Quick Guide: how to acquire single-particle tracking (SPT) imaging

Quick Guide: how to acquire single-particle tracking (SPT) imaging

1.     Start the Nanoimager and let the temperature stabilize to avoid drifts during acquisition. The stable microscope temperature is 6-7 ºC higher than room temperature (RT). To reach this stage quickly enter the desired (RT+ 6 ºC) in the temperature control panel and enable control. It will take roughly 1 to 2h to reach the desired temperature. 

2.     Channel Mapping: Focus on bead slide and run channel mapping. More details here.

3.     Focus on your sample at low laser powers. For particles in solution, you may have to go 2-3 um above the coverslip to avoid the ones attaching to the surface.

4.     Set a good Optical control and TIRF angle - changing Illumination modes (TIRF or HILO or epifluorescence). Usually go from 40 degrees (HILO) up to ~53 degrees (TIRF).

5.     Z lock - Set focus reference and press Zlock.

6.     Use low laser power for the acquisition. Start as low a possible and do small increases until the signal-to-noise ratio is decent without having photobleaching of the fluorophore.

7.    Acquisition settings: Enter exposure and number of frames. This will depend on the dynamic process to be followed. For particles moving fast, use 10-20 ms and higher power. For particles moving slowly, something between 30-50 ms and 10000 frames per channel is a good starting point.

      8.     Enter Folder name and filename.

9.     Click Acquire.

10.  To run SPT analysis please refer to this page.



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