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-> neMESYS Pumps are now installed.

Use macro SCL_automation to prepare negatively stained samples for TEM.

Before macro execution, define 4 positions on stage (be sure to use the following nomenclature, otherwise names in the macro have to be changed) and fill conditioning unit with stain

  • Washing - empty well for washing (air plug will be aspirated here, and washing steps are executed here after the experiment)
  • Sample1 - fill this well with your sample
  • Plasma1 - define a position for convenient plasma activation of grid
  • Grid1 - place grids here, align nozzle in xyz - this will be the start position for pattern writing
  • Blotting - excess sample or system liquid is blotted away to ensure reproducibility

 

Macro procedure:

  1. joystick is disabled
  2. 2 uL of system liquid are pumped thorugh the nozzle in washing position
  3. stage moves to blotting position and blotts away excess system liquid
  4. stage moves to washing position and 0.5 uL air is aspirated
  5. stage moves to sample position
  6. 0.3 uL sample is aspirated
  7. stage moves to plasma activation position
  8. sample is withdrawn into conditioning unit
  9. sample is slowly pushed through conditioning unit
  10. time for plasma activation of grid (not automated yet)
  11. after sample has passed conditioning unit, it is quickly pushed towards the nozzle tip
  12. small droplet of sample penetrating the nozzle tip is blotted away at blotting position
  13. stage moves to writing position (Grid1)
  14. pump starts infusion
  15. pattern writing is delayed for a few seconds for the sample to reach the tip
  16. sample is written on grid
  17. after writing the stage moves back to the washing position and the pump is stopped
  18. joystick is re-enabled and macro ends

 

Depending on the nozzle (inner diameter, length, taper) and the conditioning unit (length) the macro protocol has to be optimized for every newly inserted nozzle or conditioning unit, respectively. Below are the critical parameters that need to be considered:

  • Withdraw sample into conditioning unit (find the right volume to place sample plug at the rear end of conditioning unit)
  • Dialysis length: (optimize the time the sample is moving through the dialysis fiber for optimal staining conditions)
  • Move sample to nozzle tip (define the volume necessary to place the sample almost at the nozzle tip after it has passed the conditioning unit)
  • WaitTimeBeforeStart (set this time in the SCL User Interface - time befor pattern writing is started, i.e. time sample needs to pop out of the nozzle)
  • .!wait::TIME in ms (defines how long the stage will stay at position Grid1 once it has reached that position, afterwards moves to Washing position - time it in such a way that stage moves away as soon as pattern writing is completed)
  • Rule of thumb: Writing time (6 s) + WaitTimeBeforeStart (5-7 s) = .!wait::TIME 

 

Macro language:

  • define pump parameters:

    !tellmodule::SCL.nemesysPump
    .SetPumpParameters::macro::1::0.3::0

    macro is used for case selector in LabVIEW. Then follows ::FlowRate::TargetVolume::Infuse(0) or Withdraw(1)
    e.g. above 0.3 units will be infused at a flow rate of 1 unit/time unit

    Units are taken from active selection in user interface!
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