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Ensure the microscope has been switched on and initialised, and Zen Black software has been opened for at least 3 hours prior to imaging. Switch on the lasers you require at least 30 minutes before imaging.
- It is advisable to use the 100x TIRF lens to do PALM/STORM although this is not strictly necessary. Imaging in TIRF reduces the out of focus light from the background and makes PALM reconstruction easier, however TIRF only illuminates objects very close to the coverslip.
- Set up your slide / plate as you would normally. Refer to Getting Started – Putting A Specimen On And Locating It Using The Ocular for more detailed information.
- When ready to image, select the laser WF option in the light path menu. Select 'TV1', which is the Andor EMCCD for PALM/STORM imaging. It is advisable to have the 1.6 optovar selected too.
- Set up a track for your most easily located channel and select EPI. Focus on your sample in live/continuous preview and locate the field you will want to image in TIRF.
- Then change to TIRF and move the tirf slider gradually until TIRF is achieved. This is usually marked by a sudden significant decrease in background fluorescence.
- Set up a time series for ~10000-30000 images with as short as possible time interval.
- Start your experiment.
- Go to the processing tab and process your PALM / STORM acquisition with the tools available there. The SuRF facilty has a computer in Room 2.19 suitable for offline processing of these acquisitions
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