Difference between revisions of "20210325 3Dcell labnotes"

Latest revision as of 17:42, 25 March 2021

Plan for the day

Equipment

• 0.5 ml Eppendorf tubes
• tube rack for holding Eppendorfs upright
• pipette
• distilled water (or growth medium)
• low gelling temperature agarose
• small beaker
• heater
• precision balance
• microscope at 37C

Gel procedure

• start microscope heater to 37C
• put circuit and pipettes inside microscope box to heat to 37 C
• heat water bath in small beaker to 60C on hot plate
• for 0.5 ml 1 wt % agarose gel
• 0.5 g = 500 mg water = 500 ul
• weigh 5 mg agarose
• put eppendorf in water bath and mix with pipette while heating

Gel into microfluidic procedure

• Start PC and camera on microscope, choose magnification and test imaging of circuit
• connect 2 ml syringe to output of gel channel
• prepare connection of ul micropipette to input of channel
• withdraw tube/syringe tip from circuit inlet
• Fill ul pipette with agarose gel solution
• reconnect ul pipette to gel channel input
• On microscope with camera recording
  • inject gel
  • adjust counter pressure with outlet syringe to get lamella filled
  • when filled, aspire from inlet syringe and press with outlet syringe to get an open channel between lamellae

Execution

• weighed 6 mg = 0.006 g + 0.6 g water = .6 ml = 600 ul water pipetted into 1.5 ml Eppendorf
• put eppendorf in water bath on hot plate (alu paper as lid and support for eppendorf)
• cut sharp tip off an 18 gauge syringe tip
• blunted (320 gauge paper) the tip
• curved the tip to get access under condenser of microscope
• 1st try: 1.25 ml Finntip
  • the circuit filled very quickly and everything was filled and fluid coming out through all openings
  • Need more control of volume and pressure
• 2nd try: 5 ul micropipette
  • Filled quickly, but slower than with Finntip
  • hard to control by hand

Analysis

• FOV of images is 3 x 2.4 mm, see image scale.png

Images 0.11 s apart (9 fps) while filling with gel with 1.25 ml Finntip

Images 0.11 s apart (9 fps) while filling with gel with 5 ul microsyringe

Clearly the pressure was too high so the fluid escaped imto the neighbouring channel. Is the front in the main channel gelling? This may create an instability if the first fluid that enters gets a higher viscosity and then the later fluid gets into a warmed up circuit and flow faster...

Ideas for next test:

1. pressure control
   1. forward pressure
   2. suction
      1. pressure controller

I have now connected and prepared the OB1 with 2 channels
Back and forth flow?

1. 1. 1. height of connected fluid bath
2. syringe pump with microsyringe

Problems

• gelling of front more than rear fluid can create instability