Obviously clean glassware is essential to the success and reproducibility of any experiment, but even more so for single molecule fluorescence microscopy which can be extremely sensitive to stray contaminants that may be indistinguishable from single fluorophores of interest.
Our lab has more or less arbitrary, unquestionable, rules of cleaning, that often take several hours to complete, e.g. for washing sample glass vials it could take 3 x 10 min sonication in soap, then water, then ethanol, then chloroform. As you can see, this procedure takes 2 hours in sonication alone, not counting manual labor in between.
Does anybody know if there are any studies (i.e. not anecdotal beliefs and hand-me-down protocols) investigating this?
My comment on this is that, sadly, there is really no such thing as a clean surface. You have to know what is is that you are most keen to remove or exclude and then act accordingly. It sounds as if you are mainly concerned to remove organics and maybe your protocol is well-tried for that particular purpose, but one always needs to be aware that the same agent that removes may also add. A good solvent is a good solvent and it could carry in contaminants as well as remove them. In just the same way with metal contamination (a big problem with glass), EDTA is supposed to strip away bound metal ions, but, because it is good at that, it may arrive at your sample already having stripped away metal from somewhere else and then generously donating the metal to your metal-avid sample!! You just have to work out what is necessary and what is adequate for your particular application (and not assume it will work for the next application!). Life is tough.
I don't clean my glassware, I etch a fresh surface onto it with 2.5% w/v (5%concentrated HF v/v) in water (prepared in a plastic bottle)! The object to be cleaned is immersed in the dilute aqueous HF solution in a plastic vessel and gently swirled until the surface its evenly wetted. For test tubes, beakers and flasks I pour the dilute HF solution in them, cover (Parafilm M) or stopper, and shake the vessel vigorously for several minutes. The object is then rinsed with distilled water. This leaves hexafluorosilicic acid residues on the glass surface which are easily removed by immersing the object in 5%w/v KOH in 50%v/v ethanol:water for a few seconds and then rinsing with distilled water. This leaves a pure clean evenly wetted glass surface behind. Caution HF especially in the more concentrated forms is toxic, corrosive and causes severe burns. That said, in my younger days, I used to wipe the dilute HF solution around on the glass surfaces with my bare hands, with no ill effects! If you do the calculations you will find this dilute HF solution is about twice as acidic as vinegar (5% Acetic Acid). This 2.5%w/v HF solution can also be used to remove the white glazed on lettering and graduations on the outside of bottles and other glassware.
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