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vitrobot [2023/01/12 12:41] – crucifixvitrobot [2023/11/01 20:18] (current) – external edit 127.0.0.1
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 ==== Before starting ==== ==== Before starting ====
  
-  * fill a dried 5 L tank with liquid nitrogen +  * fill a dry 5 L tank with liquid nitrogen 
-  * label the grid boxes +  * label grid boxes with the name of the experiment 
-  * check a number of points: +  * check plasma cleaner is reserved 
-       * plasma cleaner is reserved+  * Assemble the necessary tools:
        * EM_grids grids are available and present in sufficient number        * EM_grids grids are available and present in sufficient number
-       * all part of the coolant container are dry +       * all part of the coolant container (make sure that they are dry) 
-       * close climate chamber's door if open +       * both vitrobot tweezers, big tweezer and blot paper clipping
-       * Paper filter clipping ring are not missing+
  
 ==== Starting up the vitrobot ==== ==== Starting up the vitrobot ====
  
-  * ++1. Switching on vitrobot|<div>+  * 1. close climate chamber's door if open 
 +  * ++2. Switching on vitrobot|<div>
   * Press the hard lock switch on the right backside of the machine   * Press the hard lock switch on the right backside of the machine
 {{ :swith-on-vitrobot.jpg?nolink |}} {{ :swith-on-vitrobot.jpg?nolink |}}
 </div>++ </div>++
-       * The Vitrobot User Interface page will appear after a few seconds.+       * ++ The Vitrobot User Interface page will appear after a few seconds.|<div>
               * Console page is dedicated to set humidity and temperature parameters               * Console page is dedicated to set humidity and temperature parameters
               * options page is dedicated to set blotting process parameters                * options page is dedicated to set blotting process parameters 
 +</div>++
                   {{ :capture2-userinterface_vitrobot.jpg?nolink |}}                    {{ :capture2-userinterface_vitrobot.jpg?nolink |}} 
  
  
-  * 2. Make sure the humidifier is operational+  * 3. Make sure the humidifier is operational
        * ++ check humificator beaker state in humidity panel|<div>        * ++ check humificator beaker state in humidity panel|<div>
 {{ :humidify-beaber-state2.jpg?nolink |}} {{ :humidify-beaber-state2.jpg?nolink |}}
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        * ++ if EMPTY refill it with distilled water |<div>        * ++ if EMPTY refill it with distilled water |<div>
 **Use ONLY distilled water ** **Use ONLY distilled water **
-     * 1. Fill a syringe with 60 ml distilled water +     * a. Fill a syringe with 60 ml distilled water 
-     * 2. inject the whole volume through the plastic tube at the bottom part of the humidifier +     * b. inject the whole volume through the plastic tube at the bottom part of the humidifier 
-     * 3. When removing the syringe from the tube, be careful not to tear it from its location+     * c. When removing the syringe from the tube, be careful not to tear it from its location
  
 {{ :capture_fill_humidif.jpg?nolink |}} {{ :capture_fill_humidif.jpg?nolink |}}
 </div>++ </div>++
  
-  * ++3. hermetically close the chamber|<div>+  * ++4. hermetically close the chamber|<div>
          * do the following dry Run:          * do the following dry Run:
              * Place new grid / continue / place ethane container              * Place new grid / continue / place ethane container
 </div>++ </div>++
         * the white latch remains in open position after swith on the power.           * the white latch remains in open position after swith on the power.  
 +
 +
 +
 +==== Set blotting process parameters ====
 +
 +This step is done on the control panel, in the "options" page 
 +
 +  * 1. 'blot total' needs to be '1' for blotting to occur
 +  * 2. check 'Humidifier off during process' and 'Skip grid transfer' in the miscellaneous' side bar
 +  * 3. Optional : set your desired blot time, force blot and waiting time (o for unsupported condition )
 +           * all these setting are checked at the begining of the freezing cycle  
                                                                          
 ==== Set humidity and temperature parameters in climate chamber ==== ==== Set humidity and temperature parameters in climate chamber ====
  
-                  This is done on the control panel, in the options page +This step is done on the control panel, in the "console" page 
  
   * 1. adjust desired temperature value   * 1. adjust desired temperature value
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                       {{ :capture_adjust-temp.jpg?nolink |}}                       {{ :capture_adjust-temp.jpg?nolink |}}
 </div>++ </div>++
-  * 3. adjust desired humidify value+  * 2. adjust desired humidify value
       * ++ 96-100 % generally |<div>       * ++ 96-100 % generally |<div>
         * the current humidity is displayed in blue         * the current humidity is displayed in blue
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                       {{ :capture2_adjust-hum.jpg?nolink |}}                       {{ :capture2_adjust-hum.jpg?nolink |}}
 </div>++ </div>++
-    * ++4. wait until desired temperature is reached to enable the humidity switchbox to start  |<div> +    * ++3. wait until desired temperature is reached then enable the humidity switchbox to start |<div> 
-press ON/OFF switch+            * a. read current temperature (red value) in panel temperature 
 +            * b. press ON/OFF switch in panel humidity
 {{ :swithon-humidif.jpg?nolink |}} {{ :swithon-humidif.jpg?nolink |}}
 </div>++ </div>++
-           * take advantages from this waiting time to start next step :  +           * take advantages from this waiting time to do the two following steps  
-   +                       * Mounting the filter papers 
 +                       * load grids inside the plasma cleaner 
 + 
 +==== Mounting the filter papers ==== 
 + 
 +  * IMPORTANT **always wears gloves** 
 +  * place blot papers onto the blotting pads with clipping ring 
 +  * support the blotting arms during assembling to prevent damage to the arms 
 + 
 + 
 ==== load grids inside the plasma cleaner ==== ==== load grids inside the plasma cleaner ====
  
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   * ++ 3. Open chamber and make sure the seal remains in place |<div>   * ++ 3. Open chamber and make sure the seal remains in place |<div>
 +{{ :open_chamber-stepbystep3.jpg?nolink |}}
        * a. unlock the rod by pushing the button on it        * a. unlock the rod by pushing the button on it
        * b. pull the rod upwards to lift the cover          * b. pull the rod upwards to lift the cover  
-       * b. move the cover on the side around its axis +       * c. move the cover on the side around its axis 
-       * c. if necessary put back the seal in place.+       * d. if necessary put back the seal in place.
              * It can either stick on the cover or come out of its channel              * It can either stick on the cover or come out of its channel
 </div>++ </div>++
-                         {{ :open_chamber-stepbystep.jpg?nolink |}}+
   * 4. ++ retrieve the metallic drawer and place it on the table |<div>   * 4. ++ retrieve the metallic drawer and place it on the table |<div>
   * the support carrying the drawer can be taken out by lifting it by the rod.   * the support carrying the drawer can be taken out by lifting it by the rod.
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             * the metallic drawer is **ALWAYS stored in the chamber**             * the metallic drawer is **ALWAYS stored in the chamber**
   * 5. close the door to prevent dust contamination inside the chamber   * 5. close the door to prevent dust contamination inside the chamber
-  * 6. ++ place EM_grids inside the metallic drawer |<div>+  * 6. place EM_grids inside the metallic drawer  
 +          * ++ EM_grids with support film facing up|<div>
       * remove the cover of the metallic drawer by sliding       * remove the cover of the metallic drawer by sliding
       * place EM_grids inside       * place EM_grids inside
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    * 11. if not yet done on vitrobot, enable the humidity switchbox to start      * 11. if not yet done on vitrobot, enable the humidity switchbox to start  
  
-==== Assemble different part of the coolant container like showing below ====+==== Assemble different part of the coolant container as pictured below ====
 {{ :coolant-container-built.jpg?nolink |}} {{ :coolant-container-built.jpg?nolink |}}
  
     * ++ legende |<div>     * ++ legende |<div>
     * A : central ethane cup     * A : central ethane cup
-    * B : grid storage device+    * B : grid box plateform
     * C : spindle     * C : spindle
     * D : Ice contamination protective sleeve (optional)      * D : Ice contamination protective sleeve (optional) 
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 ==== Cooling down the coolant container ==== ==== Cooling down the coolant container ====
  
-  * 1. Pour LN2 into both central ethan cup and outer part to faster the cooling+  * 1. Pour LN2 into both central ethan cup (A) and outer part (E) to faster the cooling
   * 2. Wait for complete evaporation of the remaining nitrogen in the central part   * 2. Wait for complete evaporation of the remaining nitrogen in the central part
   * 3. up to now continously fill the outer part to maintain the Nitrogen level   * 3. up to now continously fill the outer part to maintain the Nitrogen level
   * 4. wait 10 to 15 min for system to equilibrate   * 4. wait 10 to 15 min for system to equilibrate
   * ++ 5. then liquefy the ethan in the central cup already cooled to liquid nitrogen temperature |<div>   * ++ 5. then liquefy the ethan in the central cup already cooled to liquid nitrogen temperature |<div>
-Ethan is liquefied by flowing a stream of gas through plastic tubing connected to an ethane flask into     the central cup +             * Ethan is liquefied by flowing a stream of gas through plastic tubing connected to an ethane flask into the central cup 
 </div>++ </div>++
   * ++ 6. let ethane start to solidify for few seconds|<div>   * ++ 6. let ethane start to solidify for few seconds|<div>
-apparence of a white solid rim around the wall container indicates that ethane starts to solidify+             * apparence of a white solid rim around the wall container indicates that ethane starts to solidify
 </div>++ </div>++
-  * ++ 6. then remove the spindle|<div>  +  * ++ 7. then remove the spindle (C)|<div>  
-solidified ethan makes stick together the spindle and the ethan cup. +            * Solidified ethan makes stick together the spindle and the ethan cup. 
-Thawing the frozen ethan by placing the second ethan cup on the spindle for 10sec is the more careful way to remove it +            * Thawing the frozen ethan by placing the second ethan cup on the spindle for 10sec is the more careful way to remove it with a big tweezer
 </div>++ </div>++
 +  * 8. place grid box into the grid box platform (B)
 +          * make sure that the grid box is labelled with the experiment name
  
-==== Mounting the filter papers ==== 
  
-  * IMPORTANT **always wears gloves** 
-  * attach the blotting papers to the the blot pad by using the with clipping ring 
- 
-A way to keep the **soaking time reproducible** is do this operation always the same time before freezing 
- 
-==== Set blotting process parameters ==== 
- 
- 
-==== Set freezing condition ==== 
- 
- 
-       *  
-       * 1. press place new grid 
-       * 2. Attach Vitrobot tweezers with a grid onto the plunge rod 
- 
-* Now vitrobot is ready for freezingbeforeDo it when you are almost ready to freeze to keep the soaking time as reproducible as possible 
- 
-       ====== At this point already is ready for freezing ====== 
- 
-    At this point already is ready for freezing: 
-    climate chamber is equilibrated in temperature and humidity. 
-    Filter paper are mounted and soaking time is controlled 
-    ethane is liquefied 
-    Grids are loaded and ready to be plasma cleaned 
  
 ==== plasma clean grids ==== ==== plasma clean grids ====
  
  
-  * Grids are already loaded inside the plasma cleaner+  * 1. Grids are already loaded inside the plasma cleaner
         * ++ if not yet done|<div>         * ++ if not yet done|<div>
 see part see part
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 </div>++ </div>++
  
-  * set desired plasma cleaning parameters :gas mixture/gas flow power+  * 2. set desired plasma cleaning parameters :gas mixture/gas flow power
     * ++ commonly settings 80-20-0 are used |<div>     * ++ commonly settings 80-20-0 are used |<div>
-    * Gas mix: 80/20 oxy/Ar, power 34% +    * The setup TABS store preset parameters 
-* The setup TABS store preset parameters+    * settings 80-20-0 corresponds to Gas mix Oxy/Ar : 80/20, power 34% 
 +    * Gas Mix and plasma TABS show preset values 
 {{ :capture2-setups-tab.jpg?nolink |}} {{ :capture2-setups-tab.jpg?nolink |}}
-* Gas Mix and plasma TABS show preset values+    * Gas Mix and plasma TABS show preset values
 </div>++        </div>++       
-  * adjust the plasma cleaning time with up/down arrows head and press set+  * ++ 3. set the plasma cleaning time |<div> 
 +        * a. use up/down arrows head selectors 
 +        * b. press set buttom to load the time in the time display 
 +</div>++
         * commonly 90 s are used         * commonly 90 s are used
-  * press START to apply plasma on grids +  * 4. press START to apply plasma on grids 
-       *  +       * The gas plasma generated can be seen through the window as a pink light 
 +       * remaining time can be followed in the time display 
 +  * 5. press Vent Lock to bring the chamber under atmospheric pressure 
 +       * State is reached when message Lock At Atmosphere appears 
 +  * 6. Open the chamber 
 +  * ++ 7. Retrieve the drawer and place it on the table |<div> 
 +       * the support carrying the drawer can be taken out by lifting it by the rod. 
 +       * Be sure to keep the table horizontal so as not to slide the drawer 
 +</div>++ 
 +  * 8. close the door to prevent dust contamination inside the chamber 
 +  * 9. ++ place the plasma cleaned grids on a glass slide  |<div> 
 +      * remove the cover of the metallic drawer by sliding 
 +      * place EM_grids on the edge of the glass 
 +</div>++ 
 +  * 10. put the glass slide in a closed crystallizer to transfer safely grids to vitrobot bench  
 +  * 11. load or not new grids in the drawer and close it  
 +  * 12. put back in place the closed drawer on the support table 
 +  * 13. ++ open the door and make sure the seal remains in place|<div> 
 +      * a. if necessary put the seal back in place 
 +         * It can either stick to the cover or come out of its channel 
 +      * b.clean it just by passing your finger 
 +</div>++ 
 +   * 14. put back in place the metallic drawer inside the chamber 
 +            * on the plateform ** NOT on the inlet pump**  
 +                   {{ :support_grid-inchamber8cm.jpg?nolink |}} 
 +   * 15. ++ close the door |<div> 
 +       * a. be sure the seal is in place and clean (see part.7)  
 +       * b. move the lid around its axis to bring it over the opening of the chamber 
 +       * c. unlock the rod by pushing the button 
 +       * d. gently allow the lid to slide down   
 +</div>++  
 +   * 10. press VACCUM to bring the chamber under vaccum 
 +           * either grids are waiting ready for an additionnal run of freezing at vitrobot (step 4-to 15) 
 +           * or the fishione remains on for next user 
 +           * At the end of the day, the fishione can be switch off
  
-==== Preparation of the grid ==== 
  
  
- ++3. Bring the vitrobot to hermetically close the chamber|<div> +======Everything is ready for plunge freezing sample:======== 
-         * do the following dry Run: +    climate chamber is equilibrated in temperature and humidity. 
-  * A glow discharge grids is attached to the twezzers +    Filter paper are mounted  
-  +    ethane is liquefied 
-==== Start freezing ==== +    Grids are freshly plasma cleaned
- +
  
  
-   +==== Start freezing at the vitrobot ====
-  * ++ Do it when you are almost ready to freeze to keep the soaking time as reproducible as possible|<div> +
-Decrire +
-</div>+++
  
-==== Mounting the filter papers, IMPERATIVELY wear gloves ==== 
  
-  * ++ Do it when you are almost ready to freeze to keep the soaking time as reproducible as possible|<div> +       * 1. check that the correct freezing parameters are entered  
-Decrire+       * 2. click on "place new grid" to bring in loading position the plunge rod 
 +       * 3. Lock a Glow discharged grid on tweezers 
 +                 * Grasp as little of the grid as possible with the tweezers (but enough that it will not fall out during freezing) 
 +                 * ++ Make sure that the black clamping ring is fixed not more down to the first groves. |<div> 
 +{{ :rightclamp_twezzers.jpg?nolink |}}
 </div>++ </div>++
 +       * 4. then fix the tweezers onto the plunge rod
 +               * with the support film support on the right for right-handers and on the left for left-handers
 +       * 5. press on "continue" to bring up the tweezers into the blotting chamber
 +       * 6. place the container onto the ethane lift
 +               * Always check that ethane is at right temperature  
 +       * 7. press on to lift it
 +       * 8. click on "process"
 +       * 9. apply sample to grid
 +       * 10. click on "continue" to blot and plunge
 +       * 11. carrefully detach tweezers from the plunge rod
 +       * 12. By maintaining the grid inside the ethan cup, place the coolant container on the table 
 +       * 13. transfer grid to the grid position
 +       * 14. Dry tweezers
 +       * 15. then repeat sequence 1-14 for all glow discharged grids
 +
 +
 +
            
  
vitrobot.1673527293.txt.gz · Last modified: (external edit)