1. Core Clinic -
Structured Illumination using the
Zeiss Apotome 2
February 20th, 2015
Caterina Di Ciano-Oliveira, Erin Pardy
Research Core Facilities

2. Introduction: Structured Illumination
What is structured illumination?
• a method of optical sectioning
• i.e. “out-of-focus” light is removed from your image
Principle
• A grid is inserted into the light path: this generates a strong
signal when specimen details are in focus and a weak signal
when specimen details are out of focus
• The grid is translated in 1/3 steps (perpendicular to grid axis)
• Each site on the specimen experiences a series of
illumination intensities, which corresponds to the translation
phase of the grid
• Algorithm analyzes changes in intensity at a given pixel
location in the 3 images and creates an image of the focal
plane, where pixel locations showing the greatest rates of
change have the highest amplitude
• In-focus information is retained in the optical section,
resulting in a “confocal-like” image.
a) Example of illumination mask

3. Introduction: Structured Illumination
What is it good for?
• moderately bright specimens
• med to low mag
• e.g. tissue sections, Drosophila embryo,
nematodes, zebrafish embryo…
Advantages
• laser free
• affordable
• faster then LSCM
Disadvantages
• requires minimum of 3 images for each z-slice
• algorithm cause dynamic range to decrease by half
• slower then spinning disc
a) Example of illumination mask

4. Before you Start Acquisition: Calibrations
There are two calibrations that need to be done before:
1) Transmitted light phase calibration: (Caterina)
2) Focus Calibration: (User)
• must be done every time you launch Zen
• should be done for each wavelength used and for each objective
• e.g. for GFP, Rhod and Cy3 with a 20X and a 40X would be 6 total
calibrations.
Requirements for focus calibration:
• a uniformly fluorescent test sample
• e.g. autofluorescent tissue

5. Before you Start Acquisition: Calibrations
There are two calibrations that need to be done before:
1) Transmitted light phase calibration: (Caterina)
2) Focus Calibration: (User)
• must be done every time you launch Zen
• should be done for each wavelength used and for each objective
• e.g. for GFP, Rhod and Cy3 with a 20X and a 40X would be 6 total
calibrations.
Requirements for focus calibration:
• a uniformly fluorescent test sample
• e.g. autofluorescent tissue

6. Focus Calibration Workflow
1) Turn on system and launch Zen.
2) Make sure Apotome is pushed all the way in.
3) Put evenly distributed fluorescent calibration sample in stand.
4) Start with the first objective and wavelength you want to calibrate.
5) Look through eye piece and make sure your sample is in focus. (You
should see the grid.)
6) While in Locate tab, click ,“Acquisition” and then “Apotome Focus
Calibration Wizard”.
7) Follow the instructions
• 3 steps: Device Settings, Optical Focusing, Grid Focus Calibration
8) Repeat for each wavelength and for each objective.

7. Focus Calibration Work Flow
Notes:
• Be sure to select correct light source, filter cube, objective and
camera
- note: Other lamp = X-cite
• Use the recommended grid.
• You can adjust exposure time if needed.
• Often you must use the TFT to redirect light to camera, put in
correct filter cube and turn on RL

8. Focus Calibration Work Flow
Notes:
• You will see the sample on the screen with the grid.
• Highlight an area of sample that is evenly distributed like as shown in
figure b)
• You can start “Full Scan” and then “Local Scan
• Click finish once done scanning
• Either keep going with more calibrations or click “No” to start acquiring

9. Acquisition
• Once you have finished calibrations,
go to Acquisition tab.
• Make sure “Apotome Enabled” mode
is checked.
• Make sure correct grid is selected
• Perform acquisitions
• View image
Conventional Sectioning Optical Sectioning