Table 1 Summary of the parameters of the mounting media tested and their performance in 3D-SIM of thick samples and SMLM.

Mounting medium for SIM:	Refractive Index @589 nm, 25 °C	Viscosity^a	3D-SIM of thick samples					SMLM Alexa 647
Mounting medium for SIM:	Refractive Index @589 nm, 25 °C	Viscosity^a	Hoechst 33258	Alexa 488	Alexa 555	Cy3	Alexa 594	SMLM Alexa 647
Vectashield	1.4477	− − − −	+	+	+	+	+	+++
S65VE	1.4580	− − −	+++	+++^c*	++	+++^e*	+++	+++^f*
Sorb70VE	1.4585	− − −	++++*	+++	++*	+++^e	+++*	+++*
F80VE	1.4706	− − − − −^b	+++	++	++	+++^e	+++	++
H71VE	1.5037	− − − −	++++	+^d	+++^d	++	+++^d	+

Vectashield H-1000 was used in all experiments. Note a very low viscosity of intermediate solutions (Supplementary Table 2). Notes: ^aEstimates of the viscosity of embedding media are provided for respective temperatures of embedding. See Supplementary Table 1 for more details. Viscosity scales proportionally with the number of “−” signs. ^bHigh viscosity of fructose solutions can be decreased by an addition of urea⁴¹. This, however, may lead to DNA denaturation and impairment to Hoechst 33258 signal intensity. ^cHigh signal to noise ratio with faster bleaching. ^dSignal to noise ratio is low unless higher Vectashield vol/vol concentration in clearing media is used (Supplementary Fig. S10). ^eVery high signal to noise ratio. ^fElevating the Vectashield concentration to 40% vol/vol improves Alexa 647 photoncount (see Supplementary Fig. S14). *Recommended medium for the fluorophore. Due to fluorescence signal instability in the presence of Vectashield Alexa 647 is not recommended in SIM.

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