7Proc. Arthropod. Embryol. Soc. Jpn. 50, 7–10 (2016)Ⓒ 2016 Arthropodan Embryological Society of Japan
ISSN 1341–1527[SHORT COMMUNICATION]

Simple, Artifact-free SEM Observations of Insect Embryos:
Application of the Nano-suit Method to Insect Embryology
Mari FUJITA1, 5), Alexander BLANKE2, 5), Shûhei NOMURA3) and Ryuichiro MACHIDA4, 5)
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan
Medical and Biological Engineering Research Group, School of Engineering, University of Hull, Hull HU6 7RX, UK3)
National Museum of Nature and Science, Tsukuba, Ibaraki 305–0005, Japan
4)
Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan
5)
Current address: Sugadaira Montane Research Center, University of Tsukuba, Sugadaira Kogen, Ueda, Nagano 386–2204, Japan
E-mail: fujita@sugadaira.tsukuba.ac.jp (MF)1)
2)

Scanning electron microscopy (SEM) enables
morphological observations with a high resolution, but
unfavorable shrinkage and distortion of the materials areoften associated with the preparation of biological samples. In
insect embryological studies, which deal with fragile embryos,
the situation is grave. The fixation, dehydration and drying
are apt to lead to serious shrinkage of materials. In the laterstages when the embryonic cuticle has already been secreted,the case is more serious (Fig. 2A, B). Separating from thesurface of the embryo, the embryonic cuticle wrinkles up,
which would be a major obstacle preventing determination of
the correct morphology of the embryo. As a solution to thisproblem, Machida (2000a) proposed the use of low-vacuum
SEM of uncoated specimens, which enables us to observe thesurface structures of embryos through the wrinkledembryonic cuticle (Machida, 2000b). However, it does notalways follow that this method is very effective for every case,and the most critical point is that the required low vacuum
yields only inferior resolution.
Chambers et al. (2003) reported that some insects such as
dipteran larvae have been shown to possess special
extracellular substances (ECSs) on their body surface. Takaku
et al. (2013) found that the ECSs are polymerized by electron
irradiation into an ultra-thin tough membrane named the “nanosuit”, and that this nano-suit prevents the escape of evaporative
substances from the specimens, even in high-vacuum
conditions. They succeeded in biomimetically producing anano-suit by electron irradiation to polyoxyethylene sorbitanmonolaurate (Tween 20), which has high amphiphilic andbiocompatible properties similar to ECSs. Specimens coveredand protected by the nano-suit made from Tween 20, whichacts as a flexible barrier to the passage of gases and liquids,
can be observed under usual high-vacuum SEM with a highresolution being maintained, without any conventional
treatments such as histological fixation, dehydration anddrying (Ohta et al., 2014).
In this study, we applied the “nano-suit method” invented
by Takaku et al. (2013) to insect embryology, using grownembryos of a corydiid cockroach, Eucorydia yasumatsui
Asahina, in the later definitive dorsal closure stage, at which
the larval cuticle is yet to be secreted with the surface of theembryo only covered with the fine embryonic cuticle. The
procedure used is as follows.
1. Embryos dissected out of eggs in physiological saline weresoaked in 1% Tween 20 solution dissolved in distilled waterfor 1 h.
2. Embryos taken out from the Tween 20 solution and excess
solution were blotted with filter paper.
3. The embryos were mounted on a stub and observed with ascanning electron microscope (SEM), TOPCON SM-300,
under a high vacuum at an accelerating voltage of 5 kV.
An example of an E. yasumatsui embryo observed using
the nano-suit method is shown in Figure 1. The surface
structure of the embryo was well preserved (Fig. 1A) in termsof the details (Fig. 1B, C). Figure 1B, which is a magnified
image of the vertex, clearly demonstrates a row of pointed,
tiny egg teeth on the vertex, this being the first finding of egg
teeth in cockroaches. As shown in Figure 1C, which is amagnification around the thoracic legs, each structure is wellpreserved in natura (compare with Fig. 2B). Because thelonger exposure to and higher intensity of an electron beamwould lead to degradation of the nano-suit, the observation
should be less than 1 h, and the accelerating voltage should be
set lower than 5 kV.
After the observation using the nano-suit method, weprocessed the same embryo into the SEM specimen in theconventional methods: the specimen was detached from thestub (when the specimen is not easily detached, pour saline or
distilled water on to it), fixed with Karnovskyʼs fixative (2%paraformaldehyde + 2.5% glutaraldehyde 0.1 M HCl-sodium8M. FUJITA, A. BLANKE, S. NOMURA AND R. MACHIDA

Figs. 1, 2 Scanning electron micrographs (SEMs) of a grown embryo of Eucorydia yasumatsui Asahina in the later definitive dorsal closure
stage. The whole embryo images (Figs. 1A, 2A) were produced by merging separate photos using the software Adobe Photoshop
CS2.
Fig. 1 A. An SEM of the embryo processed using the nano-suit method. B. Enlargement of the vertex, showing the egg teeth. C.
Enlargement of a part of the thoracic region. See the text.
Fig. 2 A. An SEM of the same embryo as shown in Fig. 1A, processed in the conventional methods. B. Enlargement of a part of thethoracic region at approximately the same frame as shown in Fig. 1C. See the text.
An: antenna, Ce: cercus, Cl: clypeus, ET: egg tooth, Fe: femur, Lr: labrum, Md: mandible, MxP: maxillary palp, Sty: stylus, Ta:tarsus, Th1–3: pro-, meso- and metathorax, Ti: tibia, Tr: trochanter, Un: unguis, Vx: vertex, II-V: second to fifth abdominal segments.
Bars = 1A, 2A: 500 µm; 1B: 10 µm; 1C, 2B: 100 µm.

cacodylate buffer solution, pH 7.2) and postfixed with 1%
OsO4 solution, dehydrated through a graded ethyl alcoholseries, dried using a critical point dryer (tousimis SamdriPVT-3D), stuck to the stub and coated with gold. Figure 2A
and B are scanning electron micrographs (SEMs) of aspecimen processed in the conventional methods. We foundthat the embryo was seriously contracted and distorted (Fig.
2A), compared with the SEM of the specimen processed using
the nano-suit method (Fig. 1A): the embryo was contracted
into ca. 85% in length. Figure 2B is an enlargement of Figure
2A, approximately the same frame as for Figure 1C. The
embryonic cuticle wrinkled up due to separation form thesurface of the embryo made it difficult to observe and grasp
the correct surface structures of the appendages (Fig. 2B), thesame as the case the embryos freshly dissected out of theeggs are directly processed in the SEM specimens (data notshown).
It was revealed that the nano-suit method has a great
advantage over the conventional approach in the SEM
observations of insect embryos, especially at the later stages;
it guarantees 1) observations of high resolution, 2) with little
contraction and distortion of the surface structures of theembryos, and 3) the requisite processing is very simple,
namely, just immersing samples in the detergent Tween 20
solution for a short period. Furthermore, the nano-suit
method has another remarkable advantage. Specimens used
once for SEM observation can be reused for some histologicalobservations because the embryos used usually survive after
the SEM observation; even if they die due to the SEM
observation, their tissues would remain undamaged. We
present an example of this.
Figure 3A shows an SEM of an embryo observed using

NANO-SUIT METHOD FOR INSECT EMBRYOLOGY

9Fig. 3 Scanning electron micrography (SEM) and histological observations of a grown embryo of Eucorydia yasumatsui Asahina at the later
definitive dorsal closure stage. A. SEM using the nano-suit method, which was produced by merging separate photos using thesoftware Adobe Photoshop CS2. B. Fluorescence microscopy of the same embryo as shown in A, being stained with DAPI. C. A
histological section of the same embryo as shown in A and B. See the text.
Br: brain, E: eye, Fg: foregut, HC: head capsule, Lr: labrum, Mg: midgut, SbG: suboesophageal ganglion, Th1: prothorax, Th1, 2L:
pro- and mesothoracic leg, I, VII: first and seventh abdominal segments.
Bars = 500 µm.

the nano-suit method. After the SEM observation, the embryo
was soaked in physiological saline, removed from the stub andthen fixed with Karnovskyʼs fixative. Figure 3B shows thesame embryo as in Figure 3A, which was stained with a DNAspecific fluorescent dye, DAPI (4ʼ,6-diamidino-2-phenylindole
dihydrochloride, 10 µg/ml), and observed with a fluorescencestereomicroscope (LEICA MZ FLIII). Then, the embryo wasprocessed into methacrylate resin (Kulzer Technovit 7100)
sections according to Machida et al. (1994a, b), as shown in
Figure 3C, which were stained with 1% Delafieldʼshematoxylin overnight, 0.5% eosin G for 1 h and 0.5% fastgreen FCF 100% ethyl alcohol solution for 1 min, and observed
under a biological microscope (Nikon Optiphot-2).
As has been addressed, the nano-suit method for SEM
observations was revealed to have great advantages in insect
embryology. This method not only enables observations ofhigh resolution with little contraction and distortion on thesurface structures of embryos, but also after the SEM
observation, the same embryos can be reused for routine
histological analyses. This approach should become a standard
method for scanning electron microscopy in this field.

Prof. M. Shimomura and Dr. Y. Hirai of Chitose Institute of
Science and Technology for their kindly giving us valuableguidance and information of the nano-suit method. This study
was supported by Grants-in-Aid from the JSPS (ScientificResearch C: 2544020) to RM and from the University ofTsukuba — Deutscher Akademischer Austausch Dienst(DAAD) Partnership Program to RM and AB. This is acontribution from the Sugadaira Montane Research Center,University of Tsukuba.
References
Chambers, L., S. Woodrow, A.P. Brown, P.D. Harris, D. Phillips, M.
Hall, J.C.T. Church and D.I. Pritchard (2003) Degradation of
extracellular matrix components by defined proteinases from thegreenbottle larva Lucilia sericata used for the clinicaldebridement of non-healing wounds. British Journal of
Dermatology, 148, 14–23.
Machida, R. (2000a) Usefulness of low-vacuum scanning electronmicroscopy in descriptive insect embryology. Proceedings ofArthropodan Embryological Society of Japan, 35, 17–19.
Machida, R. (2000b) Serial homology of the mandible and maxilla inthe jumping bristletail Pedetontus unimaculatus Machida, based

Acknowledgments: We thank Prof. T. Hariyama and Dr. Y.
Takaku of Hamamatsu University School of Medicine andon external embryology (Hexapoda: Archaeognatha, Machilidae).
Journal of Morphology, 245, 19–28.

10

M. FUJITA, A. BLANKE, S. NOMURA AND R. MACHIDA

Machida, R., T. Nagashima and H. Ando (1994a) Embryonic

Ohta, I, Y. Takaku, H. Suzuki, D. Ishii, Y. Muranaka, M. Shimomura

development of the jumping bristletail Pedetontus unimaculatus

and T. Hariyama (2014) Dressing living organisms in a thin

Machida, with special reference to embryonic membranes

polymer membrane, the NanoSuit, for high-vacuum FE-SEM(Hexapoda: Microcoryphia, Machilidae). Journal of Morphology,observation. Microscopy, 63, 295–300.

220, 147–165.
Machida, R., T. Nagashima and T. Yokoyama (1994b) Mesoderm

Takaku, Y., H. Suzuki, I. Ohta, D. Ishii, Y. Muranaka, M. Shimomuraand T. Hariyama (2013) A thin polymer membrane, nano-suit,segregation of a jumping bristletail, Pedetontus unimaculatus

enhancing survival across the continuum between air and highMachida (Hexapoda, Microcoryphia), with a note on an automatic

vacuum. Proceedings of the National Academy of Science of thevacuum infiltrator. Proceedings of Arthropodan Embryological

United States of America, 110, 7631–7635.

Society of Japan, 29, 23–24. (in Japanese).