Quick Guide: How to acquire dSTORM acquisition

Quick Guide: How to acquire dSTORM acquisition

dSTORM image acquisition

  1. Start the Nanoimager and let the temperature stabilize to avoid drifts during acquisition. The stable microscope temperature is 6-7 C higher than room temperature(RT). To reach this stage quickly enter the desired (RT+ 6 C) in the temperature control panel and enable control. It will take roughly 1 to 2h to reach the desired temperature. Once the temperature has reached a desired value, you can disable the temperature control. 
  2. Channel Mapping: Focus on bead slide and run channel mapping. More details here
  3. Add a dSTORM buffer to your sample and mount it on the microscope stage. ONI dSTORM buffers are stable for 1 hour after mixing components A and B. Make to mix only the required amount and freeze the remaining part. 

  4. The following suggestions are quick guide for a dSTORM acquisition. 

  5. Focus on your sample at low laser powers. Also see the NimOS Overview for laser and acquisition controls.

  6. Set a good TIRF angle. Usually between 50-56 degree.

  7. Z lock - Set focus reference and Zlock

  8. Acquisition settings: Enter exposure and number of frames. 30ms and 10000 frames per channel is a good starting point.

  9. Enter data and acquisition tag. 

  10. Increase laser power to see blinking of dyes before clicking on the acquire tab. This step will require going to higher laser power (50- 100%) depending on the dye.

  11. If using a light program to switch lasers automatically, first click on enable light program.  

  12. Click Acquire


Channel mapping: do the channel mapping once the temperature has reached the desired value in the previous step. This is only required when you are acquiring data in two or more colors. 
For more tips and tricks on dSTORM imaging, visit our blog article 'Dos and don'ts for mastering dSTORM imaging'

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