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Chapter |

Operating Instructions

Confidential, limited rights

PN 103261 | Revision D | 23-Oct-2019

User Manual

Page

23

3.5 Preparation of the Grid

A glow-discharged grid (preferably a quantifoil or lacey carbon film grid) is attached to the
tweezer. Make sure that the black clamping ring is fixed in such a way that the grid does not
fall off in its vertical position (down to the first groves in the tweezers).

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Содержание Vitrobot Mk IV

Страница 1: ...PN 103261 Revision D 23 Oct 19 Vitrobot Mk IV User Manual ...

Страница 2: ...zard 14 2 9 Decontamination and Decomissioning 15 3 Operating 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 ...

Страница 3: ...ing 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 ...

Страница 4: ...idential limited rights PN 103261 Revision D 23 Oct 2019 User Manual 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 ...

Страница 5: ...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 ye...

Страница 6: ... 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 t...

Страница 7: ...ions 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 vitrific...

Страница 8: ... 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 gro...

Страница 9: ... 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 sa...

Страница 10: ...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 Loc...

Страница 11: ...g 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 ...

Страница 12: ...es 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 ...

Страница 13: ... 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 s...

Страница 14: ...eezing 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 temperatu...

Страница 15: ...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 envir...

Страница 16: ...lled 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 instruct...

Страница 17: ...e 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 ...

Страница 18: ...ation 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 dow...

Страница 19: ...ment 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 ...

Страница 20: ... 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 ...

Страница 21: ...d by checking the skip box In case the auto raise ethane lift option is checked the coolant container will be lifted towards the bottom of the climate chamber simultaneously with the uplift of the tweezer you can also do this in separate actions In addition all essential freezing parameters can be saved Save and loaded Load according to the type of sample or experiment that is performed Summary of...

Страница 22: ...itrification process Besides setting the proper parameter conditions the LCD lights are switched on pneumatic pressure control must be active and the blotting papers are put on either side of the blot pads The papers can be attached to the blot pads by using the white circular clipping rings For details about setting the proper parameter conditions see Starting Up Switching on the Vitrobot on page...

Страница 23: ...nual Page 23 3 5 Preparation of the Grid A glow discharged grid preferably a quantifoil or lacey carbon film grid is attached to the tweezer Make sure that the black clamping ring is fixed in such a way that the grid does not fall off in its vertical position down to the first groves in the tweezers ...

Страница 24: ...lots etc can now be set as described in Starting Up Switching on the Vitrobot on page 17 3 6 Preparation and Lifting of the Coolant Container Prior to setting the coolant container in the proper position for vitrification it needs to be pre cooled in advance To do so the outer ring of the container must be filled with liquid nitrogen The central cup can be pre cooled with liquid nitrogen before fi...

Страница 25: ...opane does not condense the metal parts are not at liquid nitrogen temperature yet To speed up the condensation and temperature decrease of liquid ethane propane the metal spindle can be positioned on top of the central cup with its feet into the pre cooled peripheral ring The central cup should be filled up to the brim with liquid ethane or propane for optimal vitrification Indicative for the opt...

Страница 26: ...ocedure Note Make sure that the metal spindle is removed from the coolant container prior to the vitrification procedure Note Working with explosive materials like ethane and propane requires working in a spark free fume hood After the coolant container has been placed on the platform ring the foot pedal switch or the Continue button in the User Interface must be pressed in order to raise the cont...

Страница 27: ...ypically 3 µl are used 3 8 Blotting and Vitrification The excess of suspension must be removed from the grid prior to plunge freezing To do so select Continue in the UI or use the foot pedal This activates a slight uptake of the grid towards the correct position between the blot pads and a subsequent blotting of the grid The blotting conditions can be set in the Options page of the UI After each b...

Страница 28: ...y frozen sample 3 9 Transfer of Vitrified Grid After vitrification the frozen grid must be transferred into a storage box or cryo holder With the semi automated grid transfer default active the grid is automatically transferred from the liquid ethane propane towards the liquid nitrogen atmosphere Prior to positioning the grid into the grid box the tweezers must be carefully disconnected from the c...

Страница 29: ...d into the grid box they are sealed with a special screw and usually stored into a somewhat larger dewar for liquid nitrogen prior to the transfer into a cryo holder and subsequently a TEM or SEM The transfer of the grid box towards nitrogen container should be done fast and swiftly as to minimize the risk of sudden and unwanted temperatur e shifts ...

Страница 30: ... pedal should be pressed again before the next step can be activated Start process Specimen preparation and vitrification will proceed according to the parameters set from the software see guidelines above Processing ends by sample vitrification and lowering the vitrified sample in the ethane for sample retrieval The humidifier will be shut off automatically at the end of the process to prevent ex...

Страница 31: ...difier Subsequently twist and pull the humidifier in order to unleash the bajonet connection Emptying the humidifier is a two step process 1 Pouring the humidifier will empty the central reservoir 2 Re connecting the syringe to the plastic tube at the bottom part and removing the water from the outside reservoir is the second essential step See the video movie for more details Note To prevent any ...

Страница 32: ...nd to increase the blotting time e g 2 3 sec before deciding for an increase in the number of blotting actions Also note the critical effects of the environmental humidity prior to a vitrification action With an unsaturated environment the actual temperature of the sample will be lower than the set temperature of the chamber dew point effect demonstrated with the shape of DPPC vesicles Evaporation...

Страница 33: ...c knowledge of the vitrification process The instrument operates only by build in PC control Check PC screen messages and connections before starting an experiment For temperature and humidity equilibration some 20 30 minutes are required also see test read outs provided with the instrument Experiments performed at high humidity require sufficient water in the water container If the humidity fails...

Страница 34: ...5 C depending on the rH of the environment and climate chamber can be reached Check the temperature of the environment Extra cooling can be attained by gently providing cool air into the backside of the VitrobotTM Extra heating can be attained by gently providing warm air The side door for application may be still open A broken grid Make sure the correct volume of the specimen is accounted for on ...

Страница 35: ...th respect to the rod catching brass part accidental dropping or undue force or alternatively 2 the blotting mechanism is misaligned with respect to the vertical movements of the forceps Check mechanical centering of the tip of the forceps and the brass part with the alignment tool alignment tool is not standard delivered with each system If this is alignment is correct and problems remain the mec...

Страница 36: ...y reproduce release perform display or disclose these technical data are restricted to those rights specified in DFARS 252 227 7015 b 2 FAR 52 227 14 g 2 Alternate II and FAR 12 211 Any reproduction of technical data or portions thereof marked with this legend must also reproduce the markings Any person other than the Government who has been provided access to such data must promptly notify the ab...

Страница 37: ...uid Nitrogen specific safety precautions 13 M Mechanical Moving Parts 11 Messages and Symbols 9 O Operating Instructions 16 P Preparation and Lifting of the Coolant Container 24 Preparation of the Climate Chamber 22 Preparation of the Grid 23 R Revision History 36 S Safety 9 Safety Data Sheets SDS 14 Sample Application 26 Shutting down Emptying the humidifier 31 Starting Up Filling the Humidifier ...

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