"In situ" means "in position" or "in the original place." But that could be interpreted in many ways. Is a complex purified from native membranes considered in situ? What about imaging them in purified organelles? Maybe... but at that point, it’s also very much ex vivo 😅. 2/n

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The trouble starts when in situ is used synonymously for all these different conditions. It creates confusion and risks leading the field to assume in situ automatically means "native". But are purified organelles really as native as looking inside a whole cell via FIB-milling? 3/n

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I would argue that "true native" is always better (of course the #TeamTomo guy says that), though not always necessary depending on the question. You just want to solve the structure of a soluble complex or membrane protein? Then purified or semi-purified is probably fine. 4/n

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However, if you want to study higher-order organization, supercomplexes, or spatial distribution... then you really don’t want to work with anything purified. A great recent example from the @cellarchlab.com lab by @wojwie.bsky.social nicely shown here: bsky.app/profile/cell... 5/n

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This work clearly shows that even within native membranes of purified chloroplast 🌿, the integrity of the organelle impacts photosystem organization, you can see a complete reorganisation. It gets tricky to draw solid biological conclusions if the starting preparation changes the architecture. 6/n

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