Getting Started – SIM Imaging & Image Processing

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.
   
   
   
   
8. Go to the Channels menu and select different Look-Up-Tables (LUTs) for the tracks. By selecting each track you can see which laser it is configured for and adjust the laser power by moving the slider. Also adjust the exposure time for each colour channel here.
   
   
   
   
9. Click on Live or Continuous to get a live preview of your specimen. Press Stop to stop the preview. Make sure to select only one track at a time and adjust the laser power and camera exposure time for each colour channel (if multiple tracks are selected the filter turret will constantly rotate). If the preview screen is dark, adjust the dynamic range of the camera by pressing min x max. If you still cannot see your sample check that all doors on the microscope enclosure are closed, since they are magnetically interlocked and will prevent the laser from turning on when open. Once you see an image re-focus if you have to and optimize the laser power and exposure time for each channel.
   
   
   
   
10. Acquire a 4 µm Z-stack of the beads. Tick the Z-stack tick box on the top left of the screen to activate the Z-stack menu, which will appear under the Multidimensional Acquisition header. Setup a stack using the Centre mode (only available if Show All in the Z-stack menu is ticked, highlighted in blue frame). Therefore focus on the centre of the beads, stop the live preview and press Centre. Turn on all laser lines in the Channels menu, then back in the Z-stack menu click smallest to optimize the step size of your stack. By changing the number of slices adjust the stack range to ~ 4 µm.
    
       
    
    
11. Make sure that all tracks you want to image are active, then press Start Experiment to acquire your bead calibration file.
    
    
    
    

12. Once you have acquired the beads you can start imaging your sample of interested using the instructions for Setting up a SIM experiment and chromatic shift correction in multicolour SIM experiments 1-9 (or 1-11 if you want to record a Z-stack of your sample). You can record 2D or 3D (z-stack) datasets by selecting Start Experiment, just make sure that the calibration beads were recorded using the same frame size and using all colour channels that you are looking at in your sample.