Direct microscopic fluorescence imaging of single molecules can provide a wealth of mechanistic information, but up to now, it has not been possible under high pressure conditions, due to limitations in microscope pressure cell design. We describe a pressure cell window design that makes it possible to image directly single molecules at high hydrostatic pressure. We demonstrate our design by imaging single molecules of Alexa Fluor 647 dye bound to DNA, at 120 and 210 bar, and following their fluorescence photodynamics. We further show that the failure pressure of this type of pressure cell window can be in excess of 1 kbar.
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450 °C is below the ageing temperature of the titanium alloy and thus the window removal process can be repeated indefinitely.
46.
While M-bond GA-61 (Vishay Micro-measurements) was the adhesive used in the experiments reported in this paper, we found that Stycast 2850FT with catalyst 23LV also worked well and has the advantage of being jet black, which may help with reducing stray light; however it tends to form air bubbles if it is not out-gassed under vacuum before use, which is rather inconvenient.
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In very recent experiments, we have found that our pressure cell design can withstand pressures approaching 1 kbar even with a standard 0.17 mm thick glass coverslip. This finding will be investigated in future work.
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