Mouse Imaging Centre OPT device:
Our system was custom built at MICe by Michael Wong and Jun Dazai. Design and implementation details can be found in their 2013 Plos ONE paper.
OPT Sample Preparation
- Please see preparation instructions here: OPT sample preparation
OPT Sample Imaging
- General imaging instructions are as follows:
- Please see above OPT Sample Preparation section for instructions on how to prepare your sample for imaging
- Once your sample is embedded in agar and has been sufficiently cleared in BABB (ie. agar and sample are transparent), it is ready for imaging
- Remove the agar-embedded sample from the vial and place it on a piece of clean gauze - pat the agar with gauze to remove excess BABB
- wrap the agar in the gauze and allow to sit for at least 30 minutes
- Take a clean metal chuck (wipe surface with methanol) and apply a small amount of gel super glue (blue container) - enough for a thin layer - over the top of the chuck. Allow to sit for 5 minutes so the glue becomes tacky.
- Place agar-embedded sample on to the top of the metal chuck - allow to set for at least an hour
- you can prepare mutliple samples/chucks at one time
- While you wait, turn on the following OPT components
- UV light source (this will take several minutes to warm up)
- microscope controller
- CCD camera
- white light source
- On OPT computer, double click the OPT Scanner 3.0 with shutter.vi icon to open the imaging software
- Open the door to the OPT system
- make sure that the glass cuvette is 80% filled with BABB. If not, it may be time to clean the cuvette and replace with fresh BABB
- On the imaging software interface, press the white arrow on the top right of the screen - you will notice that the stage will begin to move
- Set Exposure value at 25 and press the Update button
- Place the sample and chuck upside down onto the steel disk mount for the rotation stage - the metal chuck has a magnet in it and will remain secure
- Lower the sample into the BABB cuvette by adjusting the vertical position value on the OPT software (try 5 to start). Press enter.
- Once you are happy with the vertical position of your sample, adjust the zoom so that your sample takes up most of the viewing window (try 7000 to start). Press the GO box after making any changes.
- Centre your sample (using forceps - touch only the metal chuck) on the green visualization line in the middle of the viewing window.
- Change rotation value incrementally and adjust sample as needed to make sure it is centred at every angle
- Turn off white light source
- Close door to OPT system
- Change Exposure value to 500
- Adjust UV light source gain value to 100% (by pressing the 'UP' botton on the box)
- On the OPT software interface, make any necessary adjustments to the Focus (start at 18 - focus is changed automatically when this value is edited)
- Standard scanning parameters set already are:
- Step Size: 0.3
- Avg: 1
- # of views: 1200
- Enter the name of your sample in the File Name field
- When ready to scan:
- press the Acquire Dark Field button
- press the Update button
- wait for the machine to acquire 10 dark field images (the sample will be raised and then returned back to its original position)
- press Scan and then Update
- Each scan takes approximately 25 minutes
- Before your image can be reconstructed, the binary files produced by the OPT scan must be converted to tif files.