Instructions for using the CDB Microscopy Core's LSM 710 can be found here.

Load the image acquisition settings from an image in OR138.

When you start an imaging session, it's easiest to open an image from a previous session (I like to use image from the first official experiment, Job036) and "Load" the image acquisition settings. This should copy all of the image acquisition settings from that image to the current session.

When you start imaging a well, start looking for organoids in the top-left corner of the well. Systematically the well in a snake pattern and image the first 10 organoids you come across.

This prevents bias in selecting the "nice" organoids to image and it makes it easier to avoid accidentally imaging the same organoid twice. The only reasons I don't image an organoid are because (1) the organoid is too high up in the gel for me to image it without the objective bumping into the sample, (2) the organoid is only 1 cell (this is very uncommon, but if it is common for a particular experiment I make sure to note it), or (3) if the organoid is within ~1 organoid diameter of other organoids or junk.

Once you've found an organoid you want to image, center the field of view on the organoid.

Then, bring the part of the organoid where it is widest into focus and crop the image to be just larger than the organoid. This can drastically save on image time.

Set the pixel size to be between 0.35 - 0.36 um.

Once the image is cropped, the ZEN software will automatically update the pixel sizes. We want the pixel sizes to be as consistent as possible. Unfortunately, it's not possible to keep the pixel sizes exactly the same across images of different sizes. That's OK, because we can account for different pixel sizes in the code later. But, it's still wise to keep the pixel sizes relatively consistent between images. To change the pixels sizes you'll need to change the number of pixels in the image until the pixel size reads between 0.35 and 0.36 um.

Set the pixel dwell to be between 1 and 2 us.

The ZEN software will also automatically adjust the pixel dwell. This, like pixel size, cannot be kept exactly the same between images of different sizes. To change the pixel dwell you'll need to change the speed until the pixel dwell reads between 1 and 2 us.

Next, you need to set the lower and upper limits of the z-stack. I set the lower limit to just after the nuclei have disappeared. I set the upper limit to just beyond the middle of the organoid (as judged by when the organoid is largest). Thankfully, the size between pixels in z can be kept constant at 1.5 um.

Acquire the image.

Save the image as pos001.lsm in a folder with the well number and condition name formatted like well01_5uMSU6668.