The NanoSuit®, Imaging living organisms in a

scanning electron microscope

Prof. Dr. Takahiko Hariyama(Hamamatsu University School of Medicine)

Convener: Prof. Dr. Olaf Karthaus

Biomimetics: Learning from Nature for an Innovative Future 23. Japanese-German Symposium

April 20 and 21, 2018 in Frankfurt

11:00-11:30

Humans are called as a vision animal.

1600～ Light microscope Netherland

Paradigm shift life science （Organisms are composed of cells.）

Paradigm shift by new innovation.

1930～ Electron microscope 1950～ popular equipment for science.

Paradigm shift life science （Virus, Organelles）

1 nm 10 nm 100 nm 1 μm 10 μm 100 μm 1 mm 10 mm 100 mm

Naked eye

Light microscope

Electron microscope（high vacuo）

ProkaryoticEukaryoticVirus Multicellular

indispensable tool !!

Paradigm shift

1838

M.J.Shleiden

1839

T.Shwann

1986Ernst Ruska

I do want to observe the real structure of

organism in an electron microscope!

Absolutely impossible!

10-3 – 10-6 Pa

Arthropoda

Drosophila larva

Pease R.F., T.L. Hayes, A.S. Camp and N.M. Amer (1966) Electron microscopy of living insects. Science 154, 1185–1186.

Electron micrographs of living specimens of the various developmental stages of the insect Tribolium confusum have been obtained with a scanning electron microscope. In most cases the specimens resumed their normal activity after being examined with the electron microscope and under went metamorphosis into the next stage.

Red flour beetleTribolium castaneum

Young scientists encouraged me!

Observe several organism/tissures/cells in a FE-SEM.

The pressure level of the SEM which used in the former report was 10 -2 Pa.We would like to observe several living specimens with an FE-SEM (10-3-10-7 Pa).

Larva of Drosophila melanogaster in FE-SEMArthropoda

Drosophila larva

Using ECS with electron or plasma irradiation,

we could make “nanospacesuit” ・・・

100nm

“nanospacesuit”

SEM TEM

0min 60min

nano-suit+

0.3mm 1µm 200nm

0min 60min

plasma

NanoSuit+

0.3mm 1µm 200nm

l

Electron beam

control

0min 60min

0.3mm 1µm 200nm

With electron beam

Without electron beam

With plasma irradiation

When the animal possesses ECS and when irradiated by electrons or plasma, they can survive in high vacuo.

We chose amphiphilic molecules. （1% Tween20） and irradiated.

Next, we must keep living animal which donot possess ECS on their surface・・・

ECS mimetic substance

Biomimetics

Polysorbate 20（Tween 20）

500 μm

Arthropoda

200 μm

death

Mosquito larva

larva of a mosquito, Aedes albopictus FE-SEM

Without nano-siut

With nano-siut

D EC 30min

control

B 0minA

plasma

0.3mm 0.3mm 200nm1µm

30min0min

plasma

nano-suit+

G I JHF

0.3mm 0.3mm 200nm1µm

ECS mimetic substance

By only the plasma irradiation there was no NanoSuit, but with the additional amphiphilic molecules and the plasma irradiation, we found the NanoSuit on the surface of the cuticle, and the fine structure of the mosquito larvae were ordered..

nano-siut

nanospacesiut

Covered with ECS mimic solution

Irradiated by electron beam or plasma

observation↓

↓

NanoSuit method

Living organisms

↓

Need three minutes. カップラーメン Cup RAMEN noodle

nano-siut

First Fixation

Second Fixation

Dehydration

Desiccation

Covered by metal

GAand/orPFA

OSO4

Gold

OSO4

↓

↓

↓

↓

↓

observation

conventional SEM method

Need at least a whole day.

After the report of PNAS・・・

0.75cm1.0cm 2.0cm

Window

(a)

(c)(b) (d)

Self-standing Membrane

We could make self-standing ultra-thinfilms from water soluble amphiphilic molecules.

Tissue and Cells

GE

30µm 10µm

D

H

10µm

10µm

A

5mm

C

10µm

Traditional MethodNanoSuitMethod

TissueMouse

（Peritoneum）

CellHuman

( fibroblast)

＜実施例３＞ヒト胃癌_x 低倍率

Human Stomach Cancer （Front line between cancer and normal tissue）

Cancer tissue

Front line

Normal tissue

１次化学固定

100 μmNanoSuit Method

SSE / nanosuit method

Normal tissue Cancer tissue

500 μm500 μm

Human Stomach Cancer （ normal and cancer tissue）

NanoSuit 法Fixed wet sample

After the observation of the surface, we can peeled

otbecause they are wet！！

20 μm

Human Stomach Cancer

Can see the real structure

through the transparent NanoSuit.

Need trainingTakes at least a day

Few minutes

The treated structure

almost seems the real,

but not

NanoSuit®

Need at least a whole day.

The “NanoSuit®” enabled the direct observation of hydrous biological samples.

Don’t you want to seewhat you cannot see?

We can now observesubcellular size

organic substances as it is.

For Biomimetics!

Members

Vielen Dank!