Acquire 3D dSTORM image

Acquire 3D dSTORM image

  1. Start the Nanoimager using the instrument data file for a set for 3D mapping. You will not require this file is you are using a single channel for 3D imaging. (Note: Engaging 3D lens to disrupt 2D mapping and a change in camera region is required to fix mapping for 3D dSTORM. If you do not have a 3D instrument data file, please contact support. )

  2. 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 if you just started the microscope, less if the microscope was already stable. Once the temperature has reached the desired value, you can disable the temperature control. 

  3. Run 3D calibration

  4. Add a dSTORM buffer to your sample and mount on the microscope stage. ONI dSTORM buffers are stable for 2 hours after mixing components A and B. Make to mix only the required amount and freeze the remaining part. 

  5. Focus on your sample at low laser powers.

  6. Set focus reference and z-lock.

  7. Engage 3D lens

  8. Enter exposure and number of frames. 30ms and 10000 frames per channel is a good starting point for 3D dSTORM

  9. Enter data and acquisition set tag. 

  10. Increase laser power to see the blinking of dyes before clicking on the acquire tab or alternatively use the light program. Blinking 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 the light program.  

  12. Click Acquire


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