Vacuum Electron
Staining Apparatus
For safe and reproducible
electron staining on electron
microscopy samples

Filgen, Inc.

Overview

High reproducibility and safely operations

Principles
What is electron staining?
Electron staining is a method for enhancement image contrast of electron microscopy specimens which are composedwith light element (eg. polymer materials, biological specimens etc.), by binding heavy metals. For polymer specimens,
osmium tetroxide (OsO4) and ruthenium tetroxide (RuO4) are often used

A transmitted electron microscopy image of electronstained and untreated specimen, composited

What is “vacuum” electron staining?
Conventional electron staining is performed under atmospheric pressure by exposing specimen to gaseous OsO4 orRuO4 or immerses it to those solutions. However, it often causes overstaining or lack of reproducibility. Also, there is arisk for operators to expose to those staining agents which are highly poisonous.
In contrast, our patented technology, vacuum electron staining enables more precise and safer staining by performing
staining in vacuum. Staining time and gas pressure (that is, density of staining agent) in reaction chamber as well as
timing of gas introducing and exhausting are automatically controlled.

Benefits
Safe operation
Conventional

Risk for exposure to toxic staining agent withhigh sublimability. Drafter is required.

VacuumNo chance to exposure to toxic substance, withclosed system and multiple safety measure.

Effective for hygroscopic specimens

High reproducibility
Unable to control density of staining agent,

Conventional over staining occurs by remained gas.
VacuumAutomatic and precise control for staining timeand density of agent. Eliminates over staining.

Stain without atmospheric exposure

staining is performed by dry gas generated from OsO4 crystal(not aqueous solution)

Rapid and deeply staining
Less surface contamination
For determining optimal condition
Different staining time or density of agent can be set *only on 4chamber model.

➊Air isolation chamber, middle,
top-front view

Air isolation chamber, miniand main unit

Applications
For SEM/TEM samples• Enhancement image contrast for polymer specimens• Fixation for polymer specimens before ultramicrotome
sectioning• Reduction of outgassing from specimens at in electron
microscope chamber

For SEM samples• Elimination or reduction of charging effects on nonconductive specimens• Embrittilement of specimens before fracturing

Product appearance
Model# VSC4TWDH

Model# VSC1R1H
START button
Reactionchamber(s)Osmiumreservoir

Main SW
TimerDensity setting SW

High-resolution interactive touch panel controller*only on model# VSC4TWDH

Easy and institutive operation by
step-by-step visual guide

Save usage of staining agent thanks to
the function which activate/deactivatechambers individually

Easy to confirm staining settings
anytime from slide-in tab* Images are from Japanese model. English version is available.

➋Details

Operation flow

Setting

Whole process is completed by just press START button
after setting of time and gas density.

With the model# VSC4TWDH, all operation can be madeeasily by step-by-step guide on thouchpanel screen.

Place samples in chamber

Staining

Set staining time and gas density
Press START button to start staining
Sublimated OsO4 is automaticallyintroduced to chamber
OsO4 gas in chamber is automaticallyexhausted after the elapse of setting time
* Image are from Japanese model. English version is available.

Safety measuresInterlocking system with reaction chamber

Multiple safety features on Os reservoir

i.
ii.

i.
ii.
iii.
iv.

unable to open the chamber unless OsO4 is exhausted
unable to introduce OsO4 when the chamber open

Osmium absorption filter
Not require any ventilation systems. Osmium concentration in
exhaust gas is confirmed as safe level or lower by a third-party
organization

Gas-tight and robust design
Gas port integrated locking pin (see below)
Built-in ampoule cutter.
Detachable and capable to store in freezer

Failsafe system against power cut
No chance to leak OsO4 gas when power is cut and recovered

Mechanisms of OsO4 reservoir
The gas port integrated locking pin of reservoir can simultaneously control status for reservoir locking and gas
supply in safe.

Full-opened (during operation)
Reservoir： locked (undetachable) by
manually inserted locking pin ofreservoir
OsO4 gas： can be introduced(depends on solenoid status)➌Half-closed (before reservoirdetach)
Reservoir： locked (undetachable) by
manually inserted locking pin ofreservoir
OsO4 gas： blocked *residual gas in
the inlet pipe must be evacuated at
this step

Full-closed (after operation)
Reservoir： unlocked (detachable)
OsO4 gas： blocked）Process of vacuum electron staining
1253

4

1. Before processing
OsO4 is not introduced to chamber
Chamber is in vacuum
2. Gas introduction
Solenoid A is opened, and C is closed, just after start of the process
Sublimated OsO4 is introduced in chamber gradually
3. Gas density adjustment
All solenoids are closed after OsO4 density reaches to set level
4. Staining
Now staining is running in closed chamber
Solenoid C is opened and OsO4 in piping is evacuated
5. Post staining
After the elapse of set staining time, solenoid B is opened and all residual gas is evacuatedComparison: Vacuum electron staining vs. conventional
1. Before processing
2. Gas introduction
3. Gas density adjustment
4. Staining
5. Post staining

Vacuum electron staining
Inside of specimen as well as chamber aremaintained vacuum
Staining gas is immediately permeate to inside ofspecimen by high difference of pressure.
Gas density is automatically adjusted to set level(1-10 level)
Density of staining gas is maintained by gas-tightchamber with solenoids
Remaining gas inside specimen is sucked out by
negative pressure

Conventional method
Under normal pressure
Longer time is necessary to permeate staining
gas to inside of specimen.
Difficult to precise control of gas density

Staining gas is remained inside of specimen.

➍Products

Specifications
Model#Interface

Staining agent
Density of staining agent
Staining time
Reaction
Numberchamber
Inner size
Observation window
Heating system
Reservoir
NumberDetachable
Built-in ampoulecutterObservation window
Storage temp. *after
detachingGas introducing/exhausting system
Safety measure

Size
WeightVacuum pumpType
Actual pumping speed
Size
WeightAccessories

VSC4TWDH
VSC1R1HInteractive touch panel control Mechanical switches, knobs,
digital timer/temp controller,
and analog vacuum meters
OsO4 (crystals) in glass ampoule1-10 degree
1 min to 17hr
0.0-999.9 min
4186(ID) x 50(H) mm✔ (upper side)✔ (RT to 70 degC)1✔
✔
✔
-20 degC to 4 degC
Automated control with vacuum gauge, solenoids, and vacuum pump
Fully automated control system with interlocking
Residual gas trap unitGas tight reservoir with built-in ampoule cutter
610(W) x 445(D) x 510(H) mm
450(W) x 425(D) x 445(H) mm
Approx. 50 kg
Approx. 30 kg
Two-stage oil rotary vacuum pump
200 L @50Hz
170(W) x 515.5(L) x 249.5 (H)
31 kg
Gas-tight OsO4 reservoir, 1 pc.
Vacuum pump, 1 unit
Residual gas trap unit, 1 unit
Connecting parts
Cleaning kit
Instruction manual (English version, printed)

Accessories
OsO4 ampoules

Air isolation chamber
Enables electron staining under anaerobic
condition. Suitable for materials whichhave high activity or reactivity with oxygenor moisture (eg, Negative electrodematerials of Li-ion battery). 2 different
sizes are available

TEM Grid Holder

Osmium reservoir

Holds up to 8 pcs of TEM grid. Stainless Detachable, equipped with safety
steel madefeatures.

➎Transportation Container

Enable safer transportation for OsO4reservoir. Stainless steel made.

Technical data
Technical data #1
TEM images of cross sectioned ABS, OsO4 stained
Vacuum Electron Staining, 6 hrs

Vacuum Electron Staining, 65 hrs

SurfaceConventional staining, 72 hrs

Surface

Surface

Staining depth:
Approx 3 µmStaining depth:
Approx 9 µm

Staining depth:
Approx 35 µm

With Vacuum Electron Staining, deeper staining with shorter running time is possible.

Technical data #2
Reconstructed 3D images by electron tomography, RuO4 stained* Electron staining by RuO4 is available only in Japan

Vacuum Electron Staining

Conventional Electron Staining

Supportfilm

Sample thickness:
approx. 36 nm

Staining depth:
approx. 36 nm

Sample thickness:
approx. 43 nm

Staining depth:
approx. 15 nm

With Vacuum Electron Staining, uniform staining is possible.

Technical data #3
SEM images of Rubber-dispersed polymer with different blend ratio, OsO4 stained
Rubber, OsO4 stained Polymer, non-stained

Rubber : polymer
6:4

RubberPolymerRubber : polymer
4:6

RubberPolymerRubber : polymer
2:8

With Vacuum Electron Staining, dispersion of polymers can be evaluated.
➏Filgen, Inc.
Scientific Instruments Dept.
1-1409 Jonoyama, Midori-ku, Nagoya 459-8011
JAPANTEL: +81 52-624-4388
Email: si-support@filgen.jp
https://filgen.jp/(OCT 2019)