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Troubleshooting
Related Manuals for Thermo Scientific Vitrobot Mk IV
Summary of Contents for Thermo Scientific Vitrobot Mk IV
- Page 1 Vitrobot Mk IV User Manual PN 103261 Revision D • 23-Oct-19...
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Page 2: Table Of Contents
Contents Introduction ....................... Design Change ....................Design Philosophy ....................1.2.1 Theoretical analysis of specimen thermodynamics ........1.2.2 Thermodynamic analysis of specimen evaporation ........Technical Specification ..................1.3.1 Key Features .................... 1.3.2 Instrument Specifications ................. Safety ......................... General Safety ..................... Messages and Symbols ..................Mechanical Moving Parts ................... -
Page 3: Introduction
Chapter | Introduction Introduction The Vitrobot (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 plun ging, blotting and vitrification is fully automated upon placing a vial in the chamber and setting up the ethane holder. -
Page 4: Design Change
Chapter | Introduction 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. Confidential, limited rights User Manual PN 103261 | Revision D | 23-Oct-2019... -
Page 5: Design Philosophy
Chapter | Introduction 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. -
Page 6: Thermodynamic Analysis Of Specimen Evaporation
Chapter | Introduction 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 chambe r. 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. -
Page 7: Technical Specification
Chapter | Introduction 1.3 Technical Specification 1.3.1 Key Features • Temperature: software controlled, direct read out, and range: 4 - 60C. Peltier type of heating/cooling. • Humidity: software controlled, direct read out, and range: room conditions up to 100 % rH. Ultrasonic humidifier. -
Page 8: Instrument Specifications
Chapter | Introduction 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... -
Page 9: Safety
Chapter | Safety 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. - Page 10 Chapter | Safety 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.
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Page 11: Mechanical Moving Parts
Chapter | Safety 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. -
Page 12: Thermal
Chapter | Safety 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. -
Page 13: Liquid Nitrogen Specific Safety Precautions
Chapter | Safety 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). -
Page 14: Climate Chamber
Chapter | Safety • 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. •... -
Page 15: Decontamination And Decomissioning
Chapter | Safety 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. -
Page 16: Operating Instructions
Chapter | Operating Instructions 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. -
Page 17: Starting Up - Switching On The Vitrobot
Chapter | Operating Instructions 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... -
Page 18: Defining The Vitrobot User Interface
Chapter | Operating Instructions 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 –... - Page 19 Chapter | Operating Instructions 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.
- Page 20 Chapter | Operating Instructions 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 ont o the grid. In the Processes section of the screen, the parameters are displayed.
- Page 21 Chapter | Operating Instructions In the Miscellaneous box, the use of a foot pedal switch (as alternative for the stylus on the touchscreen), and the possibility to switch off the humidifier during manual application and plunge freezing can be chosen. The semi-automatic grid transfer (i.e. the automatic movement of the grid from the liquid ethane/propane towards the grid box in the liquid nitrogen atmosphere) is default activated but can be de-activated by checking the skip box.
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Page 22: Preparation Of The Climate Chamber
Chapter | Operating Instructions 3.4 Preparation of the Climate Chamber The climate chamber needs to be prepared for the vitrification 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. -
Page 23: Preparation Of The Grid
Chapter | Operating Instructions 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). -
Page 24: Preparation And Lifting Of The Coolant Container
Chapter | Operating Instructions Mount the tweezer onto the connection grove in the central axis. To do this, first select the ‘Place New Grid’ button in the Vitrobot User Interface to put the central axis in the right position for mounting the tweezer to it. The tweezers with grid are subsequently lifted into the climate chamber by selecting the ‘Start Process’... - Page 25 Chapter | Operating Instructions When the central cup is at liquid nitrogen temperature, ethane or propane can be condensed in this cup (check for remaining liquid nitrogen in the center part, wait for complete evaporation). While condensing the ethane or propane keep monitoring the peripheral ring; refilling this reservoir with liquid nitrogen might be necessary.
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Page 26: Sample Application
Chapter | Operating Instructions When the ethane/propane container is ready for vitrification the holder can be placed on the platform-ring under the Vitrobot. Note Make sure that liquid nitrogen remains present in the peripheral ring throughout the entire vitrification procedure. Note Make sure that the metal spindle is removed from the coolant container prior to the vitrification procedure. -
Page 27: Blotting And Vitrification
Chapter | Operating Instructions For manual application of the sample onto a grid, select with the foot pedal switch or the stylus ‘Continue’ to proceed. The tweezers is slightly lowered as to allow the application of suspension through the side-entry port using a pipette. As a consequence of this method only one side of the grid is inoculated with suspension. -
Page 28: Transfer Of Vitrified Grid
Chapter | Operating Instructions The blotting procedure is immediately followed by injection of the tweezers with grid into the liquid ethane or propane. The only delay between blotting and plunge freezing is determined by the drain time that can be set in the ‘Options’ page of the UI and the time required for removing the shutter from the hole in the climate chamber. - Page 29 Chapter | Operating Instructions After the grids have been transferred 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.
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Page 30: Summary - Vitrification Control Through Ui
Chapter | Operating Instructions 3.10 Summary - Vitrification Control through UI This table summarizes the instrument action in relation with the activated knobs and buttons in the Vitrobot User Interface. Console screen Instrument action Humidity disabled/enabled Humidity will be brought to the set value when enabled Place new grid Shutter opens and forceps are presented or can be placed. -
Page 31: Shutting Down - Emptying The Humidifier
Chapter | Operating Instructions 3.11 Shutting down – Emptying the humidifier After shutting down the Vitrobot PC, the remaining water in the humidifier may be removed. To do this, pull the metal ring connector downwards to disconnect the electronic cable from the humidifier. -
Page 32: Troubleshooting
Chapter | Troubleshooting Troubleshooting 4.1 Guidelines for Optimal Specimen Preparation The instrument performance has been tested with phospholipid vesicles (dipalmitoyl-phosphatidylcholine; DPPC 10mg/ml) prepared by extrusion through a 100 nm filter. The shape of these vesicles varies with temperature; spherical above 41 °C (lipid in the L phase), facetted and rippled between 37 and 41 °C (lipid in the P ’... -
Page 33: General Remarks On The Operational Procedures
Chapter | Troubleshooting The dew point (= temperature) and osmotic effects of water evaporation can be prevented by using the humidifier to obtain a relative humidity > 99 %. Concentration of particulate material, inherent to conventional cryo-preparation (and not necessarily a disadvantage), is virtually absent (or slowed down) when preparation is carried out at water saturating conditions. -
Page 34: Troubleshooting
Chapter | Troubleshooting 4.3 Troubleshooting In the following paragraph a number of possible errors are described and some points for action are considered. Problem description Solution The Vitrobot is not switching on Check if the power entry is turned on. The blot paper does not hold Check the clipring mechanism. - Page 35 Chapter | Troubleshooting Quantifoil or lacey carbon film is Centering of forceps between the blot pads is incorrect. This is likely damaged during processing the result of either one of the two causes: 1 the tip of the forceps is misaligned with 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.
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Page 36: Revision History
Chapter | Revision History Revision History Copyright The information and materials contained herein are confidential and proprietary to FEI Company, part of Thermo Fisher Scientific. They are provided for your organization’s internal use on a need to know basis. They cannot be duplicated or disseminated for any third party without the express consent of Thermo Fisher. -
Page 37: Index
Chapter | Index Index Revision History • 36 Safety • 9 Biohazard • 14 Safety Data Sheets (SDS) • 14 Blotting and Vitrification • 27 Sample Application • 26 Shutting down – Emptying the humidifier • 31 Climate Chamber • 14 Starting Up –...
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