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Measure the delivered volume with a fluorescent tracer

The typical flows in FluidFM probes are far below the detection limit of even the best flow sensors (1000x). Possible experimental methods on how to measure the flow are described here.

Additionally, the injected or spotted volume can be estimated using fluorescent tracers. 

Requirements

  • A mix of your "ink" and fluorescent tracer. The "ink" can be any liquid you want to deliver.
  • A fluorescence camera.
  • A properly calibrated microscope objective.

Now you will find the total fluorescence intensity of a reference volume. You have two options (A & B):

A. Use the probe as reference volume

  1. Fill the clip reservoir with the liquid mix.
  2. Mount and calibrate the probe according to the Prepare FluidFM workflow.
  3. Take an image of the probe filled with the fluorescent marker with exposure time t.
  4. Integrate the total fluorescence F within a certain region within the probe. This can for example be done with ImageJ. 
  5. Get the reference volume: V= 1 um x Area of region 
  6. The fluorescence per volume (fv) is now: fv(t) = F/(V*t) 

B. Use a suspended drop as reference volume

  1. Suspend a drop of your mix in optical oil.
  2. Take an image of the drop with exposure time t.
  3. Integrate the total fluorescence of the drop. This can for example be done with ImageJ.
  4. Get the reference volume: V= 4/3 π r3 
  5. The fluorescence per volume (fv) is now: fv(t) = F/(V*t) 
  6. Fill the clip reservoir with the liquid mix.
  7. Mount and calibrate the cantilever according to the Prepare FluidFM workflow.

Now we can start spotting/injecting: 

  1. Take an image of the target area/cell to get the background fluorescence. Do not move stage anymore until step 3.
  2. Inject/spot on your target.
  3. Take an image of the delivered volume with exposure time td.
  4. Integrate the total fluorescence FD of the delivered volume. Subtract the background fluorescence for increased accuracy. This can for example be done with ImageJ.
  5. Calculate the dispensed volume VD as follows: VD = FD/(fv*td)

 

 

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