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Owner`s manual and instrument guide

1. 17 Gas Requirements and Connectors for Pneumatics 17 Preparing the Nitrogen Cylinder and 2 Stage Regulator 17 Cryogenic Requirements and Safe Handling 18 Preparing the Ethane Cylinder and 2 Stage Regulator 19 Preparing the Ethane Cylinder and Regulator 19 Leak Testing the Connections 20 iv Cryoplunge3 Owner s Manual and User s Guide Adjusting the Gas Pressure 20 Leaving the Ethane Cylinder in Stand by Mode 20 Important Safety Precautions 21 Installation and Setup of Cp3 21 Unpacking and Inspection 21 Removing Cp3 and Accessories from the Shipping Container 22 Matching the Contents against the Packing List
2. 22 Connections and System Test 23 Installing and Testing Cp3 23 Preparing the Humidity Chamber 30 Preparing the Filter Paper Disks 30 Preparing the Blotters 31 Cooling the Workstation with Liquid Nitrogen 34 Condensing Ethane and Filling the Ethane Pot 35 Blotting Techniques for Freezing the Sample 40 2 sided Blotting 40 Returning Cp3 to Stand by Conditions 46 1 sided Blotting 47 Manual Multiple Blotting 48 No Blotting Method
3. 45 igure 1 38 Transfer the grid box to the storage tube filled with liquid nitrogen 46 igure 1 39 Removing the blotters 50 igure 1 40 Removing the humidity wand 51 igure 1 41 Removing the temperature humidity sensor 51 igure 1 42 Disconnect the pneumatic supply for the blotter assemblies 52 igure 1 43 Removing the humidity chamber 52 igure 1 44 Replacing the humidity chamber 53 igure 1 45 Reconnecting the pneumatic supply 53 Cryoplunge 3 Owner s Manual and User s Guide 1 Preface About this Guide This Cryoplunge 3 Owner s Manual and User s Guide provides information on the Cryoplunge3 features and functions along with procedures for installing and operating the unit Routine maintenance and troubleshooting procedures are also included in this manual The following typographical conventions are used for special comments NOTE Recommendations for getting the best performance from the equip ment CAUTION Precautionary notes and advice to avoid personal injury or damage to the equipment WARNING Advisory to call attention to
4. 49 Routine Maintenance 50 Removing the Humidity Chamber for Cleaning 50 Cleaning the Outside Surfaces of Cp3 54 Appendix A Troubleshooting 55 Cryoplunge3 Owner s Manual and User s Guide v List of Figures igure 1 1 Cryoplunge3 front view 14 igure 1 2 Cryoplunge3 side view left 15 igure 1 3 Cryoplunge3 side view back and right 16 igure 1 4 Workstation 16 igure 1 5 Pneumatics supply for Cp3 left 2 stage nitrogen regulator right 18 igure 1 6 Ethane tank and regulator 19 igure 1 7 Cp3 in the shipping crate 21 igure 1 8 Pneumatic connection 24 igure 1 9 Pneumatic mains and protective earth connections 25 igure 1 10 Safety interlock for workstation 26 igure
5. During plunger operation timer count down in process never attempt to open the guards or access the tweezers Always wait until the plunger has com pleted its operation and is at full rest Always use appropriate personal protective equipment when working with liquid nitrogen and liquid ethane such as safety glasses or face shield and temperature resistant gloves Consider all clinical specimens reagents controls calibrators etc that con tain blood and surfaces or components that have come into contact with blood as potentially infectious Wear gloves lab coats and safety glasses and follow other biosafety practices as specified in OSHA Bloodborne Patho gen Rule 29 CFR Part 1910 1030 or other equivalent biosafety procedures Instrument cleaning and decontamination Instruments must be properly decontaminated prior to servicing or shipment For users that process biologi cal samples Gatan Inc recommends contracting with a licensed firm special izing in use specific decontamination processes to meet customers specific needs Prior to servicing ensure service personnel are aware of what has been processed and ensure proper decontamination has been performed Equipment Ratings and Technical Specifications Power is applied to the units via a line cord connection Grounding is provided through this main input connection using a make first break last method Electrical Ratings and Specifications I
6. 37 igure 1 26 Pre cooling the conical tube and then pouring the condensed ethane into the pre cooled ethane pot 38 igure 1 27 Making the ethane popsicle 38 vi Cryoplunge3 Owner s Manual and User s Guide igure 1 28 Securing the specimen grid to the plunge tweezers 41 igure 1 29 Inserting the tweezers into slot at the base of the plunge rod 41 igure 1 30 Raising the plunge rod to full upward firing position 42 igure 1 31 Applying sample aliquot to the support film 42 igure 1 32 2 sided specimen blotting 43 igure 1 33 Grid plunging into the liquid ethane 43 igure 1 34 Pressing the quick disconnect push button to release the plunge tweezers from the plunge rod 44 igure 1 35 Tilting the tweezers 44 igure 1 36 Blotting excess ethane 45 igure 1 37 Disconnect the black securing clip on the plunge tweezers to free the frozen hydrated grid
7. Explosive Yes No Radioactive Yes No Other harmful substance Yes No Please list all substances gases and by products which may have been used in the instrument and supply the Material Safety Data sheets with the return Trade name Chemical name Danger Class Precautions associated Action if human contact Product name or Symbol with substance 5 Legally Binding Declaration I hereby declare the information supplied on this form is complete and accurate The shipping of equipment will be in accordance with the appropriate regulations covering Packaging Transportation and Labeling of Dangerous Substances Name of Organization ___________________________________________________________________ Date _______________ Address __________________________________________________________________________________________________ City ___________________________ State ____________________ Zip Code ________________ Country ________________ Phone __________________ FAX ____________________ Email _________________________________________________ Name ____________________________________________ Job Title _______________________________________________ Legally Binding Signature ___________________________________________________________
8. Plunge rod dampening pot damaged Check to make sure the plunge rod dampening pot is intact PROBLEM THINGS TO CHECK ACTION TO TAKE Cryoplunge 3 Owner s Manual and User s Guide 57 Ethane temperature does not display on meter TC heater connector Check to be sure this connection is secure Ethane freezes Ethane temperature setting Set the ethane temperature to a value just above its melting point of 182 C using the up down push buttons on the ethane meter until the desired temper ature is obtained PROBLEM THINGS TO CHECK ACTION TO TAKE 58 Cryoplunge 3 Owner s Manual and User s Guide Gatan Product Warranty Gatan warrants that products manufactured by Gatan shall be free of defects in materials and workmanship for the warranty period which com mences at date of shipment Gatan tests the performance of a unit as part of its final test procedure prior to shipment from its factory Gatan war rants that the unit meets Gatan s published specifications at time of shipment from its factory All product warranties provide for a period of one year after shipment to customer parts excluding all normal consumable wear and maintenance items and labor For Specimen Preparation Equipment and Specimen Holders Gatan will correct any defects in the instrument either by repair in our facility or replacing the defective part with the shipping party responsible for shipping costs For products which attach to the c
9. Quick disconnect tweezer mounting mechanism Cryo Workstation Removable liquid nitrogen cryo workstation allows transport to and from explosion proof spark proof fume hood for filling and disposal of liquid ethane Long hold temperature monitored ethane vessel Cryo grid box receptacle round and square cryo grid boxes Filter paper blot stand kept at the temperature of the FH grid within the work station allows blotting excess ethane for immediate viewing on the TEM External funnel for filling workstation with liquid nitrogen Workstation covers protect frozen hydrated grid during transfer from ethane to cryo grid storage box Description and Specifications Cryoplunge3 Owner s Manual and User s Guide 13 Liquid nitrogen cryo grid box transfer pot allows cryo grid box to be moved to a cryo holder workstation or a liquid nitrogen storage dewar while sub merged under liquid nitrogen Maintenance Preventative maintenance plan is available Description and Specifications The Cryoplunge3 Cp3 is a self contained unit as shown in the following images NOTE It might be helpful to print these images and use them as a guide when reading the instruction manual Description and Specifications 14 Cryoplunge3 Owner s Manual and User s Guide igure 1 1 Cryoplunge3 front view Ethane temperature controller Omron E5CN Specimen loading port Humidity chamber P
10. Support and Customer Service Gatan Inc provides free technical support via phone fax and electronic mail To reach Gatan technical support contact the facility nearest you or send an email to help gatan com USA Canada amp Latin America Corporate Headquarters Western USA Sales Central USA amp Latin America Sales 5794 W Las Positas Blvd Pleasanton CA 94588 Tel 1 925 463 0200 Fax 1 925 463 0204 Contact info gatan com Gatan Inc Eastern USA Sales 780 Commonwealth Drive Warrendale PA 15086 Tel 1 724 776 5260 Fax 1 724 776 3360 Contact info gatan com Asia amp Pacific Rim Nippon Gatan 3F Sakurai building 2 8 19 Fukagawa Koto ku Tokyo 135 0033 Japan Tel 81 3 5639 2772 Fax 81 3 5639 2763 Contact rabara gatan com Agency Approvals and Safety Information Cryoplunge 3 Owner s Manual and User s Guide 3 Gatan Singapore 10 Eunos Road 8 12 06 Singpore Post Centre Singapore 408600 Tel 65 6293 3160 Fax 65 6293 3307 Contact wchuang gatan com Europe Gatan GmbH M nchen Germany Ingolstadterstr 12 D 80807 M nchen Germany Tel 49 89 358084 0 Fax 49 89 358084 77 Contact mfelsmann gatan com Gatan UK 25 Nuffield Way ABINGDON OX14 1RL UK Tel 44 1235 540160 Fax 44 1235 540169 Contact ukinfo gatan com Gatan France Division de ROPER Scientific SAS 8 rue du Forez ZI petite montagne Sud CE 1702 91017 EVRY Cedex Tel 33 1 69 11 03 69 Fax 33 1
11. gas remaining in the regulator and the flexible tubing d Fully open the regulator adjusting spring handle for the second stage of the regulator turn counter clockwise until loose but not disconnected from the regulator e Close the outlet valve of the second stage of the regulator 13 Add liquid nitrogen to the workstation if needed 14 Replace the covers for the workstation 15 If necessary reset the flow rate for the fume hood 16 Carefully remove the workstation and install it on Cp3 17 Insert the TC heater connector and switch the mains ON 18 Adjust the up down push buttons on the ethane meter until the ethane is main tained just above its melting point For example if the ethane freezes when the temperature reads 172 C on the meter press the up push button to main tain a temperature just slightly above this value until the ethane melts 19 Wait several minutes for the ethane temperature to stabilize and add liquid nitrogen through the remote fill funnel as necessary to maintain the volume within the workstation 20 You are now ready to freeze your sample Blotting Techniques for Freezing the Sample 40 Cryoplunge3 Owner s Manual and User s Guide Blotting Techniques for Freezing the Sample NOTE For best results always pre treat the specimen support for example holey carbon film or continuous carbon film to render the surface hydrophilic or hydrophobic depending upon the needs of the sampl
12. s Guide 39 9 To condense the ethane open the outlet valve on the second stage of the ethane regulator such that the ethane gas flows into the bottom of the pre cooled ethane pot or the bottom of the conical tube depending on the chosen method for condensing the ethane 10 Adjust the outlet valve on ethane regulator as needed to control the rate of condensation of the ethane It takes approximately 2 minutes to directly fill the ethane pot to the upper most rim of the pot 4 mL If using the conical tube method to pour the ethane condense about 6 7 mL no more than 10 mL to allow for some potential lost ethane on pouring into the ethane pot If using the ethane popsicle method carefully pipette the ethane into the 2 mL volu metric plastic tubes and allow a few minutes for the ethane to freeze 11 When the ethane pot is full or you have a suitable quantity of ethane con densed within the conical tube close the outlet valve on the 2 stage regulator of the ethane tank and leave the ethane tank in stand by mode 12 Leave the ethane tank in stand by mode Stand by mode for the ethane tank NOTE Always close the main tank valve after use and drain the regulator of any residual ethane by following these steps a Close the main tank valve b Point the Tygon transfer tubing towards the back of the explosion proof spark proof fume hood c Open the outlet valve of the second stage of the regulator to bleed off any
13. Setup of Cp3 Cryoplunge3 Owner s Manual and User s Guide 21 1 Close the main tank valve 2 Open the outlet valve of the second stage of the regulator to bleed off any gas remaining in the regulator and the flexible tubing 3 Fully open the regulator adjusting spring handle for the second stage of the regulator turn counter clockwise until loose but not disconnected from the regulator 4 Close the outlet valve of the second stage of the regulator Important Safety Precautions The volume of the ethane pot for Cp3 is 4 1 mL to the upper rim of the pot During use do not condense more than 10 mL of ethane at one time Review the latest MSDS available on the world wide web for ethane Installation and Setup of Cp3 Unpacking and Inspection NOTE Do not discard shipping container Keep all packing materials The shipping container has been specifically designed for Cp3 and must be used in the event that the instrument must be returned to Gatan Inc for repair igure 1 7 Cp3 in the shipping crate Installation and Setup of Cp3 22 Cryoplunge3 Owner s Manual and User s Guide Removing Cp3 and Accessories from the Shipping Container NOTE All components of Cp3 are located in one shipping container To unpack Cp3 1 Cut the two securing straps that surround the outside of the box 2 Turn the lid locks as indicated on the locks to release the cover 3 Open the cover Cp3
14. a potentially hazardous situation which if not avoided could result in death or serious injury Disclaimer Gatan Inc makes no express or implied representations or warranties with respect to the contents or use of this manual and specifically disclaims any implied warranties of merchantability or fitness for a particular purpose Gatan Inc further reserves the right to revise this manual and to make changes to its contents at any time without obligation to notify any person or entity of such revisions or changes Copyright and Trademarks 2008 Gatan Inc The Gatan logo is a registered trademark of Gatan Inc Cryoplunge is a trademark of Gatan Inc All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise without the prior written permission of Gatan Inc Returns 2 Cryoplunge 3 Owner s Manual and User s Guide The text and graphics are for the purpose of illustration and reference only The specifications on which they are based are subject to change without notice Returns If there is a need to return equipment to the factory please call Gatan to obtain a Returned Merchandise Authorization Number RMA This RMA number must appear on your shipping document to help in tracking and to ensure that proper actions will be taken to repair or replace your equipment
15. above conforms to the above Directives and Standards Place Pleasanton CA Signature Date July 29 2008 Full Name Thomas G Connelly Position President and CEO Digitally signed by Thomas G Connelly DN cn Thomas G Connelly c US o Gatan Inc ou Administration email tconnelly gatan com Reason I attest to the accuracy and integrity of this document Location 5794 W Las Positas Blvd Pleasanton CA Date 2008 07 30 12 41 29 07 00 SP830100EX01_REV02 Declaration of Contamination The repair and or service of any Gatan instruments will only be performed if a properly completed declaration has been submitted please FAX this completed form prior to returning the instrument Gatan maintains the right to refuse acceptance of any equipment without a declaration Please FAX completed form to our Pennsylvania office at 724 766 3360 This declaration must be completed and signed by authorized and qualified persons 1 Description of instrument Model Serial No Ship Date 2 Reason for return 3 Equipment condition Is the equipment functioning Yes No Is the equipment free from potentially harmful substances Yes No NOTE We will not accept delivery of any equipment that has been radioactively or microbiologically contaminated without written evidence of decontamination 4 Process related contamination of equipment Toxic Yes No Corrosive Yes No Microbiological hazard Yes No
16. are met The workstation is in the innermost position safety interlock engaged The safety shield is fully closed and the safety interlock is engaged large cover should be in place on top of the workstation but the small workstation cover must be removed in order of the safety shield to close The pneumatic supply is on Ethane pot audible alarm ON OFF Ethane temperature warning lamp Blotting timer Omron H5CX Ethane temperature controller Omron E5CN Temperature humidity meter Omega iSeries Ethane pot temperature reading User preset for ethane temperature Ethane temperature up down push buttons Blot time count down window Blot time Buttons to adjust the blotting time Humidity readout Temperature readout Connections and System Test 30 Cryoplunge3 Owner s Manual and User s Guide Preparing the Humidity Chamber To prepare the chamber 1 Switch mains ON and make sure the temperature humidity sensor is inserted into the top of the humidity chamber 2 Raise the plunge rod to the full upward firing position 3 Press the RESET button to close the shutter at the base of the humidity cham ber 4 Remove the humidity wand from its receptacle on the top of the humidity chamber 5 Insert one of the compressed cellulose sponges within the three metal sup ports of the wand 6 Soak the wand sponge in hot tap water to expand the cellulose sponge 7 Remove a
17. designed plunge tweezers and lock the grid in place by sliding the black plastic securing clip until it engages the first clickstop on the tweezer shaft Blotting Techniques for Freezing the Sample Cryoplunge3 Owner s Manual and User s Guide 41 igure 1 28 Securing the specimen grid to the plunge tweezers 4 Test to make sure that the grid is held firmly in the tweezer 5 If the plunge rod is in the full upward firing position it is necessary to run a blot and plunge cycle so that the tweezer can be loaded onto the plunge rod a Remove the small workstation cover b Close the safety shield c Press RESET to close the shutter at the base of the humidity chamber d Press START to initiate the blot and plunge cycle e The plunge rod fires towards the ethane pot f Open the safety shield and replace the small cover 6 Raise the plunge rod several centimeters and insert the base of the plunge tweezer into the slot at the base of the plunge rod and press until it clicks into place taking care not to damage the specimen grid igure 1 29 Inserting the tweezers into slot at the base of the plunge rod 7 Raise the plunge rod to the full upward firing position until it clicks into place Blotting Techniques for Freezing the Sample 42 Cryoplunge3 Owner s Manual and User s Guide igure 1 30 Raising the plunge rod to full upward firing position 8 Press the RESET button to close the shutter a
18. filter paper loading jig prior to loading the filter paper Connections and System Test Cryoplunge3 Owner s Manual and User s Guide 33 1 Remove the blotters from the humidity chamber by grasping onto the outer surface of the blotter and pulling straight out from the humidity chamber see Figure 1 17 on page 31 2 Place the filter paper pin flat side down into the filter paper loading jig 3 Place a new piece of filter paper over the pin 4 Place the plastic backer on top of the filter paper igure 1 21 Loading the filter paper disk and protective plastic backer 5 Guide the blotter down the barrel of the filter paper loading jig and press down firmly to connect the pin 6 Remove the blotter from the jig and gently press down on the head of the pin to ensure that it is firmly seated and that the filter paper is flat 7 Reinsert the blotter to the humidity chamber 8 Repeat with the other blotter igure 1 22 Attaching the filter paper disk and inserting the blotter Cooling the Workstation with Liquid Nitrogen 34 Cryoplunge3 Owner s Manual and User s Guide Cooling the Workstation with Liquid Nitrogen NOTE Starting from room temperature conditions It takes approximately 20 minutes for the workstation to stabilize at liquid nitrogen temperature It is important for the ethane pot temperature to be at stable temperature below 170 C prior to condensing ethane into the ethane pot To cool
19. the workstation with liquid nitrogen 1 Have a ready supply of liquid nitrogen available along with several small dewars or liquid nitrogen transfer vessels for the next steps 2 Make sure that the TC heater connector for the ethane pot is plugged in 3 Switch on the mains supply 4 Set the ethane temperature using the up down push buttons on the ethane tem perature controller to 172 C igure 1 23 Meter for ethane temperature controller 5 Remove the covers from the workstation 6 Insert the liquid nitrogen transfer pot into the front of the workstation 7 Fill the workstation and the ethane pot with liquid nitrogen to start rapid cooldown of the workstation As liquid nitrogen is added to both the worksta tion and the ethane pot there will be rapid boil off of the liquid nitrogen Con tinue to fill both until the workstation reaches equilibrium and the ethane pot maintains at least 170 C This takes at least 20 minutes Ethane pot temperature reading User preset for ethane temperature Ethane temperature up down push buttons Condensing Ethane and Filling the Ethane Pot Cryoplunge3 Owner s Manual and User s Guide 35 igure 1 24 Initial cool down of the workstation and ethane pot 8 When the workstation is at the low temperature equilibrium prepare to con dense the ethane Condensing Ethane and Filling the Ethane Pot NOTE The following safety recommendations are summarized below for yo
20. 1 11 Workstation overflow port with Tygon tubing attached 26 igure 1 12 Insertion of Omega temperature humidity sensor 27 igure 1 13 Omega temperature humidity sensor connector 27 igure 1 14 Inserting the humidity wand 28 igure 1 15 Meters and alarms for Cp3 29 igure 1 16 Filter paper die cutter 31 igure 1 17 Inserting removing a blotter from the right hand blot assembly 31 igure 1 18 Removing the spindle 32 igure 1 19 Inserting the sponge pad 32 igure 1 20 Replacing and tightening the spindle 32 igure 1 21 Loading the filter paper disk and protective plastic backer 33 igure 1 22 Attaching the filter paper disk and inserting the blotter 33 igure 1 23 Meter for ethane temperature controller 34 igure 1 24 Initial cool down of the workstation and ethane pot 35 igure 1 25 Condensing ethane directly into the pre cooled ethane pot
21. 64 97 19 67 Contact dmonville gatan com NOTE The above information is subject to change Please consult the contact section of the Gatan website at www gatan com for the latest information Agency Approvals and Safety Information This chapter presents agency approval and certification information for the Cryoplunge3 It also provides a summary of the safety recommendations through out this manual Gatan Inc recommends following all safety precautions to pre vent harm to yourself or the equipment Please follow all warnings marked on the equipment as well Agency Approvals and Safety Information 4 Cryoplunge 3 Owner s Manual and User s Guide CE Certification The product described in this manual meets the intent of 2004 108 EC EMC Directive 2006 95 EC Low Voltage Directive 2002 95 EC Restriction of Hazardous Substances ROHS Directive 2002 96 EC Waste Electrical and Electronic Equipment WEEE Directive Compliance with RoHS and WEEE Directives As a company that has always been firmly committed to environmentally respon sible practices Gatan Inc is in complete accord with the content and spirit of Directive 2002 96 EC of the European Parliament and of the Council of 27 Janu ary 2003 on Waste Electrical and Electronic Equipment commonly referred to as the WEEE Directive The WEEE Directive applies to all products placed on the controlled market after 13 August 2005 At pr
22. Gatan Inc 5794 W Las Positas Blvd Pleasanton CA 94588 Tel 925 463 0200 Fax 925 463 0204 Cryoplunge 3 Plunge freezing Instrument for Cryo TEM Owner s Manual and User s Guide Model Number 930 Part Number 930 50500 Revision 01 ii Cryoplunge 3 Owner s Manual and User s Guide Cryoplunge3 Owner s Manual and User s Guide iii Table of Contents Preface 1 About this Guide 1 Disclaimer 1 Copyright and Trademarks 1 Returns 2 Support and Customer Service 2 Agency Approvals and Safety Information 3 Safety and Attention Symbols 5 Product Safety Information 5 Equipment Ratings and Technical Specificat
23. If using house air supply make sure that it is deliv ering the proper pressure Safety interlock for workstation Make sure that the workstation is fully pressed into the innermost position on the base plate of Cp3 and that the safety interlock for the workstation is engaged Safety shield interlock Check that the safety shield is fully closed to ensure that the safety interlock on the door hinge of the shield is engaged Plunge rod cannot be raised into humidity chamber Power was shut off with plunge rod in downward position Look at the bottom of the humidity chamber and using your fingertip gently push the shutter towards the front of Cp3 while raising the plunge rod to its full upward firing position Release the shutter The shutter is impinged upon plunge rod This conditions results when the plunge rod is in its full downward position and the power for Cp3 is switched off Plunge rod cannot be raised to full upward firing position in this condition Shutter as viewed from the base of the humidity chamber Using your fingertip press the shutter forward do not press the plunge rod forward This action will free the plunge rod Hold the shutter in this position and raise the plunge rod to the full upward firing position Release the shutter 56 Cryoplunge 3 Owner s Manual and User s Guide Blot pads do not come together to blot the grid Pneumatic supply pressure Make sure t
24. RT pushbutton 4 The sample grid fires directly into the ethane pot Routine Maintenance Removing the Humidity Chamber for Cleaning NOTE The humidity chamber can be removed for cleaning Use a mild soap and water solution to clean the chamber To remove the humidity chamber 1 Return Cp3 to stand by conditions 2 Remove the blotters igure 1 39 Removing the blotters 3 Remove the humidity wand Routine Maintenance Cryoplunge3 Owner s Manual and User s Guide 51 igure 1 40 Removing the humidity wand 4 Remove the temperature humidity sensor igure 1 41 Removing the temperature humidity sensor 5 Disconnect the pneumatic supply for the blot assemblies by pressing the outer ring of the push in fitting while removing the pneumatic supply tube Routine Maintenance 52 Cryoplunge3 Owner s Manual and User s Guide igure 1 42 Disconnect the pneumatic supply for the blotter assemblies 6 Unscrew and remove the securing screw for the humidity chamber located on the right hand side of the instrument igure 1 43 Removing the humidity chamber 7 Hold the humidity chamber and gently push downward approximately 2 cm 8 Gently pull the humidity chamber forward to disconnect To replace the humidity chamber 1 After cleaning and drying the chamber reinstall on Cp3 2 Gently press upward to engage the top of the humidity chamber with the top chamber support of Cp3
25. Routine Maintenance Cryoplunge3 Owner s Manual and User s Guide 53 igure 1 44 Replacing the humidity chamber 3 Insert the securing screw and make finger tight 4 Reconnect the pneumatic supply to the blot assemblies press the pneumatic tubing inward making sure that the connection is tight within the push in fit ting igure 1 45 Reconnecting the pneumatic supply 5 Insert the blotters 6 Insert the humidity wand 7 Insert the temperature humidity sensor 8 Maintain the instrument in stand by conditions for the next user Routine Maintenance 54 Cryoplunge3 Owner s Manual and User s Guide Cleaning the Outside Surfaces of Cp3 Use a mild soap solution on a soft cloth to clean the outside surfaces of Cp3 Do not use harsh chemicals or alcohol based cleaners as this may cause damage Cryoplunge 3 Owner s Manual and User s Guide 55 Appendix A Troubleshooting PROBLEM THINGS TO CHECK ACTION TO TAKE Meters do not illuminate Mains power supply and ON OFF switch Check connections of mains power supply and make sure electrical connection is made Switch ON OFF switch to ON position Plunge rod does not fire Pneumatic supply Make sure that the pneumatic source is on i e the main tank valve for the nitrogen gas tank which supplies the pneumatic pressure is open and set the second stage of the nitrogen gas regulator on the tank to a value of 60 and 80 psi 4 1 to 5 5 bar
26. al Specifications Cryoplunge3 Owner s Manual and User s Guide 17 Physical Specifications Gas Requirements and Connectors for Pneumatics Cp3 requires a pneumatics supply The high pressure tubing connection to the pneumatic inlet at the back of the instrument is a 6mm outer diameter push in fitting It is recommended that a nitrogen gas cylinder is used to provide the pneu matic supply to operate Cp3 A compatible 2 stage regulator should be fitted and the second stage of this regulator must be capable of delivering 100 psi 7 bar of pressure in order to achieve the operating pressures for Cp3 60 min to 80 max psi of pressure 4 1 to 5 5 bar Two thread to push in fittings for the second stage of the 2 stage regulator are provided Choose the one with the proper diameter that fits your regulator 1 4 inch or 1 2 inch are provided with Cp3 Alternatively a dry oil free compressed air supply that can deliver 60 80 psi of air pressure output 4 1 to 5 5 bar can also be used Preparing the Nitrogen Cylinder and 2 Stage Regulator NOTE The 2 stage regulator not provided must be suitable for use with nitrogen gas The second stage of the regulator should be able to deliver 100 psi 7 bar with an outlet fitting 1 4 inch or 1 2 inch that accepts one of the thread to push in fittings provided The following instructions are intended as guidelines 1 If you are unsure as to how to connect a 2 stage reg
27. amber to air dry 14 Open the specimen loading door 15 Remove the blotters optional 16 Allow the humidity chamber to equilibrate to room temperature humidity conditions 1 sided Blotting To freeze the sample using 1 sided blotting NOTE Follow the instructions for freezing the sample using 2 sided blotting with the following exceptions 1 Remove one of the blotters and insert the blotter blanking plug 2 Apply a small aliquot of the sample suspension to the pre treated surface of the support grid using an accurate volumetric pipetting device 3 Orient the grid for 1 sided front or backside blotting a 1 sided backside blotting blots the aliquot of the sample droplet through the holes on the holey carbon film Blotting Techniques for Freezing the Sample 48 Cryoplunge3 Owner s Manual and User s Guide This method is helpful to use when for example the sample concen tration is very low b 1 sided frontside blotting blots the aliquot of the sample directly onto the filter paper 4 Because only one blot pad is being used to remove excess solution from the surface of the support film set the blotting timer for two times the blotting time that would normally be used for 2 sided blotting a For example if you add 3 microliters of your sample and normally blot for 4 seconds with 2 sided blotting use an 8 second blot time for 1 sided blotting b Upon blotting the bulk of the solution is re
28. and all accessories are packaged within several layers of sturdy foam 4 Remove the layers of foam packing material to expose the contents within the packing crate 5 Remove the accessories box and the workstation 6 Remove addition layers of foam packing material to fully expose Cp3 7 Remove Cp3 by carefully and gently lifting it out of the box and placing it in the designated area where it will be used for example a laboratory bench top 8 Replace all of the foam layers 9 Close the box lock the lid and store the shipping container in a suitable loca tion Do not discard the shipping box Matching the Contents against the Packing List Make sure that these items are included in the box Cryoplunge3 Instruction manuals Cryoplunge3 instruction manual Digital blotting timer instruction manual Omron H5CX Digital thermohygrometer instruction manual Omega iSeries Digital temperature controller manual Omron E5CN Cryoplunge3 accessories kit Blot assembly O ring 2 004 Buna Tubing for pneumatic supply 5 meters 1 8 inch NPT thread to push in fitting 6 mm 1 4 inch NPT thread to push in fitting 6 mm Standard workstation Cryoplunge3 pot assembly Liquid nitrogen filling funnel Humidity wand to retain cellulose sponges Humidity wand sponge 2 Chamber assembly includes 2 blotter assemblies Conne
29. ating sur faces are clean and free of any particulate matter Second stage pressure gauge on ethane regulator Pipette tip affixed to the end of the flexible tubing Gas pressure regulator adjusting spring handle Outlet valve Conical tube for condensing the ethane Styrofoam box for precooling the conical tube prior to condensing the ethane Flexible tubing Ethane tank Safety chain Main tank valve First stage pressure gauge on ethane regulator Preparing the Ethane Cylinder and 2 Stage Regulator 20 Cryoplunge3 Owner s Manual and User s Guide 6 Wrap the regulator s threaded mating surface with Teflon tape and screw securely into place on the ethane tank 7 Close the outlet valve on the second stage of the regulator and open the sec ond stage gas pressure regulating adjusting spring handle fully i e no gas pressure to the second stage 8 Affix a short length 60 to 90 cm of narrow bore flexible tubing Tygon for example to the outlet port on the second stage of the ethane regulator so that it can reach the vessel within which the ethane will be condensed 9 Attach a small pipette tip to the other end and cut approximately 3 mm to widen the opening at the tip of the pipette Leak Testing the Connections 1 Open the main tank valve This fills the regulator with gas but no gas should escape through the flexible tubing because the outlet valve of the 2 stage reg ulator is closed from th
30. condense the ethane 1 When the pre cooled workstation has reached its low temperature equilib rium switch mains OFF 2 Disconnect the ethane pot TC heater connector 3 Place the workstation in the explosion proof fume hood 4 Remove the small workstation cover 5 Top off the workstation with liquid nitrogen via the filling funnel 6 Remove any residual liquid nitrogen that might be in the ethane pot Given time the liquid nitrogen evaporates on its own Alternatively one can speed up this process by inserting a small piece of clean Tygon tubing into the ethane pot Be sure to face the opening of the Tygon tubing away from you to prevent injury 7 Adjust the gas pressure for the 2 stage regulator on the ethane tank a Check to be sure that the outlet valve of the second stage of the regulator is closed so that no gas can escape b Open the main tank valve c Adjust the pressure of the second stage of the ethane regulator to deliver the ethane at about 2 to 4 psi 0 14 to 0 28 bar by turning the regulator adjusting spring handle clockwise d At this point the system is now ready to use for condensing ethane 8 There are several methods that are commonly used to condense the ethane Three examples of these methods follow NOTE Make sure that the containers receiving the ethane are free of any particulate matter Method 1 Insert the nozzle not provided at the end of the Tygon tubing direct
31. ctions and System Test Cryoplunge3 Owner s Manual and User s Guide 23 Blotter assembly Cp3 tweezers 2 Filter paper pin pack pack of 10 Filter paper pack of 100 disks Whatman 1 Backing plastic disk pack of 20 Blotter cushioning foam pack of 20 Cryo grid box square Grid box handling rod Grid transfer storage tube Conical tube for condensing ethane Cryo grid box transfer pot Filter paper loading tweezers Tygon tubing for workstation overflow port 1 meter Blotter blanking plug Workstation cover small Workstation cover large filter paper loading jig Blot pad spindle wrench Connections and System Test Installing and Testing Cp3 1 Place Cp3 onto a stable work surface away from drafts and open flames and oriented so that you can easily see the connection points at the back and right hand side of the instrument 2 Suggested supply for the pneumatic operation of Cp3 is a clean dry nitrogen source If using nitrogen gas ensure that the nitrogen tank is secured follow local safety regulations for securing the tank and that the main tank valve is closed before proceeding Operating pressure for Cp3 is 60 min to 80 max psi of pressure 4 1 to 5 5 bar Pressure should not exceed 80 psi 5 5 bar when the valve is fully open during use If using house air supply ensure that the s
32. d to the support grid set the blot time for 4 seconds therefore volume plus one second 15 Remove the small workstation cover and close the safety shield 16 Press START a The blot pads come together and blot the specimen for the time indicated on the timer igure 1 32 2 sided specimen blotting b The grid is plunged into the liquid ethane igure 1 33 Grid plunging into the liquid ethane To remove the frozen hydrated grid from the ethane pot NOTE The freshly frozen hydrated grid is protected while immersed in the liquid ethane It is also protected while transferred within the cryogenic environment of the workstation Prior to transferring the frozen hydrated grid from the ethane refresh the positive outflow of cryogenic nitrogen gas from the chamber by adding a small volume of liquid nitrogen via the filling funnel Do not breathe into the workstation when transferring the frozen hydrated grid as this will damage the grid NOTE Make sure that the cryo grid box is at liquid nitrogen temperature and ready to receive the frozen hydrated grid If necessary top off the liquid nitrogen level within the liquid nitrogen transfer pot Blotting Techniques for Freezing the Sample 44 Cryoplunge3 Owner s Manual and User s Guide NOTE If you wish to blot excess ethane from your frozen hydrated grid prior to storing it within the cryo grid box insert a piece of filter paper into the paper clip within the workstatio
33. e NOTE The plunge tweezers are specially designed and aligned to provide optimal contact with the blot pad assemblies NOTE The black plastic securing clip for the plunge tweezers has a small and a large rectangular opening The small opening should always be placed onto the tweezer shaft from the blunt end and the first detent position on the shaft of the tweezer should always be used to secure the specimen grid first detent furthest from the tweezer tip Pressing the securing clip further down on the tweezer shaft can damage the tip of the tweezer or cause the specimen grid to disengage prematurely during the plunging cycle NOTE During the process of plunging the sample grids it is necessary to monitor and or adjust the ethane temperature humidity of the chamber and the volume of the liquid nitrogen in the workstation and in the liquid nitrogen transfer pot NOTE The sample concentration sample volumes and blotting time generally have to be determined for each new specimen that will be plunge frozen in order to produce optimal results The following recommendations are guidelines 2 sided Blotting To freeze the sample using 2 sided blotting 1 Pre cool the cryo grid box by placing it inside of the liquid nitrogen transfer pot within the workstation 2 Pre cool a second cryo grid box in the staging receptacle located on the metal mesh base of the workstation 3 Secure the specimen support grid to the specially
34. e previous step above The first stage of the regulator indicates the pressure of the gas coming directly from the tank 2 Using a dilute soap solution leak test the fitting between the ethane tank and the 2 stage regulator to make sure that the connection is tight and that no gas is escaping from this connection if gas is escaping at the connection bubbles form at the connection site If this occurs close the main tank valve tighten the connection and then repeat the procedure until the connection no longer indicates a leak Adjusting the Gas Pressure 1 Check to be sure that the outlet valve of the second stage of the regulator is closed so that no gas can escape 2 Open the main tank valve 3 Adjust the pressure of the second stage of the ethane regulator to deliver the ethane at about 2 to 4 psi 0 14 to 0 28 bar by turning the regulator adjusting spring handle clockwise 4 At this point the system is now ready to use for condensing ethane However if you are not going to immediately condense ethane for freezing your sam ples the ethane tank and regulator should be left in stand by mode Leaving the Ethane Cylinder in Stand by Mode Leave the ethane tank in stand by mode until you are ready to condense ethane for freezing your samples When the ethane is not being used always close the main tank valve after use and drain the regulator of any residual ethane by following these steps Installation and
35. e workstation guide plate Slide the workstation onto the guide plate until it stops When the workstation is pressed fully into position the safety interlock is engaged Pneumatic connection Mains power connection Protective earth connection Connections and System Test 26 Cryoplunge3 Owner s Manual and User s Guide igure 1 10 Safety interlock for workstation NOTE There are two safety interlocks on Cp3 one is on the safety shield and the other is on the liquid nitrogen workstation 13 Place the large cover over the top of the workstation with the detent for the small cover to the right Place the small cover on top of the large cover so that it engages the detent in the large cover With the small cover in place the safety shield cannot close This is done purposefully so that one cannot acci dentally plunge the tweezers into the small cover 14 Connect a piece of Tygon tubing provided to the overflow port of the work station such that it extends beyond the edge of the base plate igure 1 11 Workstation overflow port with Tygon tubing attached 15 Connect the ethane pot TC heater connector to its receptacle on the back right hand side of Cp3 there is only one orientation for this connector 16 Insert the Omega temperature humidity sensor into the large hole on the top left hand side of the humidity chamber Insert the electrical connection for the sensor into its receptacle on the right hand side of the c
36. ective earth post at the back panel of Cp3 and attach the opposite end to a ground source 5 Connect the power supply to the mains cable 6 Connect the cable to the mains receptacle on back of Cp3 Inner rim of quick disconnect fitting Push high pressure pneumatic tubing firmly into the quick disconnect fitting Outer rim of quick disconnect fitting Connections and System Test Cryoplunge3 Owner s Manual and User s Guide 25 igure 1 9 Pneumatic mains and protective earth connections 7 Connect the plug to the house electrical supply 8 Orient Cp3 to a comfortable working position on the laboratory bench 9 If using nitrogen gas for the pneumatics make sure that the second stage of the regulator is not pressurized at this point 10 Open the main tank valve on the nitrogen gas tank and note pressure on the first stage of the regulator This is the tank pressure 11 Adjust the second stage pressure to at least 60 psi approximately 4 1 bar by turning the regulator adjusting spring handle clockwise NOTE Make sure that all connections are secure to avoid personal injury When fully pressurized the high pressure pneumatic tubing carries 60 80 psi 4 1 to 5 5 bar to Cp3 As a safety precaution it is recommended that the remainder of the tubing is secured to a stationary object 12 Place the liquid nitrogen workstation onto the base of Cp3 and align the groove at the base of the workstation with th
37. een tested by an independent accredited third party and has been found to be in compliance with applicable CE Marking requirements per the EMC Directive and the Low Voltage Directive See the Declaration of Con formity at the end of this manual for applicable standards and tests The most recent Declaration of Conformity is available by request from Gatan Inc SYMBOL NAME DESCRIPTION Danger An immediately hazardous situation which if not avoided will result in death or serious injury Danger is limited to the most extreme situations Warning A potentially hazardous situation which if not avoided could result in death or serious injury Caution A potentially hazardous situation which if not avoided may result in minor or moderate injury Caution may also be used to alert against unsafe practices Electrical hazard A potential electrical or electric shock hazard Pinch point A potential pinch point mechanical hazard Cold A potential cold surface hazard Flammable A potential flammable material hazard Biohazard A potential biological hazard Product Safety Information 6 Cryoplunge 3 Owner s Manual and User s Guide Warnings Before you attempt to install or operate this equipment for the first time please make sure that you are aware of the precautions that you must take to ensure your own safety Caution read this section first This section is only to be used for guidance Please consult the Prod
38. esent any Gatan Inc products sold within the European Union may be returned to Gatan for proper disposal We have placed great emphasis on creating a truly easy to use return process for our many customers around the world To return a product for disposal customers simply need to contact our customer ser vice department to obtain an RMA number authorizing the return We are pleased that our current disposal process is a relatively streamlined one Of course we are still busy evaluating other options that will further reduce the transport overhead of recycled products Every day we are actively working with design engineers vendors and additional key partners to enact even more effi cient end of life strategies for our products This product is compliant with Directive 2002 96 EC This product is compliant with 2002 96 EC as Category 9 equipment The Declaration of Conformance is available from Gatan Inc or from your authorized distributor Safety and Attention Symbols Cryoplunge 3 Owner s Manual and User s Guide 5 Safety and Attention Symbols You must be aware of safety when you install and use this system This guide pro vides various procedures that require careful attention to precautions The follow ing symbols appear in this document as referenced from ANSI Z535 1 4 Product Safety Information CE Marking The Cryoplunge3 is designed to meet the regulatory standards and CE require ments This product has b
39. ess the yellow ethane ON OFF button on the main support column of Cp3 to silence the alarm NOTE There is one audible alarm and two visual alarms for the ethane pot temperature NOTE The cut off temperature for the audible visual alarm is preset to 150 C at the factory 22 Set the blotting timer to 4 seconds using the toggle switches on the front of the timer Connections and System Test Cryoplunge3 Owner s Manual and User s Guide 29 igure 1 15 Meters and alarms for Cp3 23 Check to make sure that the plunge rod is in its full upward firing position then press the RESET button to close the shutter located at the base of the humidity chamber 24 Press the START button The blotters come together for 4 seconds and then the plunge rod fires towards the ethane pot 25 Raise the plunge rod to the full upward firing position NOTE Remember to return the plunge rod to its full upward firing position before pressing the RESET button or depressurizing the pneumatic supply to Cp3 Failure to do this causes the shutter at the base of the humidity chamber to close against the plunge rod This situation can be corrected by manually moving the shutter towards the front of the instrument while raising the plunge rod to the full upward firing position Refer to the Troubleshooting section of this manual for further instructions if necessary NOTE The plunge rod does not fire unless the following preconditions
40. first returning the plunge rod to its full upward firing position and then pressing RESET The humidity wand sponge is too dry to maintain humidity Re soak the wand with water to increase the humidity within the chamber Plunge tweezers do not hold grid The black securing clip is not properly inserted on the tweezers shaft Make sure that the smaller of the two openings on the securing clip is inserted onto the tweezers shaft such that it faces the tip of the tweezers and locks onto the first detent furthest from the tweezers tip The tweezers tips are splayed apart due to misuse Remove the black securing clip and with the tweezers opened gently press each tip of the tweezers against a solid surface to re align tips If necessary the tips of the plunge tweezers can be gently filed against the surface of a sharpening stone as needed to improve performance and longevity Grid disconnects from plunge tweezers during plunge cycle Black securing clip not properly inserted on tweezers shaft Make sure that the smaller of the two openings on the securing clip is inserted onto the tweezers shaft such that it faces the tip of the tweezers and locks onto the first detent furthest from the tweezers tip Tweezers tips are splayed apart due to misuse Remove the black securing clip and with the tweezers opened gently press each tip of the tweezers against a solid surface to re align tips
41. g exceptions 1 Remove the blotters 2 Insert a blanking plug into the right and left hand blot assemblies 3 Apply a small aliquot of the sample suspension to the pre treated surface of the support grid using an accurate volumetric pipetting device 4 Press RESET to close the shutter 5 Set the blotting timer for the desired time for example 10 minutes 6 There should be no need to orient the grid with the blot axis because the grid is not blotted 7 Press the START pushbutton The specimen slowly dries to a thin film in the high humidity of the chamber prior to plunging into the ethane The exact time for this to occur must be determined based on the humidity in the cham ber Method 2 Immediate plunge with no blotting NOTE In some cases the specimen aliquot is applied in such small volumes that blotting or drying is not required An example of this method would be spraying a small volume of the sample directly onto the surface of the support film within the high humidity environment of the chamber NOTE Follow the instructions for freezing the sample using 2 sided blotting with the following exceptions Routine Maintenance 50 Cryoplunge3 Owner s Manual and User s Guide 1 Apply a small volume of the sample directly to the surface of the support film using a spray technique 2 Set the blotting timer to zero with the blotting timer set to zero the blotters do not fire 3 Press the STA
42. hat the pneumatic source is on i e the main tank valve for the nitrogen gas tank which supplies the pneumatic pressure is open and set the second stage of the nitrogen gas regulator on the tank to a value of 60 and 80 psi 4 1 to 5 5 bar If using house air supply make sure that it is deliv ering the proper pressure The blotters are not fully inserted into the blot assembly at one of the four detent positions for blotting Press the blotters to their fully in and align with one of the four detent positions Ethane warning lamp does not illuminate Mains power supply and ON OFF switch Make sure the connector for the temperature humidity sensor is inserted in the proper orientation and that it is secure and the mains power is switched ON The bulb is burned out Replace the bulb Contact Gatan Inc for part number Ethane alarm ON OFF pushbutton does not illuminate Mains OFF Check to make sure that the electrical connection is made and that the ON OFF switch is switched ON The bulb is burned out Replace the bulb Temperature humidity not indicated on meter Temperature humidity connector Check to be sure that the connector for the temper ature humidity sensor is inserted in the proper orien tation and that it is secure Loss of high humidity condition within humidity chamber The shutter and or specimen loading port is open Close the shutter and or specimen loading port by
43. igure 1 36 Blotting excess ethane 8 Position the tweezers such that the grid is in the liquid nitrogen of the cryo grid box transfer pot and at this point disconnect the black plastic securing clip for the plunge tweezers taking care not to drop the grid igure 1 37 Disconnect the black securing clip on the plunge tweezers to free the frozen hydrated grid 9 The grid can now be placed within one of the receptacles of the cryo grid box 10 If the cryo grid box has a rotating cover secured with a screw position the slot of the cover and tighten the screw such that the grids cannot fall out of the box when it is transported for the next step To remove the frozen hydrated grids from the workstation NOTE This step requires that you have a ready supply of liquid nitrogen a liquid nitrogen transfer dewar a liquid nitrogen storage dewar and large tweezers to manipulate the liquid nitrogen transfer pot containing the blue cryo grid box Blotting Techniques for Freezing the Sample 46 Cryoplunge3 Owner s Manual and User s Guide NOTE Add liquid nitrogen to the workstation through the filling funnel as necessary to maintain the temperature of the workstation NOTE There are many methods for storing the frozen hydrated grids prior to viewing on the cryo TEM A grid transfer storage tube conical tube provided can be used to contain the blue cryo grid boxes This tube has holes drilled near the cap to allow liquid nitroge
44. ind whether based on warranty contract tort or any other legal theory The limits of Gatan liability in any dispute shall be the price received from the purchaser for the specific equipment at issue The laws of the state of Pennsylvania apply to all aspects of this warranty Part Number 001 02113 Rev 1 According to ISO IEC GUIDE 22 and EN 45014 DECLARATION OF CONFORMITY Manufacturer s Name Gatan Inc Manufacturer s Address Pleasanton CA 94588 U S A 5794 W Las Positas Blvd DECLARES THAT THE PRODUCT Product Name CryoplungeTM3 Model Number 930 CONFORMS TO THE FOLLOWING EUROPEAN DIRECTIVES 2006 95 EC Low Voltage Directive 2004 108 EC EMC Directive 2002 96 EC Waste Electrical and Electronic Equipment WEEE Directive 2002 95 EC Restriction of Hazardous Substances RoHS Directive Exempt as Category 9 Monitoring and Control instruments In accordance with the following standards Safety and laboratory use Part 1 General requirements EN IEC 61010 1 2001 2nd Edition Safety requirements for electrical equipment for measurement control EMC EN55011 Radiated Emissions Class A EN 61326 1 2006 Emissions and Immunity EN61000 4 2 ESD Immunity EN61000 4 3 RF Immunity EN61000 4 4 EFT Immunity EN61000 4 5 Surge Immunity EN61000 4 6 Conducted Immunity EN61000 4 8 Magnetic Field Immunity EN61000 4 11 Voltage Dip Immunity I the undersigned hereby declare that the equipment specified
45. ing space around the products for servicing by Gatan authorized service representatives 6 Provide access to and use of all information and facilities determined necessary by Gatan to service and or maintain the products Insofar as these items may contain proprietary or classified information the customer shall assume full responsibility for safe guarding and protecting them from wrongful use Repairs and Replacements Gatan will at its option either repair or replace defective instruments or components with conforming goods Repair or replacement of products or parts under warranty does not extend the original warranty period With the exception of consumable and maintenance items the replacement parts or products used on instruments out of warranty are themselves warranted to be free of defects in materials and workmanship for 90 days Any products part or assembly returned to Gatan for examination or repair shall have Gatan s prior approval with the customer requesting a Returned Material Authorization RMA approval This RMA and the associated RMA number may be obtained through Gatan Service or directly from Gatan s Warrendale facility at 724 776 5260 If the item is not under warranty to obtain an RMA the customer must provide a Purchase Order PO for the repair If the item is under warranty and the customer is requesting an expedited exchange as may be the case for a printed cir cuit board a PO will be required A credit agains
46. ions 8 Assembly Location and Mounting Requirements 9 Cryoplunge3 Owner s Manual and User s Guide 11 Overview 11 Features 11 Easy Installation 11 Safe Operation 12 Humidity Chamber 12 Specimen Blotting 12 Plunge and Tweezer Securing Mechanism 12 Cryo Workstation 12 Maintenance 13 Description and Specifications 13 Physical Specifications
47. ld materials objects that are soft and pliable at room temperature such as rubber or plastic become hard and brittle and are easily broken at these extremely low temperatures NOTE For this next procedure you will be working in an explosion proof fume hood No flames or explosive chemicals are allowed in this hood NOTE If the fume hood has an exceptionally high draw rate you might have to minimize the draw for this procedure and then restore the draw rate at the close of the procedure NOTE Have a ready supply of liquid nitrogen available to maintain the workstation at the low temperature equilibrium NOTE Always wear eye protection when working with liquefied ethane or any cryogens Important The volume of the ethane pot for Cp3 is 4 1 mL to the upper rim of the pot Do not condense more than 10 mL of ethane at one time Always make sure that the containers that will receive the condensed ethane do not contain any residual liquid nitrogen In a closed environ ment and upon evaporation the liquid nitrogen pushes the liquid ethane out of the vessel at a high rate and can cause injury Always position the ethane vessel away from you Ethane vaporizes at 88 6 C so it is very important to maintain the level of liquid nitrogen within the workstation of Cp3 when the ethane pot is filled Condensing Ethane and Filling the Ethane Pot Cryoplunge3 Owner s Manual and User s Guide 37 To
48. lunge rod in firing position Temperature humidity sensor Liquid nitrogen workstation Temperature humidity meter Omega iSeries Blotting timer Omron H5CX Base plate START push button RESET push button Mains ON OFF Ethane pot TC heater connector Safety shield Pneumatic supply for blotters Right side blotter Securing screw for humidity chamber Humidity wand with sponges attached Temperature humidity sensor connector Ethane temperature audible alarm ON OFF Left side blotter Ethane temperature warning lamp Description and Specifications Cryoplunge3 Owner s Manual and User s Guide 15 igure 1 2 Cryoplunge3 side view left Plunge rod dampening pot Plunge rod plunge tweezers not attached Liquid nitrogen overflow Push in connector for blot assembly pneumatics Workstation cover large Specimen loading port Humidity wand with sponge attached Liquid nitrogen funnel Description and Specifications 16 Cryoplunge3 Owner s Manual and User s Guide igure 1 3 Cryoplunge3 side view back and right igure 1 4 Workstation Pneumatic push in connection Protective earth stud Mains connection Cryo grid box transfer pot with square cryo grid box inserted Cryo grid box staging area Ethane pot Filter paper clip for pre blotting frozen hydrated grid prior to storage Liquid nitrogen overflow port Liquid nitrogen filling port with funnel attached Physic
49. ly into the pre cooled ethane pot and hold it at the bottom of the pot The nozzle being used in this method is made from a volumetric pipette tip that is commonly found in the laboratory Clip the end of the pipette tip to allow an opening of approximately 2 3 mm igure 1 25 Condensing ethane directly into the pre cooled ethane pot Condensing Ethane and Filling the Ethane Pot 38 Cryoplunge3 Owner s Manual and User s Guide Method 2 Pre cool a conical tube provided in a small bath of liquid nitrogen Condense 6 7 mL of ethane in the bottom of the tube Carefully pour the condensed ethane into the pre cooled ethane pot igure 1 26 Pre cooling the conical tube and then pouring the condensed ethane into the pre cooled ethane pot Method 3 Pre cool a conical tube provided in a small bath of liquid nitrogen and pre cool three or four 2 mL volumetric tubes Pipette the ethane into the pre cooled tubes and allow them to freeze When frozen partially thaw the ethane by touching the outside of the plastic tube to a warm surface such as the counter top of the fume hood Invert the tube over the ethane pot and allow the ethane popsicle to slide out Exercise caution when using this method as the 2 mL volumetric tubes must be dry no liquid nitrogen prior to filling with liquid ethane igure 1 27 Making the ethane popsicle Condensing Ethane and Filling the Ethane Pot Cryoplunge3 Owner s Manual and User
50. moved within approximately the first four seconds after which time the grid and filter paper disengage The remaining four seconds allow the solution to slowly evaporate in the high humidity of the chamber to the desired thickness before it is finally plunged into the ethane pot Manual Multiple Blotting To freeze the sample using manual multiple blotting NOTE When the safety shield is left slightly ajar pressing RESET closes the shutter but pressing START blots the specimen without plunging the specimen grid into the ethane pot This feature allows the user to perform manual multiple blots of the specimen grid prior to plunging it into the ethane pot NOTE This technique is useful when multiple reagents or additional sample aliquots are to be added to the specimen grid prior to plunging NOTE Because each blot cycle is manual for multiple blotting different blotting times can be entered on the blotting timer to accommodate the constraints of the experiment being performed NOTE Follow the instructions for freezing the sample using 2 sided blotting with the following exceptions 1 Apply a small aliquot of the sample suspension to the pre treated surface of the support grid using an accurate volumetric pipetting device 2 Close the sample loading port to maintain the high humidity environment within the humidity chamber 3 Rotate the plunge rod 90 degrees to the left or right to engage one of the four detent position
51. n NOTE The specimen grids are fragile and must be handled with care to avoid damage bending of the grid or breakage of the support film 1 Hold the plunge tweezers with one hand 2 Press the blue quick disconnect pushbutton on the plunge rod with your other hand igure 1 34 Pressing the quick disconnect push button to release the plunge tweezers from the plunge rod 3 Push the plunge rod upwards approximately 3 cm to clear the top of the plunge tweezer 4 Tilt the plunge tweezer slightly while keeping the frozen hydrated grid immersed within the center of the liquid ethane pot igure 1 35 Tilting the tweezers 5 Gently lower the plunge piston to its bottom most position and leave it at this position for the next steps 6 Remove the tweezer from the ethane pot keeping it well within the worksta tion to prevent devitrification of the frozen hydrated grid Blotting Techniques for Freezing the Sample Cryoplunge3 Owner s Manual and User s Guide 45 7 OPTIONAL If you want to view your grid on the same day that it is plunged it is helpful to blot off any excess ethane that is sticking to the grid To blot the excess ethane place the grid just below the filter paper such that the tines of the plunge tweezers contact the filter paper without touching the grid This action draws away any excess ethane that is trapped between the tines of the plunge tweezers and subsequently from the surface of the grid
52. n to flow freely in and out of the tube Affixing a piece of string to this tube through the holes provided allows transport of the storage tube containing the cryo grid box with the frozen hydrated grids into a large liquid nitrogen storage dewar for example 35 liter dewar 1 The frozen hydrated grids should be secured within the grid box 2 Remove the small workstation cover and ensure that the cryo grid box transfer pot is filled with liquid nitrogen 3 Prepare a liquid nitrogen transfer dewar by filling with liquid nitrogen and waiting several minutes for the temperature of the dewar to stabilize 4 Remove the cap on the grid transfer storage tube conical tube provide and immerse the tube into the dewar of liquid nitrogen 5 Using a pair of large tweezers remove the cryo grid box transfer pot from the workstation and place inside of the transfer dewar 6 Transfer the grid box to the storage tube using the large tweezers igure 1 38 Transfer the grid box to the storage tube filled with liquid nitrogen 7 Return the cryo grid box transfer pot to the workstation 8 Refill the remove the cryo grid box transfer pot and the workstation with liq uid nitrogen to continue freezing more grids If you no longer wish to freeze more grids return Cp3 to stand by for the next user Returning Cp3 to Stand by Conditions To return Cp3 to stand by conditions for the next user 1 Clean and dry the plunge tweezers a
53. nd store them with the protective cap in place 2 Clean and dry the filter paper loading tweezers and store them in their protec tive sleeve Blotting Techniques for Freezing the Sample Cryoplunge3 Owner s Manual and User s Guide 47 3 Clean and dry the filter paper loading jig and store it in its protective plastic bag 4 Close the main tank valve on the nitrogen gas supply pneumatics supply to Cp3 5 Raise the plunge rod to its fully upward firing position within the humidity chamber 6 Open the safety shield 7 Press RESET several times to bleed the nitrogen gas from the pneumatic lines the shutter opens and closes during this process 8 Switch the mains OFF 9 Unplug the TC heater connector for the ethane pot 10 Remove the workstation and place in the explosion proof spark proof fume hood 11 Remove the small and large workstation covers 12 Close the protective screen on the fume hood to allow the cryogens to vent out of the hood When the workstation is dry all of the cryogens have vented off it can be returned to Cp3 for the next user WARNING Never use a heat gun to speed up the drying process These instru ments produce very high concentrated heat that damages melts the workstation If you wish to dry the workstation when all of the cryogens have vented off it is acceptable to use a hair dryer with a low heat set ting 13 Remove the humidity wand from the humidity ch
54. nect the plunge tweezers from the plunge rod The frozen hydrated grid can then be moved to a pre cooled cryo grid storage box within a removable liquid nitrogen filled transfer pot The specimen grid can then be safely transferred to a liquid nitrogen storage vessel or to the workstation of the cryo transfer holder for low electron dose imaging on the trans mission electron microscope Features Easy Installation Everything is included for fast easy set up Light weight and portable Small footprint Features 12 Cryoplunge3 Owner s Manual and User s Guide Safe Operation Safety interlocks protect the user during the blotting and plunging cycle Humidity Chamber Safe guards the specimen from drying during the blotting process Temperature humidity metering within the specimen blotting chamber 98 RH within 15 minutes 3 entry points for specimen loading right left and front Specimen Blotting 1 or 2 sided blotting Single and multiple blotting Filter paper loading jig to minimize contact with filter paper while loading onto blot assemblies Electronic blotting timer accurate to 0 1 sec Interchangeable blot assemblies Adjustable blotting pressure Any filter paper type available can be used Plunge and Tweezer Securing Mechanism Pneumatically assisted multi positional plunge piston Plunge speed 1 7 m sec
55. ng pin Disconnect all external power connections before servicing Should a leak occur remove power Use paper towels or Kimwipe to clean up the spill Cryoplunge3 makes use of liquid ethane To ensure your safety ethane gas must only be ordered in consultation with the Laboratory Services Manager and site Safety Officer Please follow your laboratory and site Safety Officer s recommendations for storage and use The size of ethane gas cylinder should be restricted to lecture theater size or the smallest size that is available for the purity rating required at least CP grade which is 99 pure fitted with a 2 stage regulator that is suitable for use with ethane Always check the ethane cylinder for any signs of corrosion prior to use Product Safety Information Cryoplunge 3 Owner s Manual and User s Guide 7 Liquefaction of ethane must only be carried out using purpose built equip ment designed to be used in conjunction with liquid nitrogen as the primary coolant The ethane should be condensed in an explosion proof spark proof fume hood The ethane cylinder should be supported in an approved stand within the fume hood The fume hood must not contain any flammable or harmful substances and should be clear of any unnecessary equipment that could impede air flow Other sources of ignition such as electrical equipment including ovens must also be removed fr
56. nput Voltage 100 240V Input Power 1 35A Frequency 47 63Hz Output Voltage 24VDC Output Current 2 08A max NOTE Use only with a Gatan specified power supply Assembly Location and Mounting Requirements Cryoplunge 3 Owner s Manual and User s Guide 9 Environmental Storage Shipping Operating Conditions Non operating relative humidity non condensing 25 85 Storage Temperature Range 0 33 C Operating Humidity Range 0 100 Operating maximum thermal gradient 15 C hr Operating temperature range ambient 4 24 C Ventilation do not block or cover ventilation holes Assembly Location and Mounting Requirements Prior to Unpacking the Instrument The carton comprising the shipment should be inspected for any signs of damage before unpacking If any severe damage is visible the following procedures should be carried out Photograph the extent of the damage Digital photographs sent by email are often the best method Gatan Inc info gatan com Describe the extent of the damage to Gatan Inc to estimate possible damage to the enclosed equipment and decide whether an insurance assessor and engineer need to be present before further unpacking Installation Cryoplunge3 is intended only for use in laboratory applications and is designed specifically for workstation table top installations The product is intended for operation in a c
57. ny excess water by blotting against absorbent paper NOTE Soak the sponge in hot water to rapidly raise the chamber humidity 8 Replace the humidity wand 9 Wait approximately 15 minutes for the humidity chamber to reach 98 RH 10 Resoak the humidity wand as needed to maintain 98 RH within the humid ity chamber Preparing the Filter Paper Disks NOTE The die cutter for preparing filter paper disks comes pre cleaned from the factory but should be cleaned periodically to keep the filter paper disks free from contaminants To clean the die cutter wear protective gloves and gently wipe the upper and lower cutting surfaces with a cotton swab or lint free cloth moistened with ethanol Clean the support surface with a lint free cloth moistened with ethanol Never touch the surface of the filter paper disks with your bare hands Always wear protective gloves to ensure that the disks remain contamination free To prepare the filter paper disks 1 Place the die cutter on a firm clean surface 2 Clean the cutting surfaces to ensure that the filter paper disks are free from contaminants 3 Prepare a pertri dish to capture the freshly cut filter paper disks 4 Wear protective gloves for example RNAse free Nitrile gloves 5 Insert the desired type of filter paper into the die cutter and press the lever downward to cut the disk 6 Move the cut disk to the petri dish Connections and System Test Cryoplunge3 O
58. olumn Cameras DigiScan GIF and PEELS travel of up to 100 miles from a Gatan authorized repair center Pleasanton CA Warrendale PA Munich GmbH and Corby UK is included Travel expenses for service beyond 100 miles will be charged for Instruments parts and accessories not manufactured by Gatan will be warranted by Gatan for the specific items and periods in accordance with and provided by the warranty received by Gatan from the Original Equipment Manufacturer All such accessory warranties extended by Gatan are limited in accordance with all the terms conditions and other provisions stated in this Original Equipment Manufacturer warranty Gatan makes no warranty whatsoever concerning products or accessories not of its manufacture except as noted above Customer Responsibilities The customer bears the following responsibilities with regard to maintaining the warranty The customer shall 1 Perform the routine maintenance and cleaning procedures at the required intervals as specified in Gatan s operating manuals Failure to per form specified maintenance will automatically void warranty 2 Use Gatan replacement parts Failure to use the specified replacement parts will automatically void warranty 3 Use Gatan or Gatan approved consumables 4 Provide Gatan authorized service representatives access to the products during normal Gatan working hours during the coverage periods to perform service 5 Provide adequate and safe work
59. olumn for Cp3 The connector for the sensor is configured such that it can only be inserted in one orientation Safety interlock Workstation slides over glide plate Connections and System Test Cryoplunge3 Owner s Manual and User s Guide 27 igure 1 12 Insertion of Omega temperature humidity sensor igure 1 13 Omega temperature humidity sensor connector 17 Insert the humidity wand into the small hole on the top right hand side of the humidity chamber Connections and System Test 28 Cryoplunge3 Owner s Manual and User s Guide igure 1 14 Inserting the humidity wand 18 Remove the small workstation cover and fully close the workstation safety shield This engages the magnetic safety interlock In this position the plunge rod can fire into the ethane pot NOTE If the safety shield is open pressing the RESET push button closes the shutter at the base of the humidity chamber If the START push button is pressed when the safety shield is opened the blot pads activate but the plunge rod does not fire into the ethane pot 19 The instrument is delivered with the plunge rod in its full upward firing position i e it is positioned within the humidity chamber of Cp3 20 Place the right and left hand blotters into their receptacles on the humidity chamber 21 Switch mains ON The meters for ethane temperature blot time and temperature humidity illuminate If the ethane alarm sounds pr
60. om the fume hood before using ethane The flow of ethane gas through the liquefaction apparatus must be such that only liquid ethane is produced thus avoiding any unnecessary venting of ethane gas into the fume hood Use caution in high flow fume hoods as they tend to draw the ethane out of the hood before it can become condensed into liquid form To ensure your safety do not condense more than 10 mL of liquid ethane at one time When sufficient ethane has been liquefied the ethane cylinder valves should be closed and the 2 stage regulator should be drained of any residual ethane The liquid ethane must be maintained at near liquid nitrogen temperature to prevent boil off into the atmosphere The workstation of Cryoplunge3 incorporates a liquid nitrogen bath that maintains the liquid ethane at an operating temperature just above its melting point The boil off of the liquid nitrogen produces an inert blanket of gas over the ethane preventing oxygen from the atmosphere condensing on the surface of the ethane to form an explosive mixture The liquid ethane must be kept cov ered by nitrogen gas at all times that are practicable to prevent dangerous oxy gen ethane mixtures from forming Maintaining the level of liquid nitrogen within the workstation of Cryoplunge3 will ensure that a layer of nitrogen gas will enshroud the ethane vessel at all times Ensure that all equipment is adequately earthed The in
61. ontrolled work environment laboratory or clean room environ ment It is recommended the unit not be blocked from general room ventilation Connection to the Power Prior to plugging the unit in read all safety precautions contained within this man ual and ensure the power switch on the unit has been turned off I refers to ON and O refers to OFF Apply all applicable connections detailed above Use only the power cord provided with this product and proper electrical power as specified on the equipment rating label Ensure the outlet is equipped with a proper ground pin A separate ground wire is required for connection at the main PE terminal at the back of the main column and designated by the IEC 60417 5019 symbol Should there be any questions as to how to connect the ground contact a licensed electrician Assembly Location and Mounting Requirements 10 Cryoplunge 3 Owner s Manual and User s Guide When power is applied to unit the meters on the front panel illuminate indicating that the unit is on If Cp3 is going to be moved or stored close the pneumatic supply nitrogen gas cylinder or house air supply connection and vent any residual pressure in the pneumatic lines by first ensuring that the plunge piston is in its full upward fir ing position and then repeatedly pressing the RESET push button Disconnect the pneumatic supply line from Cp3 Switch the ON OFF switch to OFF and unplug the mains connection Over
62. op layer of ethane within the ethane pot This Condensing Ethane and Filling the Ethane Pot 36 Cryoplunge3 Owner s Manual and User s Guide in turn can trap ethane gas which can explode through the frozen layer of ethane showering the surrounding area ASPHYXIATION Asphyxia is a potential hazard with all cryogens if they are allowed to warm and produce large volumes of gas Because the gases produced by ethane and nitrogen are colorless and odor less they can escape into the atmosphere undetected and quickly reduce the concentration of oxygen below the level necessary to sup port life lt 19 5 v v Victims might be unaware At low concentra tions ethane can cause narcotic effects and symptoms can include dizziness headache nausea and loss of coordination COLD BURNS Because of extremely low temperatures cryogens are capable of producing severe cryogenic burns and frostbite When spilled on a surface they tend to cover it completely and therefore cool a large area The vapors from these liquids are also extremely cold and can produce burns Exposure which might be too brief to affect the skin of the face or hands can damage delicate tissues such as the eyes Flesh sticks to extremely cold materials such as non insu lated pipes or metallic vessels containing cryogenic liquids Even non metallic materials are dangerous to touch at low temperature In addi tion to the hazards of frostbite or flesh sticking to co
63. s the grid is now aligned with the blot pad s 4 Set the timer for the desired blotting time 5 Remove the small workstation cover and leave the safety shield slightly ajar to override the safety interlock the shield is still in a protective position 6 If necessary press RESET to close the shutter Blotting Techniques for Freezing the Sample Cryoplunge3 Owner s Manual and User s Guide 49 7 Allow the sample aliquot to adsorb onto the grid substrate for the desired amount of time for example 5 to 15 seconds 8 Press START The blot pad s blots the specimen for the time indicated on the timer but the grid does not plunge into the ethane 9 Press RESET The shutter closes to maintain the high humidity environment of the chamber 10 Add additional sample or reagent to the grid if desired 11 Close the safety shield completely to engage the safety interlock 12 Press START The specimen grid is blotted and plunged into the ethane pot No Blotting Method Method 1 Forming the thin film by slow evaporation under high humidity conditions NOTE In some cases the experiment might require that the grid not be blotted at all There are two methods to accomplish this with Cp3 NOTE Cp3 is delivered with one blanking plug Additional blanking plugs are available as an accessory item at an additional charge NOTE Follow the instructions for freezing the sample using 2 sided blotting with the followin
64. strument must always be connected to an electrical ground when it is being used to reduce the risk of electric shock The ground wire green yellow in the instrument power cable must be connected to the laboratory electrical ground Only use exten sion cables if they have an earth conductor Do not disconnect the protective ground inside or outside the instrument and do not have external circuits con nected to the instrument when its protective ground is disconnected The instrument does not stop working if the earth wire is not connected and there is no indication that you might be in danger Make sure that it is checked regularly and at least annually Equipment Ratings and Technical Specifications 8 Cryoplunge 3 Owner s Manual and User s Guide WARNING To avoid electrical hazards heat shock and or fire hazard do not make connections to terminals outside the range specified for that ter minal See the product user manual for correct connections Electronic components on printed circuit boards are extremely sensitive to static electricity Ordinary amounts of static electricity generated by your clothing or work environment can damage the electronic equipment When installing the board in a system you must use anti static grounding straps and anti static mats to prevent damage due to electrostatic discharge To avoid injury fire hazard or explosion do not operate this product in an explosive atmosphere
65. t the base of the workstation 9 Position the blot pads There are four blotting positions as indicated on the blot assemblies Each blotter has a white index line Start by positioning the index line on each blotter so that it is aligned with the 12 o clock uppermost position on the blot assembly Remember to rotate the blotters after each plunge cycle so that each grid being blotted is exposed to an unused area of the filter paper for example after each blot plunge cycle rotate the blotters towards the back of the instrument 10 Open the specimen loading port on the front of the humidity chamber 11 Apply a small aliquot of the sample suspension to the pre treated surface of the support grid using an accurate volumetric pipetting device 3 micro liters is a good starting point igure 1 31 Applying sample aliquot to the support film 12 Close the specimen loading port to maintain the high humidity environment within the humidity chamber 13 Rotate the plunge rod 90 degrees to the left or right so that the grid is now aligned with the blot pads Blotting Techniques for Freezing the Sample Cryoplunge3 Owner s Manual and User s Guide 43 There are four detent positions for the plunge rod to allow accurate align ment of the grid with respect to the blot pads 14 Set the timer for the desired blotting time For example for two sided blotting where a 3 mL volume is applie
66. t this PO will be issued by Gatan upon receipt of the item as returned in accordance with the RMA instructions The returned item should be shipped prepaid by the customer with the RMA number clearly marked on the exterior of the shipping container and on the enclosed shipping documents If the returned item is under warranty return transportation will be prepaid by Gatan If the returned item is not under warranty return transportation will be charged to the customer Warranty Limitations The warranty does not cover 1 Parts and accessories which are expendable or consumable in the normal operation of the instrument 2 Any loss damage and or instrument malfunction resulting from shipping or storage accident fire flood or similar catastrophes normally covered by insurance abuse alteration misuse neglect or breakage or abuse of parts by User 3 Operation other than in accordance with correct operational procedures and environmental and electrical specifications 4 Performance to specifications or safety of use including X ray emissions if the unit is physically installed on used in conjunction with or used as part of a third party s equipment and is not installed by a Gatan service engineer 5 Performance to specifications or safety of use including X ray emissions as a result of the use of Gatan s equipment with that of a third party due to the third party s product design 6 Modification of or tampering with the sys
67. tem 7 Improper or inadequate care maintenance adjustment or calibration by User 8 User induced contamination or leaks 9 Any loss damage and or instruments malfunction resulting from use of User supplied software hardware interfaces or consumables other than those specified by Gatan Warranty Exclusions In the course of normal use and maintenance certain parts have finite lifetimes For this reason the consumables wear and maintenance parts as specified in Gatan s operating manuals carry a 90 day warranty unless otherwise specified Post Warranty Period Support and Product Obsolescence After the expiration of the warranty period described above Gatan will provide service support for Gatan manufactured products at Gatan s service labor rates and parts pricing in effect at the time of the repair Gatan will continue to provide billable service support for the products for a period of three years after discontinuance or design obsolescence by Gatan After this three year period service support will be offered at the sole discre tion of Gatan Liability Limitations This warranty is in lieu of and excludes all other expressed or implied warranties including but not limited to warranties of merchantability of fitness for a particular purpose Under no circumstances will Gatan Inc or Gatan International be liable for any direct indirect special inciden tal or consequential damages including lost profit or loss of any k
68. uct Safety and Assembly Location and Mounting Requirements sections of this manual prior to installation of this instrument If unsure consult your Safety Officer Safety Precautions Review the following precautions to avoid injury and prevent damage to this product or any products to which it is connected To avoid potential hazards use the product only as specified Read all safety information provided in the compo nent product user manuals and understand the precautions associated with safety symbols written warnings and cautions before accessing parts or locations within the unit Save this document for future reference Follow all warnings and instruc tions marked on the equipment Ensure that the voltage and frequency of your power source matches the voltage and frequency inscribed on the equipment s electrical rating label Never push objects of any kind through the openings in the equipment Dangerous voltages might be present Conductive foreign objects could produce a short circuit that could cause fire electrical shock or damage your equipment If in doubt contact your local Safety Officer for guidance Disconnect power before replacing fuses and only use the value specified on the product s rating label Do Not Operate Without Covers To avoid electric shock or fire hazard do not operate this product with any removed enclosure covers or panels Always use a power cable with a grounded plug third groundi
69. ulator to a compressed gas tank please seek assistance from your on site Safety Officer or laboratory advisor 2 The tank must be secured to a wall stand or cart using a safety chain so that it cannot tip over during use 3 The main tank valve should be closed as indicated on the top of the valve SPECIFICATION VALUE Weight 12 25 kg Dimensions length x width x height 41 L x 31 W x 75 H centimeters Power input 24V 2 08 max Total weight Cp3 and shipping crate 58 lb Shipping crate dimensions 102 L x 64 W x 48 H centimeters 40 L x 25 W x 19 H inches Cryogenic Requirements and Safe Handling 18 Cryoplunge3 Owner s Manual and User s Guide 4 Prior to attaching the 2 stage regulator to the tank make sure the mating sur faces are clean and free of any particulate matter 5 Wrap the threaded end of one of the thread to push in fittings with Teflon tape and attach to the exit port of the second stage of the regulator 6 Press one end of the pneumatic tubing provided fully into the thread to push in fitting There is an o ring within the fitting that the tubing must go through When fully inserted check by pulling the tubing away from the fitting If the tubing does not disengage from the fitting it is properly installed The oppo site end of this tubing fits into the push in fitting on the back panel of Cp3 igure 1 5 Pneumatics supply for Cp3 left 2 stage nitrogen regulator right Cr
70. upply valve is closed before proceeding Operating pressure for Cp3 is 60 min to 80 max psi of pressure 4 1 to 5 5 bar Pressure should not exceed 80 psi 5 5 bar when the valve is fully open during use 3 Connect high pressure tubing to the quick disconnect fitting for the pneumatic supply on the back panel of Cp3 Connections and System Test 24 Cryoplunge3 Owner s Manual and User s Guide Simply press the end of the tubing fully into opening see red arrow in image There is a slight resistance as the tubing goes through the o ring within the fitting Connect the opposite end of the high pressure tubing to the thread to push in fitting on a 2 stage nitrogen regulator or to the outlet for the house air supply The connection to the to the thread to push in fitting on the 2 stage nitrogen regulator was describe previously in Preparing the Nitro gen Cylinder and 2 Stage Regulator igure 1 8 Pneumatic connection NOTE In the event that you need to remove the tubing always make sure the main nitrogen tank valve or house air valve is fully off and while pressing the outer rim of the quick disconnect fitting in towards the inner rim of the fitting pull the high pressure tubing out There will be slight resistance from the o ring connection located within the fitting Upon disconnecting the high pressure tubing the residual gas in the tubing is released 4 Connect the protective earth cable to the prot
71. ur convenience You are strongly advised to review the latest MSDS available on the world wide web for safe handling procedures for liquid ethane and liquid nitrogen WARNING REVIEW AND REMEMBER THESE IMPORATANT SAFETY PRE CAUTIONS WHEN HANDLING THE LIQUID CRYOGENS USED IN THIS PROCEDURE FIRE AND EXPLOSION Fire and explosion are the primary hazards associated with flammable liquefied gases such as ethane Flamma ble gases can be ignited by static electricity or by a heat source such as a flame or any hot object Cryogens including nonflammable nitro gen which achieve temperatures below 183 C can liquefy oxygen from the surrounding environment to form potentially dangerous oxy gen mixtures which support violent combustion EXPLOSION DUE TO EXPANSION At temperatures above boiling point all liquid cryogens expand rapidly by factors in excess of 700 times Any cryogen contained or trapped in a sealed compartment or vessel is a potential explosion risk if allowed to warm up SPILLAGE DUE TO FAILURE OF STORAGE VESSELS Glass vac uum dewars can shatter when liquid cryogens are introduced causing a spill and potential lacerations from glass shards Take care when using glass vacuum dewars If the vacuum integrity of the dewar is in doubt use stainless steel vessels SPILLAGE OF ETHANE DUE TO NITROGEN ENTRAPMENT If the liquid nitrogen used to cool the ethane vessel overflows into the ethane it can freeze the t
72. view Cryoplunge3 Owner s Manual and User s Guide 11 Cryoplunge3 Owner s Manual and User s Guide Overview Cryoplunge 3 Cp3 is a semi automated plunge freezing instrument used for the preparation of frozen hydrated specimens for cryo transmission electron microscopy Cp3 incorporates a humidity chamber and timed single or multiple blot capabilities for either 1 sided or 2 sided blotting The specimen grid clamped between the tines of plunge tweezers and containing generally 3 microliters of the specimen suspension is blotted to produce a thin aqueous film It is then plunged into a temperature monitored ethane bath to produce a uniformly thin layer of vit reous ice with the specimen embedded in it A liquid nitrogen workstation with a remote fill funnel and two removable covers maintains the temperature of the liq uid ethane vessel which can be held at a temperature just above the melting point of liquid ethane 182 C As liquid nitrogen is added to the workstation a blanket of cold inert nitrogen gas fills the workstation chamber minimizing condensation of atmospheric oxygen onto the surface of the ethane and providing a protective cryo interface for transferring the frozen hydrated grid within the workstation The workstation covers are positioned to allow the operator easy access to the fro zen hydrated specimen grid One press of the quick disconnect on the plunge rod allows the operator to quickly and easily discon
73. wner s Manual and User s Guide 31 7 Continue until the suitable quantity of filter paper disks have been produced for your blotting needs igure 1 16 Filter paper die cutter Preparing the Blotters To mount the blotting foam onto the blotters NOTE Cp3 is shipped without the filter paper or foam attached to the blot assembly 1 Remove the blotters from the humidity chamber by grasping onto the outer surface of the blotter and pulling straight out and away from the humidity chamber There may be a slight resistance from the o rings on the shaft of the blotter igure 1 17 Inserting removing a blotter from the right hand blot assembly 2 Using the blot pad spindle wrench unscrew the top spindle Connections and System Test 32 Cryoplunge3 Owner s Manual and User s Guide igure 1 18 Removing the spindle 3 Insert one sponge pad over the outer spindle making sure it is properly aligned with the spindle core igure 1 19 Inserting the sponge pad 4 Replace the top spindle and tighten using the spindle wrench 5 Make sure the foam pads are uniform and flat when the spindle is tightened 6 Repeat for the other blot assembly igure 1 20 Replacing and tightening the spindle Loading the filter paper disk onto the blotters NOTE To prevent contamination of the filter paper disks wear protective gloves or handle the disks with pre cleaned tweezers NOTE If necessary clean and dry the
74. yogenic Requirements and Safe Handling Liquid nitrogen Please refer to the latest MSDS on the world wide web for safe handling information Liquid ethane Please refer to the latest MSDS on the world wide web for safe handling information Stage 2 Stage 1 Thread to push in fitting for Cp3 pneumatics Cp3 pneumatic tubing Regulator adjusting spring handle Nitrogen gas tank secured in support stand Cylinder valve handle 2 stage regulator Preparing the Ethane Cylinder and 2 Stage Regulator Cryoplunge3 Owner s Manual and User s Guide 19 Preparing the Ethane Cylinder and 2 Stage Regulator igure 1 6 Ethane tank and regulator NOTE The 2 stage regulator not provided must be suitable for use with ethane gas The second stage of the regulator should be able to deliver 40 psi 3 bar with an outlet fitting that accepts narrow bore Tygon tubing not provided Preparing the Ethane Cylinder and Regulator 1 Locate the ethane tank in an explosion proof spark proof fume hood 2 If you are unsure as to how to connect a 2 stage regulator to a compressed gas tank please seek assistance from your on site Safety Officer or laboratory advisor 3 The tank must be secured with a safety chain such that it cannot tip over dur ing use 4 The main tank valve should be closed as indicated on the top of the valve 5 Prior to attaching the 2 stage regulator to the tank make sure the m

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