Table S2.

Theoretical versus observed uncertainty calculations

	PaGFP purified protein		PaGFP-Utrophin in HEK cells
Variables	RT (PaGFP Protein)	Cryo (2.6N and 1.1S and 2b)	RT (PaGFP-Utrophin)	Cryo (2.4N and 1.2S and 1.4b)
S	166.91	196.84	305.80	366.22
N	157.56	414.53	209.01	506.41
b	5.07	22.09	5.05	7.08
Uncertainty, nm	24.15	20.97	44.24	33.83
RT/Cryo uncertainty, nm	15% decrease (3.18 nm)		31% decrease (10.41 nm)

We used the freely available ImageJ plugin ThunderStorm to localize and fit molecular positions as well as reconstruct “superresolved” images. ThunderStorm takes into account EMCCD gain and photon/AD count and calculates localization uncertainty ($\sigma$) based on this equation (8): $\sigma =\sqrt{\left(S^2/N\right)+\left(a^2/12N\right)+\left(8\pi s^4{b}^2/a^2{N}^2\right)}$, where S is the SD of the Gaussian fit, N is the number of photons captured from a given fluorescent molecule, a is the pixel size of the EMCCD detector, and b is the SD of the background signal. The table shows the median values we obtained in our PaGFP purified protein experiments and PaGFP-Utrophin experiments for S, N, and b at RT and cryogenic temperature. Increases in S and b at cryogenic temperature are responsible for limiting the effect of N on uncertainty. Hence, the observed values for uncertainty are fully accounted for by the changes in background signal and Gaussian fit at cryogenic temperature. We suspect that by adding an insulator that cools down faster than our closed-cell styrofoam, and rapidly accounting for changes in focus while imaging, uncertainty at cryogenic temperatures could be further reduced.