the muscle fibers were
clearly indicated (Fig. 3C). Details of the surface structure of one of
the macrophages were further observed at high resolution of the
SEM (8000  ) (Fig. 3D). Thus the FL-SEM allows spatial localiza-
tion of a cell via its molecular expression and its 3-D character-
ization at higher magnification. At the same time, this new
instrument has also solved the problem of locating the site of
interest for SEM observation, through the process of prescreening
with a fluorophore-labeled structure.
Next, the bulk of the Fluolid-labeled fractured kidney, in which
an ion-etching technique was applied to delineate the subcellular
structure, was observed (Fig. 4A–B). In the merged image, the
labeling was detected in the apical region of the epithelial cells
within the proximal tubules (Fig. 4A, an arrow). The enlargement
of the labeled site by the SEM demonstrated that the Fluolid-
labeled site almost corresponded to the thick brush border
consisting of tall microvilli that were sharply delineated by the
ion-etching (Fig. 4B). In the thick bulk sample, we sometimes
encountered non-specific fluorescent sites (Fig. 4A, arrowheads).
This is probably because the entire sample was excited indis-
criminately and most of the fluorescent photons arose from out-
of-focus fluorophores [1]. Therefore, a thin section of the kidney in
which ion-etching was also carried out was further observed. The
fluorescent image was dramatically improved; the labeling was
restricted to the apical region of the epithelial cells (Fig. 4C),
which corresponded to the microvilli (Fig. 4D). Thus, by using a
fluorophore-labeled section through the cells, the FL-SEM is also
capable of analyzing correlations between the FM and the SEM
images of the intracellular structure.
The necessity of a dual mode SEM equipped with an optical
microscope was stressed by Yamada et al. [18] who characterized
the constituent distribution of food tissues utilizing a color SEM
image obtained from two separate instruments through digital
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Fig. 4. FL-SEM imaging of the PNA-stained fractured bulk (A and B) and a thin section (C and D) of the rat kidney that have been treated with an ion-etching technique to
delineate the subcellular structure. A–B: Merged images (A) and an enlarged SEM image (B) of the Fluolid-W-orange-labeled PNA-positive site (orange) indicated by an
arrow in (A). Arrowheads indicate non-specific fluorescent sites. (C–D): The merged image (C) and the SEM image (D) of a cross-section of the renal proximal tubule. Note
that the labeling (orange) is precisely superimposed onto the microvilli. Asterisks indicate the nuclei of the corresponding endothelial cells within the proximal tubule.
Scale bar: (A): 10 lm, (B): 1 lm, and (C–D): 10 lm.

image processing. Boyde et al. [19] analyzed bone tissues with the
correlation of qualitative and quantitative BSE-SEM imaging with
confocal scanning light microscopy imaging modes using their
own overlapping software package tailored to their needs. An
advantage of the FL-SEM is that both the FM and the SEM images
of the identical area of a single specimen can be quickly obtained
without moving the specimen once it is set up in the device.
The FL-SEM may be applied to the samples reported, since both
tissues reported were thin-sectioned for analysis and the FL-SEM
is suitable for observations of flat and even specimens, such as
flat tissues of the diaphragm (Fig. 3A–D) and thin sections of the
kidney (cf. Fig. 4C and D). In particular, the bone samples are
assumed to be practically available under the FL-SEM without any
structural change, since a BSE mode is attached to the microscope.
In the current study, by employing an ion-etching technique
on thin sections, we showed the possibility that the FL-SEM
could also work as fluorescence TEM, which is expected to
be engineered in the future. We are currently collaborating to
improve the FL-SEM and its sample preparation techniques with a
view to obtain a higher resolution.
4. Conclusion
For the first time we have developed a fluorescence SEM
(FL-SEM). This new instrument is an advanced type of SEM with
a prescreening tool and it could be applied to correlative study
using both FM and SEM in biological research. The development
of the FL-SEM has been accompanied by the use of an organic
EL fluorophore ‘‘Fluolid’’ and an ion-etching technique for the
preparation of the FL-SEM sample. This hybrid instrument could
be a practical tool with a high throughput for biological research.
Acknowledgements
We thank A. Togo, K. Yamashita, and N. Yamaguchi for their
technical support; Dr. F. Ishikawa for helpful suggestions; and Dr.
T. Kondo and Dr. K. Ohta for their helpful discussions. This work
was supported by a grant-in-aid from the Fukuoka Industry,
Science and Technology Foundation (Fukuoka IST) and Fukuoka
Prefectural Bio-Industry Center Conference. The English used in
this manuscript was revised by Miss K. Miller (Royal English
Language Center, Fukuoka, Japan).
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