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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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.

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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.

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CAUTION!
Pinch hazard! Hand surface injuries can occur when the Ethane container lift is
raised or lowered. Keep hands away below or above the Ethane container lift when it
is operating.
Location: Below Ethane container lift.
DANGER!
Indications that there are lethal voltages present in the electronics compartment.
Take necessary precautions when removing the covers.
Location: In electronics compartment.
CAUTION!
Protect ESD sensitive devices from electrostatic discharge.
Always work with:
• An ESD wristband.
• An ESD table mat.
•ESD-safe gloves.
Location: On ESD bags of spare parts.
2.3 Mechanical Moving Parts
CAUTION!
Pinch hazard! Hand surface injuries can occur when the Ethane container lift is
raised or lowered. Keep hands away below or above the Ethane container lift when it
is operating.
•Always keep hands away from the hole below the climate chamber. A pinch hazard can
occur if the ethane container is missing on the ethane lift, tweezer is mounted and a
plunging cycle is performed.
•During the regular use of the Vitrobot, mechanical parts will move. The chamber door
needs to be closed for the vertical metal rod to operate. The rod will be plunged at high
speed in the vertical downward direction. Until the metal rod is decelerated and completely
stopped in its most downward position, keep your hands away from the space right
underneath the Vitrobot chamber.
•Vitrobot tweezers have a pointy, sharp tip; care must be taken to handle the tweezers with
care, as direct contact with the sharp tip will result in a skin puncture and cut. Handle the
Vitrobot tweezers as any other sharp object.
•If there is any suspected malfunction of any of the mechanical moving parts, do not
attempt to fix it yourself. Always turn off the instrument and contact the Thermo Fisher
Scientific service to schedule an Thermo Fisher Scientific service engineer on-site visit.
2.4 Electronics

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DANGER!
Indications that there are lethal voltages present in the electronics compartment.
Take necessary precautions when removing the covers.
Note Users are not allowed to take of any cover. Lethal voltages are present inside the
Vitrobot.
•Users should notify Thermo Fisher Scientific service immediately when any electrical error
occurs.
Fuses
Only trained service personnel should replace fuses. Replace only with fuses of the same
type, voltage rating, and current.
2.5 Thermal
CAUTION!
Cold substances could be present. Extremely low temperatures are reached using
Liquid Nitrogen. Precautions must be taken where this sticker is visible.
Metallic parts of Vitrobot liquid nitrogen container
The Vitrobot will be provided with metallic parts, which are cooled with liquid nitrogen during
use. During normal use, these get extremely cold (-196 °C). These metallic accessories, once
cold, should not be handled by hand directly, but with appropriate tweezers covered with an
insulating material, to avoid skin burn.
Liquid Ethane/Propane
When used in the appropriate small container provided with the Vitrobot accessories, a small
volume (~4 ml) of liquid ethane/propane is formed by cooling the gases at liquid nitrogen
temperature; there is a risk of skin burns upon direct contact with liquid ethane or liquid
propane or a mixture of both. Use protective eyewear during the entire session.
•Ethane and Propane gases are highly flammable and, along with Nitrogen, pose an
asphyxiation hazard. They should only be used in a well-ventilated, spark-free
environment. It is recommended to use the Vitrobot in a fume hood, installed in a lab
where there is an Oxygen level detector.
•It is recommended that a small Ethane/Propane gas bottle is used in the lab where the
Vitrobot is installed; usually 2.5 l –5.0 l; the reduced dimensions should facilitate storage
of the gas bottle in a fire-proof cabinet when not in use.
•A pressure regulator with two dedicated gauges (100 psi to 5 psi) is needed to extract the
gas in a regulated, constant and safe manner.

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Liquid nitrogen (LN2)
Liquid Nitrogen is a cryogen (-196 °C) and should be handled with care and with appropriate
personal protective equipment (eye protection, cryo gloves,lab coat). Direct contact with liquid
nitrogen will cause severe frostbite of skin and eyes. A 4.0 l container for the Liquid Nitrogen
is recommended to work near the Vitrobot, larger volumes are usually not required for a
Vitrobot session aiming at freezing 4-12 samples (= 4 to 12 grids).
The Vitrobot has a LN2container with a cup filled with liquid ethane to vitrify samples. Only
authorized personnel should carry out replenishment and the SDS for liquid Nitrogen (refer to
SDS overview) should be read and understood. The use of liquid Nitrogen can give a risk of
asphyxiation. Be careful when handling pressurized gas of any kind. Thermo Fisher Scientific
advises to install a separate oxygen detector in the room. Most cryogenic liquids are odorless,
colorless, and tasteless when vaporized. When cryogenic liquids are exposed to the
atmosphere the cold boil-off gases condense the moisture in the air, creating a highly visible
fog.
2.5.1 Liquid Nitrogen specific safety precautions
Wear safety goggles or a full faced mask and cryogenic gloves when handling Liquid
Nitrogen. Special clothing may be advisable. It is preferable to wear trousers outside of boots
or work shoes. Cryogenic gloves are for indirect or splash protection only, they are not
designed to protect against immersion into cryogenic liquids!
•Always handle these liquids carefully to avoid skin burns and frostbite. Dewars must be
moved carefully. Sloshing liquid into warmer regions of the container can cause sharp
pressure rises. Carry the vessel with both hands and as far away from your face as
comfortably possible.

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•Use a cryogenic glove and/or tongs to handle any object going into or out of the Liquid
Nitrogen.
•Dewar flasks are under vacuum to provide insulation and can collapse from thermal shock
or slight mechanical shock.
•Before usage, the inside of the Dewar flask should be inspected for etched surfaces,
cracks or pinholes which could cause an explosion. When damage is observed the flask
should be replaced. If there is a small leak in the flask e.g. developed when it is frozen
and some liquid “enters” the flask vacuum compartment it will expand when the flask is
warmed up, (expansion ratio is 696:1) and cause an explosion.
•If cryogenic liquid or cold boil off contacts a worker's skin or eyes, frozen tissues should
be flooded or soaked with tepid water (60-100 F, 16-38 °C). DO NOT USE HOT WATER.
Cryogenic burns which result in blistering or deeper tissue freezing should be seen
promptly by a physician.
Note For more information about possible dangers, causes and recommendations when
using a Dewar flask, see manufacturer’s safety instructions.
2.6 Climate Chamber
CAUTION!
Heated surfaces. Extreme temperatures may be present. Take necessary precautions.
The Vitrobot chamber can be operated at a temperature range between 4 °C and 60 °C; when
used at hot temperatures, care should be taken not to touch hot surfaces to avoid skin burn.
2.7 Safety Data Sheets (SDS)
For Safety Data Sheets, please refer to your chemicals supplier.
2.8 Biohazard
•When used in a ML-II (BSL-2) or higher category lab, especially when working with
infectious biological material, the Vitrobot pads can be protected to avoid contamination by
covering them with a piece of Parafilm M® plastic sheet, carefully placed between the
sponge material of the pads and the filter paper disc used for blotting the samples prior to
the plunging session.
•After use, both the filter paper disc and the Parafilm M® sheet should be disposed
according to the EH&S rules and regulations in vigor depending on the BioSafety level and
local officer guidelines.

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2.9 Decontamination and Decomissioning
Decontamination and Decommissioning
Some of the system construction materials are recyclable. The system is comprised mainly of
steel, aluminum, copper, and lead. Standard PVC insulation is used on most cables.
Additionally, the system contains components included in standard electronic equipment.
Consult your local environmental agencies for decontamination and cleaning procedures and
for recycling and disposal of electrical equipment. Process chemicals must be disposed of
according to local environmental requirements.
According to the Waste Electrical and Electronic Equipment (WEEE) directive, these products
of Thermo Fisher Scientific are classified as “monitoring and control instruments”
(WEEE-category 9). In accordance with the requirements of the WEEE-category classification,
the following applies:
For its products sold in the European member States after 13 August 2005, Thermo Fisher
Scientific guarantees the users of Thermo Fisher Scientific equipment that collection,
treatment, removal, and environmentally sound disposal of this product at end of life is part of
the transaction.
Go to https://www.fei.com/recycling for details.
Unless Member State legislation requires a different approach, Thermo Fisher Scientific policy
for older equipment that was on the market before 2005-08-13 (referred to as “historical
waste”) is that all costs shall be borne by the users.
Customer is responsible for materials, compound and biological materials which are analyzed
with the system. The proper decontamination is in responsibility of device user.

Chapter | Operating Instructions
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3 Operating Instructions
In this chapter, the complete operation of the Vitrobot, both hardware and software is
stepwise explained and where possible visualized.
3.1 Starting Up –Filling the Humidifier
Prior to activation of the Vitrobot, the humidifier must be filled with distilled water. For this, the
syringe needs to be filled with 60 ml of water before filling the humidifier through the plastic
tube at the bottom part. After injection of the water into the humidifier, it is important that an
“under-vacuum” is created inside the syringe (by “de-filling or pulling” the syringe while
keeping attached to the plastic tube). By pulling out “air” from the humidifier, the humidifier is
properly filled with water (see also the instruction movie for further details).
The humidifier is bajonet-attached to the bottom of the climate chamber. By turning and
pulling, the humidifier can be removed. For further details on the exchange or refreshment of
the water see:
on page 30

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3.2 Starting Up –Switching on the Vitrobot
Prior to switching on the Vitrobot by using the hard lock switch on the backside of the Vitrobot,
make sure all cables and wires are properly connected.
Note Be aware to fill the humidifier beaker with sufficient distilled water prior to enabling
the ultrasonic humidification!
Note The humidifier has to operate on distilled water. Any other liquids used may cause
damage to the VitrobotTM.
After activation of the hard lock switch the embedded PC with Linux operating system is
automatically starting up. The Vitrobot User Interface page will appear after a few seconds.

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3.3 Defining the Vitrobot User Interface
The Vitrobot User Interface consists of two pages: the Console and the Options screen. In
both pages, a variety of vitrification parameters can be set.
In the console screen the temperature can be set towards any value between 4 °C and 60 °C
with the + and –buttons or drag the black marker up or down. The actual temperature
read-out is displayed in red.
Similarly, the humidity –displayed in green - in the climate chamber can be set. Select the
desired humidity value of choice with the + and - buttons or drag the black marker up or down
and enable the humidity switchbox to start the evaporation (On/Off switch).

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The light in the climate chamber can be switched off as well (“Light is On” changes into “Light
is Off” upon activation).
A chronometer is added to record experimental times. Once a specific time is set, the
chronometer starts to count down displaying a counterclockwise movement of the clock.
In the Options screen, additional vitrification parameters can be set. For example –under the
section Process Parameters - the relaxation time before the blotting (‘wait time’) and the
intermediate time between blotting and plunge freezing (drain time).
Parameters that affect the blotting process are the number of blottings (Blot Total), the time of
each individual blot (‘blot time’) and the force of the blotpads onto the grid (‘blot Force’). The
latter determines the force at which the excess of fluid is removed from the grid.
The last possibility is to skip application of sample.

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One of the features is the option to do repetitive sample application onto the grid and
subsequent blotting prior to plunge freezing. To activate this function press Add and define
the application parameters of the first substance that is to be applied onto the grid. In the
Processes section of the screen, the parameters are displayed. By again pressing ADD, the
application parameters for the second substance can be defined –as displayed in Processes.
Up to 20 application cycles can be added in this way (1,2,3,4….20).
The Console Screen also gives the opportunity to control the mouse sequence of the
complete vitrification process (‘Place New Grid’, ‘Start Process’, etc) or to Exit the interface.
After 16 sequential blottings, the “reset blot paper” button becomes “Red” pointing out that the
blot papers need to be replaced.
The memobox on the left side of the interface functions as an event logger. All major actions
and warnings are displayed there.
By pressing Delete selected application parameters can be removed from the sequence list.