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Setting up a SIM experiment and recording beads for chromatic shift correction in multicolour SIM experiments

 

  1. For SIM imaging make sure to select the 63x oil objective. You can also use the 100x oil or the 63x water lens, but the 63x oil lens should be used preferentially.

  2. The first thing you should do every day when doing multicolour SIM imaging is to record a TetraSpeck (100 or 200 nm) bead stack to allow you to correct for chromatic shifts between the different colour channels. This is not required when you are doing one-colour imaging. Make sure to acquire your bead and sample images using the same objective lens and zoom settings.

  3. Therefore place the Channel Alignment bead sample (top drawer in the desk) on the microscope stage and focus on the beads as described in the section GETTING STARTED – PUTTING A SPECIMEN ON AND LOCATING IT USING THE OCULAR.

  4. Go to the Acquisition tab and in there expand Laser menu and check the lasers you want to use are turned on (available laser lines: 405, 488, 561 and 642 nm).

  5. Then expand the Light Path menu below the Laser menu.

  6. At the top you have the option to select between WF (widefield), SIM (Structured Illumination Microscopy) and Laser WF (laser widefield). Select SIM. In this acquisition mode TV2 (sCMOS camera) will be automatically selected as default camera and the 1.6 x Optavar lens and SIM lens and gratings (highlighted in red) will be inserted in the light path. You can still configure the light path manually as well, but here the recommended settings are shown.



  7. Configure tracks with the laser lines for excitation (click on the laser icon and select) and matching emission filters (see images below). The matching grating for each wavelength will be inserted in the light path automatically as soon as you select a laser (e.g. 28.0 µm at 488 nm; manually adjust the grating for fast imaging or when imaging thick specimen use larger grid at expense of resolution). New tracks can be added by pressing the +-button and removed by pressing the trash-button next to it.

 

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