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Happy New Year, Alby and All!
My general take on sample preparation: whatever we do to study live cells (not talking about fixation artifacts), affects them in some ways. To begin with, putting them on a flat glass is already a big deviation from their natural environment. But they manage to grow on a glass coverslip in an artificial solution or to withstand modification of proteins or tolerate many other abuses. Perhaps what we are often investigating are the possibilities of life rather than cells' natural morphology or their behavior in the organism. And we tend to be satisfied as long as we get an overall consistent picture of things.
More specifically to your labeling example. An increase in molecular size (if that's your concern), may have an effect. First, the overall mass/volume concentration of large molecules ("macromolecular crowding") seems to be a very important and highly conserved quantity. All eukaryotic cells have macromolecular density close to 0.2 g/ml in the cytosol (higher in some organelles); if the density increases, cells die by apoptosis; and if it decreases, I am not sure at this time what happens then (it's actually quite difficult to keep cells in a diluted state for a long time). But if you are only labeling one single type of protein, the overall crowding will not be greatly affected, but the mobility or the biological activity of that protein can very well suffer of course.
But your assumption that all nuclear proteins become bigger while the nuclear volume stays the same (if I understood you correctly) may not be realistic. As cells synthesize more protein material, they accumulate water and ions and just grow. If you start stuffing them with more or larger proteins, that will disrupt the osmotic balance and, since the membranes are permeant to water, everything will readjust.
Mike
-----Original Message-----
From: Confocal Microscopy List <[log in to unmask]> On Behalf Of Alberto Diaspro
Sent: Sunday, January 1, 2023 9:36 AM
To: [log in to unmask]
Subject: EXT: Alby...new year a civil duty and a technical question to start...
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Friends,
First of all accept my best wishes for a great 2023. Many things to say about 2022, one that I want firmly say, again, for 2023 is: “Verità per Giulio Regeni - Truth for Giulio Regeni” (*) My technical question is simple: do you know/have any paper reporting about a mistake in understanding cell morphology and function due to wrong sample preparation or due to the use of fluorescent molecules perturbing in a certain way the sample? (*) All the best Alby
(*) Giulio Regeni (Italian pronunciation: [ˈdʒuːljo reˈdʒɛːni]; 15 January 1988 – 25 January 2016) was an Italian University of Cambridge graduate who was abducted and tortured to death in Egypt.Regeni was a PhD student at Girton College, Cambridge, researching Egypt's independent trade unions, and a former employee of the international consulting firm Oxford Analytica.
Regeni's mutilated and half-naked corpse was found in a ditch alongside the Cairo-Alexandria highway on the outskirts of Cairo on 3 February 2016. His recovered body showed signs of extreme torture: contusions and abrasions all over from a severe beating; extensive bruising from kicks, punches, and assault with a stick; more than two dozen bone fractures, among them seven broken ribs, all fingers and toes, as well as legs, arms, and shoulder blades; multiple stab wounds on the body including the soles of the feet, possibly from an ice pick or awl-like instrument; numerous cuts over the entire body made with a sharp instrument suspected to be a razor; extensive cigarette burns; a larger burn mark between the shoulder blades made with a hard and hot object; a brain hemorrhage; and a broken cervical vertebra, which ultimately caused death… https://nam11.safelinks.protection.outlook.com/?url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FMurder_of_Giulio_Regeni&data=05%7C01%7Cmmodel%40KENT.EDU%7C0b82ff5216ef4433d8af08daec059ddb%7Ce5a06f4a1ec44d018f73e7dd15f26134%7C1%7C0%7C638081806119111164%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=A5O%2FjX1Dz5fCXZBi5eRZ0xwg1JidjB%2BNOicEy0M77Q8%3D&reserved=0
(**) lets imagine to study the traffick of a certain protein in the cell nucleus. Its movements could be reasonably influenced by the presence of other macromolecules. Now, imagine to label with fluorescent molecules the very same proteins or other macromolecules in the nucleus with flags having a molecular weight ranging from 300 Da up to 37kDa, assuming that the volume offered by the nucleus remains the same, the question is: Is the syetm invariant with respecto to changes of molecular density in the nucleus?
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