Thermo Scientific Vitrobot Mk IV User manual

PN 103261
Revision D • 23-Oct-19
Vitrobot Mk IV
User Manual

Contents
1Introduction.......................................................................................................3
1.1 Design Change....................................................................................................4
1.2 Design Philosophy...............................................................................................5
1.2.1 Theoretical analysis of specimen thermodynamics...................................5
1.2.2 Thermodynamic analysis of specimen evaporation ..................................6
1.3 Technical Specification........................................................................................7
1.3.1 Key Features............................................................................................7
1.3.2 Instrument Specifications .........................................................................8
2Safety.................................................................................................................9
2.1 General Safety.....................................................................................................9
2.2 Messages and Symbols.......................................................................................9
2.3 Mechanical Moving Parts...................................................................................11
2.4 Electronics.........................................................................................................11
2.5 Thermal.............................................................................................................12
2.5.1 Liquid Nitrogen specific safety precautions.............................................13
2.6 Climate Chamber...............................................................................................14
2.7 Safety Data Sheets (SDS) .................................................................................14
2.8 Biohazard ..........................................................................................................14
2.9 Decontamination and Decomissioning...............................................................15
3Operating Instructions ...................................................................................16
3.1 Starting Up –Filling the Humidifier.....................................................................16
3.2 Starting Up –Switching on the Vitrobot..............................................................17
3.3 Defining the Vitrobot User Interface...................................................................18
3.4 Preparation of the Climate Chamber..................................................................22
3.5 Preparation of the Grid.......................................................................................23
3.6 Preparation and Lifting of the Coolant Container................................................24
3.7 Sample Application............................................................................................26
3.8 Blotting and Vitrification .....................................................................................27
3.9 Transfer of Vitrified Grid.....................................................................................28
3.10 Summary - Vitrification Control through UI................................................30
3.11 Shutting down –Emptying the humidifier..................................................31
4Troubleshooting..............................................................................................32
4.1 Guidelines for Optimal Specimen Preparation ...................................................32
4.2 General Remarks on the Operational Procedures..............................................33
4.3 Troubleshooting.................................................................................................34
5Revision History..............................................................................................36
6Index ................................................................................................................37

Chapter | Introduction
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Page 3
1 Introduction
The VitrobotTM (Vitrification Robot) is a fully PC-controlled device for vitrification (= rapid
cooling) of aqueous samples. Minimal, yet essential, training is required when operating the
instrument for research purposes in a laboratory environment. The process of plunging,
blotting and vitrification is fully automated upon placing a vial in the chamber and setting up
the ethane holder. Operation is strictly controlled by dedicated hardware and software,
implying that parameters and the results are reproducible. This enables a high throughput of
vitrified samples, with an easy and straightforward control of the vitrification process. The
parameters to be influenced are: temperature, humidity, the number of blots, and a number of
critical time settings. The instrument will consistently produce excellent specimens for
cryo-electron microscopy. Research can now rely on specimen preparation allowing the
investigator to concentrate on structural studies of the specimen.

Chapter | Introduction
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Page 4
1.1 Design Change
In 2017 the design of the Vitrobot Mk IV was updated to the Thermo Scientific branding. There
are no mechanical or functional changes.

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1.2 Design Philosophy
Sample preparation for cryo-electron microscopy involves a few steps; application of the
sample to a specimen grid, blotting away excess liquid and shooting the thin (about 100 nm)
sample into liquid ethane.
In the study of thermal behavior of liposomes and intact cells by cryo-EM we found
unpredictable thermal and osmotic effects. These effects were ascribed to the dual effect of
evaporation; cooling the specimen to the dew point and concentration of solutes. Theoretical
analysis as well as experimental evidence indicated that the heat and mass exchange are
rapid processes in an aqueous thin film with a high surface to volume ratio (see figures
below). Time constants involved are a few tenth of a second or shorter. The environmental
humidity is a key parameter for sample evaporation. After preliminary experiments with
environmental controlled chambers it was decided to design and construct a fully
self-contained system that included sample application, blotting and rapid cooling into ethane.
A first prototype was built and automatic blotting was one of the key features of this PC
controlled prototype. After 4 years of experience with this prototype it became clear that the
cryo-EM world was in demand of reliable specimen preparation unit. The VitrobotTM as
presented should fulfill this demand. The Vitrobot (patent pending) is the result of a
complete redesign, involving hardware, electronics and software. The instrument is tested in
research groups with an established reputation in cryo-EM.
1.2.1 Theoretical analysis of specimen thermodynamics
The visualized experiment describes the behavior of a thin film of an aquous specimen at
various temperatures in an environment of 40 °C and a relative humidity of 40 %. Thickness of
water layer is as indicated.

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1.2.2 Thermodynamic analysis of specimen evaporation
The underlying graph illustrates that the evaporation velocity is independent of film thickness
but that the evaporation depends on the relative humidity in the reaction/climate chamber. A
constant ice thickness is therefore easier to achieve at higher humidity. Furthermore it is clear
that thin film preparation under room conditions (e.g. 20 °C and a relative humidity of 40 %)
may result in an appreciable loss of water; in the two seconds of a typical preparation 80 nm
of the thickness of the thin film is evaporated. This evaporation has not only an effect on the
temperature (preceding paragraph) but also on the solute concentration (N.B. osmotic
effects!) and the concentration of the suspended particles.
Inside the climate chamber of the Vitrobot, a small micro-thermocouple is present which
measures the temperature in the chamber. In the figure below the temperature of the
specimen was measured during the vitrification process i.e. during dipping in a vial,
withdrawal, blotting and the last moments before the freezing. The thermal history was
estimated with an environmental humidity of 40 % (blue) and > 99 % (magenta) respectively
while the chamber temperature was at 38°C. Also note the slow/insufficient equilibration of the
temperature of the sample in the vial.

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1.3 Technical Specification
1.3.1 Key Features
•Temperature: software controlled, direct read out, and range: 4 - 60C. Peltier type of
heating/cooling.
•Humidity: software controlled, direct read out, and range: room conditions up to 100 % rH.
Ultrasonic humidifier.
•Sample application: software controlled dipping/withdrawing from a vial. Small sample
volumes can be applied manually through a side port allowing access to a pipette. The
time between sample application and blotting (“wait time”) is controlled by software.
•Blotting: removal of excess liquid by a controlled blotting action using filter paper on
(rotating) foam pads. The number of blotting actions (1 - 16) as well as the duration of the
blotting action is software controlled. Time between blotting and vitrification (“drain time”)
can be set.
•Vitrification: smooth acceleration of the thin film to the liquid ethane container. A shutter
opens the chamber automatically to give the specimen access to the liquid/solid ethane
(-178C). Tailored damping action prevents bouncing of the specimen during vitrification.
A lift for the ethane container brings the coolant as close as possible to the shutter to
ensure optimal vitrification. The carriage of the vitrified specimen is coupled to the ethane
lift and by lowering this assembly the vitrified specimen can be retrieved and stored for
further investigations.
•Process control: the entire vitrification process is controlled by a built in PC. Dedicated
software is used for process control and this program allows tailoring of relevant
parameters to fit the specimen and the design of experiments.

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1.3.2 Instrument Specifications
The Vitrobot is designed and is strictly meant for use in a fume hood exclusively and is to be
operated by trained personnel only. Users of the Vitrobot are required to have basic
knowledge of the vitrification process.
Power supply
Voltage
100 - 240 Vac
50 - 60 Hz
Fuse
6.3 mains fuse (90 - 150 V)
3.15 mains fuse (150 - 260 V)
Full load
0.25 kVA
Conditions of use
Ambient temperature
18 - 25 C
Storage temperature
- 40 - 70 C
Relative ambient humidity
lower than 85 % rH, not condensing
Dimensions and connections
Power cable
90 - 250 V, euro female entry
Wiring configuration
2 wires + ground, single phase
Weight
31 kg
Dimensions (h x b x d) in mm
890 x 260 x 413
Footprint in mm
310 x 450

Chapter | Safety
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2 Safety
2.1 General Safety
•One should abide by the rules of the laboratory and local authorities to secure safe
working conditions.
•The Vitrobot has been designed for use in a fume hood exclusively and is to be operated
by trained personnel only.
•Working with explosive materials like ethane and propane requires working in a spark free
fume hood. Working with these materials is at own risk!
•Only use the Humidifier with demineralized, distilled water. Any other liquids may cause
damage to the Vitrobot.
•To prevent any formation of bacteria in the pre-heated water of the humidifier, it is
advisable to dispose the water in the humidifier at the end of each working day!
•Working with Biohazard materials is at the user’s own risk and responsibility!
•Do not perform internal trouble shooting unless you are a trained service person and
unless another person, capable of rendering first aid or resuscitation, is in the immediate
vicinity.
•Be aware of the location of the nearest phone.
•Be aware of the emergency services number.
•Always ensure a safe environment when performing service or maintenance.
•Make sure your hands are dry and that you are standing on a dry, insulated surface,
capable of withstanding the accessed voltages.
•Make sure the Vitrobot has been leveled to avoid the chance of toppling over.
•FEI cannot be held responsible for the consequences of improper handling!
2.2 Messages and Symbols
Messages
The following messages are used throughout the manuals to highlight information.
Note Text
A Note message indicates the information in it requires special attention.

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CAUTION!
Text
A Caution message indicates a potentially hazardous situation that, if not avoided, may result
in moderate or minor injury. It may also be used to alert against unsafe practices.
WARNING!
Text
A Warning message indicates a potentially hazardous situation that, if not avoided, may result
in death or severe injury.
DANGER!
Text
A Danger message indicates an imminently hazardous situation that, if not avoided, will result
in death or severe injury.
Explanation of warning symbols
CAUTION!
Cold substances could be present. Extremely low temperatures are reached using
Liquid Nitrogen. Precautions must be taken where this sticker is visible.
Location: On the LN2container.
CAUTION!
Heated surfaces. Extreme temperatures may be present. Take necessary precautions.
Location: In climate chamber.