====== Vitrobot procedure ======

The VitrobotTM (Vitrification Robot) is a fully PC-controlled device for vitrification (= rapid cooling) of aqueous samples

{{ :capture_vitrobot-description.jpg?nolink |}}

==== Before starting ====

  * fill a dry 5 L tank with liquid nitrogen
  * label grid boxes with the name of the experiment
  * check plasma cleaner is reserved
  * Assemble the necessary tools:
       * EM_grids grids are available and present in sufficient number
       * all part of the coolant container (make sure that they are dry)
       * both vitrobot tweezers, big tweezer and blot paper clipping

==== Starting up the vitrobot ====

  * 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
{{ :swith-on-vitrobot.jpg?nolink |}}
</div>++
       * ++ The Vitrobot User Interface page will appear after a few seconds.|<div>
              * Console page is dedicated to set humidity and temperature parameters
              * options page is dedicated to set blotting process parameters 
</div>++
                  {{ :capture2-userinterface_vitrobot.jpg?nolink |}} 


  * 3. Make sure the humidifier is operational
       * ++ check humificator beaker state in humidity panel|<div>
{{ :humidify-beaber-state2.jpg?nolink |}}
</div>++
       * ++ if EMPTY refill it with distilled water |<div>
**Use ONLY distilled water **
     * a. Fill a syringe with 60 ml distilled water
     * b. inject the whole volume through the plastic tube at the bottom part of the humidifier
     * c. When removing the syringe from the tube, be careful not to tear it from its location

{{ :capture_fill_humidif.jpg?nolink |}}
</div>++

  * ++4. hermetically close the chamber|<div>
         * do the following dry Run:
             * Place new grid / continue / place ethane container
</div>++
        * 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 ====

This step is done on the control panel, in the "console" page 

  * 1. adjust desired temperature value
      * ++: to any value between 4 and 60° |<div>
        * the actual temperature value is displayed in red
        * set to any desired value  with + and buttons in temperature panel
                      {{ :capture_adjust-temp.jpg?nolink |}}
</div>++
  * 2. adjust desired humidify value
      * ++ 96-100 % generally |<div>
        * the current humidity is displayed in blue
        * set to desired value  with + and - buttons in humidify panel
                      {{ :capture2_adjust-hum.jpg?nolink |}}
</div>++
    * ++3. wait until desired temperature is reached then enable the humidity switchbox to start |<div>
            * a. read current temperature (red value) in panel temperature
            * b. press ON/OFF switch in panel humidity
{{ :swithon-humidif.jpg?nolink |}}
</div>++
           * 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 ====


  * ++ 1. switch on plasma cleaner if not started|<div>
         * If stopped, press the hard lock switch on the backside of the machine,
         * Press CLEAR to initialyze the vaccum system
          {{ :switching_on_fish.jpg?nolink |}}
</div>++
         * fishione started with chamber locked under vaccum
         * READY / HIGH VACCUM messages
           {{ :fishione_on-small2.jpg?nolink |}}

  * 2. press Vent Lock to bring the chamber under atmospheric pressure
       * State is reached when message Lock At Atmosphere appears 
          
From now on IMPERATIVELY ** wears gloves**

  * ++ 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
       * b. pull the rod upwards to lift the cover  
       * c. move the cover on the side around its axis
       * d. if necessary put back the seal in place.
             * It can either stick on the cover or come out of its channel
</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.
  * Be sure to keep the table horizontal so as not to slide the drawer
</div>++
            * the metallic drawer is **ALWAYS stored in the chamber**
  * 5. close the door to prevent dust contamination inside the chamber
  * 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
      * place EM_grids inside
      * generally sets of 4 to 8 **IDENTICAL** grids are loaded simultaneously  
      * close the drawer by putting back in place the cover
      * put back in place the closed drawer on the support table
</div>++
          * generally sets of 4 to 8 **IDENTICAL** grids are loaded simultaneously
   * 7. ++ 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>++
   * 8. put back in place the metallic drawer inside the chamber
            * on the plateform ** NOT on the inlet pump** 
                   {{ :support_grid-inchamber8cm.jpg?nolink |}}
   * 9. ++ 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
        *  State reached when message "HIGH VACCUM" appears
   * 11. if not yet done on vitrobot, enable the humidity switchbox to start  

==== Assemble different part of the coolant container as pictured below ====
{{ :coolant-container-built.jpg?nolink |}}

    * ++ legende |<div>
    * A : central ethane cup
    * B : grid box plateform
    * C : spindle
    * D : Ice contamination protective sleeve (optional) 
    * E : outer nitrogen container
</div>++
 
      

==== Cooling down the coolant container ====

  * 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
  * 3. up to now continously fill the outer part to maintain the Nitrogen level
  * 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>
             * Ethan is liquefied by flowing a stream of gas through plastic tubing connected to an ethane flask into the central cup 
</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
</div>++
  * ++ 7. then remove the spindle (C)|<div> 
            * 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 with a big tweezer
</div>++
  * 8. place grid box into the grid box platform (B)
          * make sure that the grid box is labelled with the experiment name



==== plasma clean grids ====


  * 1. Grids are already loaded inside the plasma cleaner
        * ++ if not yet done|<div>
see part
==== load grids inside the plasma cleaner ====
</div>++

  * 2. set desired plasma cleaning parameters :gas mixture/gas flow power
    * ++ commonly settings 80-20-0 are used |<div>
    * 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 |}}
    * Gas Mix and plasma TABS show preset values
</div>++       
  * ++ 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
  * 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



======Everything is ready for plunge freezing sample:========
    climate chamber is equilibrated in temperature and humidity.
    Filter paper are mounted 
    ethane is liquefied
    Grids are freshly plasma cleaned


==== Start freezing at the vitrobot ====


       * 1. check that the correct freezing parameters are entered 
       * 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>++
       * 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