Live Cell Imaging-Basic Start-up
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| Live Cell Imaging-Basic Start-up
If you have booked the confocal for live cell imaging, please indicate so on the calendar 1. To heat stage, ensure all perspex sliding doors are closed. turn on the blower on the shelf above the confocal. underneath the confocal, switch on the right-hand controller (set @ 37°C).
This should be done 30mins prior to imaging! (it is advisable to heat up the universal stage at the same time-if confocal is in use, leave the required stage at bottom of perspex incubator) 2. Humidification there is a blue glass bottle lid inside the perspex incubator, this should be filled with water (tap water will do) and placed carefully within the incubator
push in the DIC rings above condensor (or they'll get damaged!) push head back-gently! ensure lenses are in 'empty' position remove stage by pushing in bottom left corner (red dot) and lifting the opposite corner insert new stage by pushing in 'red' corner and lowering in opposite corner
gently lower head back into position close the sliding doors-ensuring transmitter flex passes through the silicon rubber exit (you will need to guide it) move DIC rings out of light path If phase/DIC is required you may need to redo Kohler alignment
The microscope must be returned to the normal imaging setup as soon as you have finished imaging (if someone is booked immediately after you, allow time within your booking to do this) 1. Replace slide stage This is done as described above, please ensure: You take care when moving the head back (see above-2) The stage is clean, and any oil is cleaned off with tissue You return the stage to the cupboard DIC rings are left out of light path
as the blower above the confocal 3. Kohler Alignment Kohler alignment must be re-done after live cell imaging as it is affected by the movement of the head; leave in empty objective position. 4. Clean up Empty water out of blue lid and put all lens tissue and blue roll etc in bin provided If you are unsure of any part please contact Rachel or Liz immediately (Rachel: 2804, Liz: 2063) Live Cell Imaging-C02 Required You will require the heated block stage (found at righthand side of perspex incubator) and its lid (found in pink bubble-wrap bag in cupboard-has glass components so be careful with it). This is connected to the heater control, so will automatically warm up with incubator. Connecting the C02 Ensure cylinder is connected and C02 water bottle has enough distilled water to cover the block Feed the rubber tube from the C02 water bottle completely into the rear of perspex incubator-this allows the air to be heated in the incubator Insert the C02 slide lid into incubator through front panels and attach to C02 tube Start C02 Feed Switch on the C02 controller underneath microscope table (lefthand side) at the back (air will bubble through the water in the bottle). Leave for 10 minutes Unscrew the valve on the C02 tubing. Place cells into stage and close the lid Press 'valve' on C02 controller and wait for red light to turn green and display to change from 0.0-0.1 to 5 Happy cells and happy imaging! Shutting down Press 'valve' on C02 controller and wait for green light to turn red. Turn off CO2 controller at rear Disconnect lid from C02 feed tube and replace in bubble wrap in the cupboard Remove C02 feed tube from inside perspex and leave neatly at the side. Replace in corner of incubator as found. Follow normal live-cell shut down protocol, ensuring microscope alignment ok, ready for less adept users. Thank you! If you are unsure of any part please contact Rachel or Liz immediately (Rachel: 2804, Liz: 2063) |