Community collective: essential optimisation

Posted by , on 29 June 2023

Microscopy images have the ability to wow us, and the data we can extract from them can help answer our research questions, as well as leading us to pose new ones. Advances in sample preparation and microscopy technology means we have never been in a better position to use imaging in biology but is it really as simple as doing one experiment and collecting the data from the microscope? Of course, the answer is (usually) no, and optimisation remains key to producing useful (and beautiful!) images.

Jessica Henty-Ridilla (aka @cytoskeletown) asked for advice on how to convey to new users that planning, optimising and experimentation at all stages of the experiment is essential to collecting the best data.

You can read the full Twitter thread here:

Some of the most liked suggestions include Jen-Yi Lee and Maiko Kitaoka’s guide to rigor and reproducibility in imaging published in MBoC, ‘A biologist’s guide to the field of quantitative bioimaging’ from Senft, Diaz-Rohrer, et al. and the MyScope training material from Microscopy Australia.

We asked Jessica for some more details about her request.

What prompted you to ask for advice?

I train a lot of different students to use advanced microscopes (particularly TIRF and confocal systems). As time goes on and with each new trainee, I am starting to see several common misconceptions: 1) imaging is easy; 2) imaging (the act of taking one beautiful image) somehow solves the problem; 3) all microscopes look the same therefore they are. Someone that has done a lot of microscopy knows each of these misconceptions to be true. I figured someone had already solved this problem and I just wasn’t clued into the answer. It was extremely validating to see I was not the only one that has noticed these problems and I think as a whole we will all have to work harder to value what goes into making a beautiful image (lots of optimization!!!) and what can be learned from data collected from microscopes (so many useful and quantitative things!!).

How do you usually approach this problem?

My usual training approach involves several imaging sessions with samples prepared by the user as well as ones that I have prepared and imaged previously. We start with the lightpath, how to turn on the microscope, and then how to acquire an image with specific modalities. I try to tailor my approach to the specific experiments a student may be interested in addressing and guide them to resources (some of which were linked in the thread) to help them. Often I end up serving as an advisor on many of these student’s committees.

Is there any nugget of advice or resource that you’ll be using going forward?

I am so grateful to the community for all those resources. I have already linked them to a training request document used internally here! I really like the Lee and Kitaoka MBoC paper and the Senft preprint because they both cover a lot of ground and are not specific to any one modality (but still provide even more resources for interested learners).

If you have requested (or seen) any community advice that you would like to have a permanent home on FocalPlane, contact us at or on our social media pages. And if you are not on social media but would like to solicit community advice, then drop us and email and we’ll ask on your behalf.

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