The new Atmospheric Scanning Electron Microscope
enables observation of cells in buffer under normal
atmospheric pressure
H. Nishiyama1), M. Suga1), T. Ogura2), Y. Maruyama2, A. Ohtani3), M. Koizumi 1), K. Mio 2), S.
Kitamura1), T. Shiga3), C. Sato2)
1) JEOL
Ltd., Tokyo, Japan. 2) AIST, Tsukuba, Japan. 3) University of Tsukuba, Tsukuba, Japan.
- ASEM（Atmospheric Scanning Electron Microscope） is able to observe aqueous
cells directly in atmospheric pressure at high resolution.
- Quasi-simultaneous imaging with ASEM and optical microscope.
- The microscope’s open specimen chamber allows drug administration.
We have developed a brand new ASEM (Atmospheric Scanning Electron Microscope) with an open culture dish. The SEM had to be redesigned to
have an inverted organization: the gun is set at the bottom and the SiN airlock-windowed culture dish (ASEM Dish) at the top. Although the
thickness of the SiN film is only 100 nm, the film withstands a 1-atmospheric pressure gap and is transparent to electrons. The ASEM dish alone,
when filled with medium of a few ml, can be used for prolonged cultivation of cells in a CO2 incubator. After fixation of the cultured cells, the dish
is loaded onto the ASEM. An electron beam is projected upward through the SiN film to the cells, and the electrons backscattered through the film
are captured by a detector positioned below for ASEM imaging. Above the dish, an optical microscope with immersion lens is set to realize quasisimultaneous observation. In this system, the enormous preprocessing, including dehydration, for the electron microscope observation is not
required. Furthermore, administration of drugs is easy because of the open system. This system is named ‘ClairScope’.
1. ASEM Configuration
OM
(Optical Microscope)
Drug
Normal atm
administration
Fixed
Medium (2ml)
薄膜ﾃﾞｨｯｼｭ cells
Backscattered
electrons
Open
system
Detector
Electron beam
Vacuum
OM
OM
ASEM
ASEM dish
SiN thin film
φ 35 mm (thickness: 100 nm or less)
ASEM
ASEM
Dish is removable
for cell culture
ClairScope (JASM-6200)
View from
under-side of dish
ASEM
Dehydration
(2 h)
Dry
(5 h)
Metal
evaporation
Metal stain
(1.5 h)
Fix
Metal stain
Conventional SEM
(example)
Fix
2. High-throughput pretreatment
Observation
in vacuum
High-throughput observation in atmospheric pressure
Only exchange reagent
Pretreatment time is about 10 minutes
3. Applications
3-1. Microbe
Escherichia coli K12
Rhodospirillum rubrum NBRC 3986
(1)Apply Poly-L-Lisine solution to
ASEM dish and incubate for one hour
at room temperature.
(2)Remove solution and wash three times
with double distilled water.
(3)Remove water.
(4)Apply 5 uL of microbe suspension into
ASEM dish and incubate for one hour
at room temperature.
(5)Fix with 1% Glutaraldehyde for 10
min.
(6)Stain with Phosphotungstic acid and
PtB solution for 30 min.
Yeast
Budding
The ASEM can acquire high-resolution images of
internal structures at high throughput rates.
3-2. Neuronal network (primary culture)
1 day culture
ASEM
14 day culture
(1)Apply polyethylenimine solution to the ASEM dish.
(2)Remove solution and wash with double distilled water.
(3)Culture or rat cerebral cortex, one day.
(4)Fixation with 1 % Formaldehyde.
(5)Stain with rhodamine-phalloidin, OM observation.
(6)Stain with platinum blue, ASEM observation.
OM (F actin)
Long term cell culture
(ASEM dish in CO2 incubator)
ASEM
- Fluorescent microscopy identifies the location of F-actine.
- ASEM captures high resolution images.
3-3. OM observes cell dynamics to determine the timing for fixation (Endocytosis)
Pretreatment
(1) Culture of COS7 cells on the SiN membrane.
(2) Administration of Qtracker non-targeted
quantum dots (Invitrogen cat#Q25041MP) for
endocytosis
(3) Fix with 1% glutaraldehyde.
(4) Stain glycan: Qdot® wheat germ agglutinin
conjugate (Invitrogen, cat# Q12021MP) for OM
(5) Stain with 1 % phospho tungstic acid for ASEM
Drug administration
OM
薄膜ﾃﾞｨｯｼｭ
OM
Normal atm
Vacuum
x2000 10 µm
ASEM
ASEM
OM: Live cell imaging
ASEM: High resolution (after fixation)
Acknowledgement
The authors would like to thank to Dr. Yoshiyuki
Watanabe (Yamagata Research Institute of Technology)
for manufacture of SiN membranes.
This work was supported by matching funds from
the National Institute of Advanced Industrial Science and
Technology (AIST) and JEOL Ltd., Japan; by a Grant-inAid for Science Research on Priority Areas, Structure of
Biological Macromolecular Assemblies.