January 2005
JEOL User’s Meeting
JEOL (Europe) S.A.
Observation of Wet Specimens
Simple Freeze Dry and
LV CRYO Technique
Takeo Suzuki
JSM-6060LV
One of the latest Low Vacuum SEM from JEOL
Plant and Insect
in Low Vacuum SEM with no specimen preparation, 1993
Flower with a bug with no fixation(BSE)
Method
1. Fixation
2. Rinse in water
3. Freeze a specimen outside the LV SEM
4. Freeze dry in a LV SEM
5. Observation without conductive coating in LV
mode
6. Observation with conductive coating in HV mode
7. Store the specimen for future study
Comparison of Methods
Freeze drying in LV SEM
Conventional methods
Collection
Collection
Cleaning
Cleaning
Primary fixation
Primary fixation
less than
1h
Rinse in water
Secondary fixation
Rinse in buffer
over 24hrs
Dehydration
Replacement to
T-Butyl alcohol
Replacement to
Amyl acetate
Freezing
Quick freezing
Freeze drying
Critical point drying
Freeze dry in LV SEM
Coating
Coating
Observation
Observation
Observation
Development of the Technique
1. Observation of fresh water plankton - Drinking water supply quality control, 1993
2. Observation of plant, 1993
3. “A new drying method:
Low vacuum SEM freeze drying and its application to plankton observation”,
T. Suzuki, T. Toda et al, Bulletin of Plankton Society of Japan, 1995
4. “A simple Scanning Electron Microscopic Technique for Observing House Mites”,
M. Ujiie, T. Suzuki, M. Yoshikawa, House insects, 1995
5. “A Method for Identification of Cryptosporidium parvum from River Water using
Low-Vacuum SEM”,
J. Suzuki, T. Suzuki, et al, Japan Journal of Food Microbiology, 1998
6. “ Observation of Cysts of Acanthamoeba spp. by Low-Vacuum SEM”
H. Kasahara, T. Suzuki, T. Tsuruhara, Tokyo Gakugei Univ., 1999
7. “Observation of Waterborne Protozoan Oocysts Using a Low-Vacuum SEM”
Jun Suzuki, Rie Murata, Takeo Suzuki, Iwao Murata, The Tokyo Metropolitan Research Lab of Public Health, 2002
8. “Observation of Ciliate living in dead mosquito larva, its multiplication and cyst formation”
T. Takahashi*, T. Miyoshi*, T. Suzuki, R. Jotaki, T. Sunahara**, *Nishi-Kyushu Univ, **Saga Medical Univ. 2002
9. “Observation of nematode Caenorhabditis Elegans in Low-Vacuum SEM”
Kondo, E., Koga, T., Saga University; Suzuki, T., JEOL LTD, 2003
Sea Water Plankton
by Freeze Drying in Low Vacuum SEM
“A new drying method: Low vacuum SEM freeze drying and its application to plankton observation”,
Takeo Suzuki, Kazuaki Tanaka, Tatsuki Toda et al, Bulletin of Plankton Society of Japan, 1995
Ice being removed by sublimation.
Marine copepod Acartia specimen fixed in glutaraldehyde (BSE)
Freeze Drying in Low Vacuum SEM
Ice is removed by sublimation.
Marine copepod Acartia specimen fixed in glutaraldehyde
Plant
in Low Vacuum SEM with no specimen preparation, 1993
Pollen grain (cabbage) with no fixation(BSE)
Plant Cross Section
by Freeze Drying in Low Vacuum SEM, 1993
Cross section of leaf without fixation (BSE)
Red Blood Cell
by Freeze Drying in Low Vacuum SEM, 1994
Red blood cell with fixation and coating (SE)
Sea Water Plankton
by Freeze Drying in Low Vacuum SEM
“A new drying method: Low vacuum SEM freeze drying and its application to plankton observation”,
Takeo Suzuki, Kazuaki Tanaka, Tatsuki Toda et sl, Bulletin of Plankton Society of Japan, 1995
Abstract
A new preparation method for examining the micro structures of soft phyto- and zooplankton,
low-vacuum SEM freeze drying, is described through its application to observations of the marine
copepod Acartia sp. and the fresh-water blue-green algae Anabaena spiroides.
Copepod specimens fixed in glutaraldehyde solution are transferred to distilled water and rinsed.
After hydration with this water, specimens are dropped onto a specimen stub to which a piece of
double-sided adhesive carbon tape has been attached.
Specimens on the stub with their associated water are then rapidly frozen using liquid nitrogen.
The stub is then attached to a specimen holder for use in low vacuum SEM, and freeze dried in its
specimen chamber.
For fresh-water organisms, it is possible to omit the fixation step. Specimens collected from fresh
water are transferred directly into distilled water, rinsed, and then freeze-dried as above.
Scanning electron micrographs revealed that the drying method preserved the micro structures of
copepods and blue-green algae well.
Dehydration in a graded ethanol series is not necessary in the new method.
Low-vacuum SEM freeze drying is, therefore, a simple, time-saving and reproducible method for
scanning electron microscopy that is applicable to various aquatic micro organisms covered in soft
tissues or exoskeletons.
Sea Water Plankton
by Freeze Drying in Low Vacuum SEM
“A new drying method: Low vacuum SEM freeze drying and its application to plankton observation”,
Takeo Suzuki, Kazuaki Tanaka, Tatsuki Toda et al, Bulletin of Plankton Society of Japan, 1995
Alexandrium tamarence fixed with glutaraldehyde (BSE)
House Mite
by Freeze Drying in Low Vacuum SEM
“A simple Scanning Electron Microscopic Technique for Observing House Mites”,
Masayuki Ujiie, Takeo Suzuki, Midori Yoshikawa, House insects, 1995
Summary
A new simple technique for examining microstructures of house mites, low-vacuum SEM freeze drying,
was applied for observation of Dermatophagoides puteronyssinus, Chelacaropsis moorei and Tyrophagus
putrescentiae, although those mites had been previously examined by a complicated SEM technique.
Water with a surface active agent was dropped on a specimen stub to which a piece of double-faced carbon
tape was attached. A living or dead mite was dropped into the water on the stub and frozen by pouring
liquid nitrogen around the stub. The stub is then set to a specimen holder in low-vacuum SEM to
dehydrate the specimen in a chamber. Mites could be observed at this stage. Besides, the specimen could
be observed in high vacuum SEM after it was evaporated using gold.
This new technique shortened the time for dehydration with less failure in specimen-preparation, and
showed favorable observation compared with previous complicated techniques.
House Mite
by Freeze Drying in Low Vacuum SEM
“A simple Scanning Electron Microscopic Technique for Observing House Mites”,
Masayuki Ujiie, Takeo Suzuki, Midori Yoshikawa, House insects, 1995
specimen courtesy of Tokyo Metropolitan Research Laboratory of Public Health
Mite Dermathophagoides Pteronyssinus without fixation (BSE)
House Mite
by Freeze Drying in Low Vacuum SEM
“A simple Scanning Electron Microscopic Technique for Observing House Mites”,
Masayuki Ujiie, Takeo Suzuki, Midori Yoshikawa, House insects, 1995
specimen courtesy of Tokyo Metropolitan Research Laboratory of Public Health
Mite Tyrophagus Putrescentiae without fixation (BSE)
Cryptosporidium Parvum
by Freeze Drying in Low Vacuum SEM
“A Method for Identification of Cryptosporidium parvum from River Water using Low-Vacuum SEM”,
Jun Suzuki, Iwao Murata, Rie Murata, Satoshi Murozumi, Takeo Suzuki, Japan Journal of Food Microbiology, 1998
Abstract
In J une 1996, the outbreak of waterborne cryptosporidiosis in Saitama prefecture, J apan caused
prolonged diarrheal illness in more than 8,000 people who drank tap water contaminated with
Cryptosporidium parvum oocysts.
The Ministry of Health and Welfare had to introduce a provisional determination method of
Cryptosporidium consisting of an immunofluorescence assay and Nomarski differential interference
contrast microscopy. For river water samples, however, this method cannot distinguish C. parvum from
other C. parvum-like organisms such as free-living protozoan because of non-specific fluorescence and
structural resemblance to C. parvum oosysts.
In this study, the microstructure of C. parvum oocysts of a sample was directly observed using a
low-vacuum scanning electron microscope (SEM). The sample used for observation river water to which
concentrated C. parvum oocysts were previously added. The sample was filtered on a specimen stub,
washed with distilled water, then frozen with liquid nitrogen.
As a result of this method, C. parvum oocysts could be observed at a >2,000x magnification and the
fine microstructure such as oocysts-wall and four sporozoites could be seen at 10,000x magnification.
Therefore, these were concluded that the low-vacuum SEM method is useful for discrimination and
identification of C. parvum oocysts.
Cryptosporidium Parvum
by Freeze Drying in Low Vacuum SEM
“A Method for Identification of Cryptosporidium parvum from River Water using Low-Vacuum SEM”,
Jun Suzuki, Iwao Murata, Rie Murata, Satoshi Murozumi, Takeo Suzuki, Japan Journal of Food Microbiology, 1998
Cryptosporidium parvum with no fixation (BSE)
Cryptosporidium Muris
“Observation of Waterborne Protozoan Oocysts Using a Low-Vacuum SEM”,
Jun Suzuki,, Rie Murata, Takeo Suzuki, Iwao Murata, JEOL News Vol.37E, 2002
The Tokyo Metropolitan Research Laboratory of Public Health
By direct immunofluorescence assay
By freeze dry in Low Vacuum SEM
By Normarski differential
interference contrast microscopy
Cryptosporidium muris
fixed with 10% neutral formalin (BSE)
Acanthamoeba spp.
by Freeze Drying in Low Vacuum SEM
“ Observation of Cysts of Acanthamoeba spp. by Low-Vacuum SEM”
Kasahara, H., Suzuki, T., Tsuruhara, T., Tokyo Gakugei Univ., 1999
Abstract
The life cycle of Acanthamoeba is divided into two distinct phases, trophozoite
and cyst. And the classification of Acanthamoeba species is now mainly based on
the cyst morphology. In order to get further information for the cysts, we carried
out the precise morphological changes of cysts of various Acanthamoeba spp. using
a low-vacuum scanning electron microscope (SEM) freeze-drying method. The use
of low-vacuum SEM mode permits observation of specimens which have not been
coated with a conductive material such as gold or carbon, and so this mode is useful
for examining both surface and intra-cyst structures.
Acanthamoeba spp.
by Freeze Drying in Low Vacuum SEM
“ Observation of Cysts of Acanthamoeba spp. by Low-Vacuum SEM”
Kasahara, H., Suzuki, T., Tsuruhara, T., Tokyo Gakugei Univ., 1999
1. The specimen was dropped on the SEMpore membrane filter.
2. The specimen was fixed in 0.05% glutaradelyde & 1.5% paraformaldehyde
with 0.1M cacodylate buffer (pH 7.2)
3. The specimen was dehydrated and rinsed through 50, 70, 90, 100%
t-butyl alcohol.
4. The specimen was frozen in liquid nitrogen.
5. The specimen was freeze dried in the low vacuum SEM.
6. The specimen was observed at the pressure between 20 and 60 Pa.
Acanthamoeba spp.
by Freeze Drying in Low Vacuum SEM
“ Observation of Cysts of Acanthamoeba spp. by Low-Vacuum SEM”
Kasahara, H., Suzuki, T., Tsuruhara, T., Tokyo Gakugei Univ., 1999
Cysts of Acanthamoeba culbertsoni and Trophozoite coming out from Cysts
Simple CRYO Technique in LV SEM
1.
Most of specimens can be observed with the simple freeze dry in LV SEM.
2.
Some specimens are fragile in the vacuum and shrinks during observation.
3.
These specimens can be observed easily using a large heat sink.
4.
A specimen is placed in the heat sink and is frozen in liquid nitrogen in the same
way as the simple freeze dry.
5.
Insert the specimen with the heat sink into the LV SEM chamber. Use a cap over the
specimen to minimize condensation of frost over it.
7.
Low vacuum observation:
Keep the pressure at about 30Pa.
The specimen can be observed in 20 to 30 minutes with all the frost removed by
sublimation.
Simple CRYO Technique in LV SEM
Heat sink
Anti-frost cap
Specimen
holder
Specimen mounting block
Only simple tools are required for the simple CRYO method.
Simple CRYO Technique in LV SEM
Top view
Specimen mounting block
Bottom view
Heat sink
Insulator
The large heat sink keeps a specimen frozen for over 30 minutes.
Procedure of Cryo-Observation of Mold
Procedure of Low Vacuum Cryo-Observation of Mold
1.
2.
3.
4.
5.
6.
7.
Cultivation of Mold:
Agar medium of 3 to 5mm thick
Collection:
Cut it out by a knife into about 3mm x 3mm x 1mm
Specimen mounting:
With using starch glue stick the cut out specimen on a piece of carbon double-side
sticky tape on the cooling holder.
Freezing specimen:
After putting the anti-frost cap over the specimen, cool the holder with liquid nitrogen.
Insert the cooled holder in the 51mm specimen holder quickly.
Insert the specimen holder into an SEM specimen chamber and remove the cap right
before pressing EVAC button.
Low vacuum observation:
Keep the pressure at about 30Pa.
The specimen can be observed in 20 to 30 minutes with all the frost removed by
sublimation.
Note: A specimen, which shrinks easily, requires pre-treatment with vapor fixation
by using Osmium Tetroxide.
Mold
by Freezing in Low Vacuum SEM, 1998
Specimen courtesy of Tokyo Metropolitan Research Laboratory of Public Health
Penicillium oryzae without fixation (BSE)
Mold
by Freezing in Low Vacuum SEM, 1998
specimen courtesy of The Flower Center 21 in Aomori
Powdery mildew of Babys breath Oidium sp. without fixation (BSE)
Apple
by Freezing in Low Vacuum SEM
Cut surface of apple with the simple CRYO in LV SEM (BSE)
Caenorhabditis Elegans
by Freeze Drying in Low Vacuum SEM
Kondo, E., Koga, T., Saga University; Suzuki, T., JEOL LTD, 2003
specimen courtesy of Prof.Kondo,E., Saga University
7 degree tilt
Stereo pair
0 degree tilt
The specimen (nematode) was fixed and dehydrated. Alcohol was then replaced with t-butyl alcohol.
The specimen was frozen in liquid nitrogen and freeze dried in the Low Vacuum SEM.
Caenorhabditis Elegans
by Freeze Drying in Low Vacuum SEM
Kondo, E., Koga, T., Saga University; Suzuki, T., JEOL LTD, 2003
specimen courtesy of Prof.Kondo,E., Saga University
The specimen was fixed and dehydrated. The specimen was dried with Critical Point Dryer.
The specimen was coated and observed with JEOL FESEM.
Nematode on Root
by Freeze Drying in Low Vacuum SEM
Specimen courtesy of
Prof. Dr. Abdul Rahman Razak,
University Putra Malaysia
Nematode:
Root:
Tylenchulus semipenetrans
Citrus Reticulata
Tetrahymena spp.
by Freeze Drying in Low Vacuum SEM
“ Observation of Tetrahymena spp. by Low-Vacuum SEM”
Suzuki, T., Takahashi,T, Nishikyushu University., 2002
BEI
Ciliate living in dead mosquito larva
Tetrahymena spp. with fixation
SEI
SEI
Tetrahymena spp.
by Freeze Drying in Low Vacuum SEM
“ Observation of Tetrahymena spp. by Low-Vacuum SEM”
Suzuki, T., Takahashi,T, Nishikyushu University., 2002
X6,000
SEI
X4,000
Ciliate living in dead mosquito larva
Tetrahymena spp. with fixation (SEI)
Summary
Advantages of
Simple CRYO Technique in LV SEM
• Specimens, which shrinks, can be observed without
damage with the freeze dry method.
• This is a simple and quick method.
• This technique does not require specialized tools,
such as cooling stage in a Low Vacuum SEM.
SEM Pore
• The SEMpore specimen mount is made with a Polycarbonate (PC)
filter’s membrane, supported on hard conductive resin stub.
• SEMpore is used for observation of microorganisms and
microparticles.
• The PC membrane pores are 0.6 µm of diameter.
• SEMpore: 15 mm dia.
• Filter: 10 mm dia.
• Package 50 pieces
SEMpore specimen mount (7801 31801)
SEM Pore
Red blood cells to which are
stuck Staphylococcus aureus
• Fitted to an
injector,
SEMpore carry out
filtering easily.
Red blood cells
SEM Pore
Low Vacuum mode
High Vacuum mode
Sample: Diatoms not coated
SEM Pore
Sample: Diatoms not coated with FEG SEM
SEM Pore
0.2 kV x 100.000
Sample: Diatoms not coated with FEG SEM
SEM Pore
• A wide variety of specimen holders and accessories is available.
Thank you for your kind attention!
JSM-6380LA
JSM-6380LA
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Electron Optics Division
JEOL LTD
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JSM-6380LV
JSM-6380LA
Mini cup
EDS
detector
SEM monitor
EDS monitor
SEM and EDS in one PC
The new product JSM-6380LA is a High resolution analytical SEM with EDS.
Smile Shot
The optimum operation condition is automatically set by simply
checking the kind of specimen and its condition.
Operation GUI
The operation GUI is simple and easy to understand.
It is not necessary to look for functions hidden under the live image area.
Operation GUI
The 10 icons in the area marked with a red box can be customized
from 40 icons.
Smile View
Smile View is a versatile and flexible report creation software. A
report with SEM images can be edited quickly.
Accelerating Voltage
20 ｋ V
１ｋ V
５ｋ V
The adjustment of the electron gun after changing voltage is fully automated including
the alignment and the gun bias voltage.
The astigmatism correction is almost done with the JEOL unique stigma-memory.
Probe Current Control
Focus change after changing the probe current is minimal due to
the JEOL unique zoom condenser lens.
Thank you for your kind attention!
Cryptosporidium Cayetanensis
“Observation of Waterborne Protozoan Oocysts Using a Low-Vacuum SEM”,
Jun Suzuki,, Rie Murata, Takeo Suzuki, Iwao Murata, JEOL News Vol.37E, 2002
The Tokyo Metropolitan Research Laboratory of Public Health
By direct immunofluorescence assay
By freeze dry in Low Vacuum SEM
By Normarski differential
interference contrast microscopy
Cyclospora cayetanesnsis
fixed with 10% neutral formalin (BSE)
Isospora Belli
“Observation of Waterborne Protozoan Oocysts Using a Low-Vacuum SEM”,
Jun Suzuki,, Rie Murata, Takeo Suzuki, Iwao Murata, JEOL News Vol.37E, 2002
The Tokyo Metropolitan Research Laboratory of Public Health
By direct immunofluorescence assay
By freeze dry in Low Vacuum SEM
By Normarski differential
interference contrast microscopy
Isospora belli
fixed with 10% neutral formalin (BSE)
Tetrahymena spp.
by Freeze Drying in Low Vacuum SEM
“ Observation of Tetrahymena spp. by Low-Vacuum SEM”
Suzuki, T., Takahashi,T, Nishikyushu University., 2002
specimen courtesy of Prof.Takahashi,T., Nishikyushu University
(SEI)
Tetrahymena spp.
by Freeze Drying in Low Vacuum SEM
“ Observation of Tetrahymena spp. by Low-Vacuum SEM”
Suzuki, T., Takahashi,T, Nishikyushu University., 2002
specimen courtesy of Prof.Takahashi,T., Nishikyushu University
(SEI)
Ciliate living in dead mosquito larva