Hello all. I have a Leica Thunder Imager with TIRF, 100x N.A. 1.47 and 4 laser lines with power between 100-250mW. I’m thinking to try dSTORM using the system.
The camera on the system is Leica K8 sCMOS, 6.5um pixel, 95fps, QE95%, read noise of 1.8e. I also have a spare EMCCD DU-897D, 16um pixel. Would you recommend using the K8 with 100x TIRF lens or would you suggest EMCCD with 100x TIRF lens plus 1.6x mag changer?
Separately, Leica suggest me to buy their 160x TIRF lens to fit my EMCCD for dSTORM. The main argument is that dSTORM will ‘burn’ my 100x TIRF lens. Is it a valid argument? Thank you for sharing.
A 100x objective with an EMCCD camera was the configuration in many of the first STORM papers so that will work fine. The sCMOS camera sounds like it will also work, but it would be better paired with a 60x objective. Ideally you want a pixel size (in the focal plane) of around 100nm. There are at least a few papers out there that discuss optimal pixel size for STORM. These might be worth a read to get an understanding of how pixel size affects resolution.
What are the laser colors? In my experience you won’t easily burn an objective with a longer wavelength laser such as a 250mW 647 laser. A short wavelength laser like a 250mW 405nm laser might be more of an issue. That said I don’t think that 100x vs 160x would make any difference in this respect. In both cases for TIRF you are going to be focusing the lasers somewhere near the back focal plane of the objective and likely into or near the final optical elements in the objective. So unless a 160x has a radically different design the danger would seem to me to be about the same.
Thank you for sharing. I don’t have any luck searching for literature discussing the optimal pixel size ~100nm. Would you mind giving me some help?
I have 405, 488,561,638nm laser. For now, I’m only interested in using 638nm ~150mW. Yes, I had a hard time understanding the “burning” part as well.
This paper discusses this, see in particular figure 3:
Localization Accuracy in Single-Molecule Microscopy
Raimund J. Ober, Sripad Ram, E. Sally Ward
Biophysical Journal, Volume 86, Issue 2, P1185-1200, 2004.
I think you should be safe with 638nm at that power. I’d be more nervous about the 405nm laser if it can reach a similar power, and you plan to use a power like that.
Thank you so much for your help.