3D mapping of nanoscale crosslink heterogeneities in microgels† †Electronic supplementary information (ESI) available: Detailed synthesis and molecular characterization of compounds; hydrogels and microgels preparation details; additional DLS and SEM results; video of dye diffusion from hydrogel matrix. See DOI: 10.1039/c8mh00644j

The majority of gels exhibit nanoscale spatial variations in crosslink density. We present the first 3D super-resolution microscopy images of dye tagged cross-link distributions in microgels and hydrogels. The morphology of nanoscale features never imaged previously in microgels, are revealed.

Double distilled H 2 O was filtered through 0.2 um GHP syringe filter (Pall Laboratory) prior use.

B. Methods
Proton ( 1 H) NMR spectra were recorded with an Agilent 500 MHz instrument. Electrospray ionization/mass spectroscopy (ESI-MS) was carried out with a Thermo Scientific LTQ Orbitrap Dry hydrogel thin films were reconstituted in STORM buffer (containing β-mercaptoethanol, glucose oxidase and catalase) prior to imaging with the W-4PiSMSN system (equipped with two silicone oil immersion objectives, Olympus, NA = 1.35). Microgel samples for imaging were prepared by casting 40 ul of 0.01% w/v dispersion on polylysine coated circular glass coverslips.
The particle coverage was optimum at 15 min of deposition, after which particles not adhered to the surface were washed away by gently rinsing with ultrapure H 2 O. After drying, microgels were reconstituted in STORM buffer for at least 1 h. Samples were imaged at 200 fps at a laser (642 nm) intensity about 15 kW/cm 2 and the data analysis was done as previously described 1 .
Calculation of the resolution for the imaged samples and assessment of the potential impact of multiple blinking is presented in F1.
Clusters and particles were identified from the 3D localization data by using a DBSCAN algorithm. For hydrogels, the threshold size (epsilon) and number (minpts) were identified by varying epsilon and minpts until the number of clusters was maximized, while always keeping epsilon above 25nm. This resulted in epsilon and minpts values of 45 nm and 30 points respectively. Individual microgels were isolated using DBSCAN by setting epsilon and minpts at 500 nm and 1000 points respectively. XY, YZ and XZ projections of the microgels were mapped and 67 relatively non-oblate microgels identified. An ensemble microgel, comprising all the localizations from the 67 microgels, was constructed. Localizations within 30 nm thick XY, XZ & YZ slices passing through the center of the ensemble microgel were identified and each replaced with a 2D Gaussian probability distribution with a FWHM determined by the Cramer-Rao lower bound value associated with each localization. Each peak value was adjusted such that the probability under each Gaussian remained equal to 1. From the resulting localization probability map, the probability per unit area for 10 nm wide rings was plotted against distance from the center.
Nanoclusters within the individual relatively non-oblate microgels were identified using the DBSCAN algorithm. The value of epsilon was set at 25 nm. For each microgel, minpts was identified as the minimum value at which the number of clusters within the microgel is maximized, while also keeping the ratio of minpts to total number of localizations in the respective microgel within the range 0.027±0.0027 (see figure S3). For the radial localization density plot of the clusters, a 3D Gaussian probability distribution with a FWHM determined by the Cramer-Rao lower bound value was associated with each localization. Each peak value was adjusted such that the probability under each Gaussian remained equal to 1. From the resulting localization probability map, the probability per unit volume for 5 nm thick spherical shells was plotted against distance from the center.
Transmittance measurements were taken using a green LED illuminating source (ThorLabs) and power meter (Newport 2931-C). Samples were placed between the source and detector and two sets of measurements were realized after disassembling the holder and exchanging coverslips.
Dark background was measured ~30 nW and 100% transmittance was set at ~70 μW.

C1. Molecular synthesis
Scheme S1. BMA-NH 2 synthesis overview previously cleaned in triplicate with acetone, ethanol and DI water. The holder was transferred with the aid of a cool box to a thermostated water bath at the desired temperature and left for 1 h, after which it was disassembled. The hydrogel stubs were removed, purified with frequent exchange of DI water and stored at the dark in a solvent excess. Upon reaching swelling equilibrium, the stubs were cut into 25 mm discs with the aid of a punch and mounted in the sample holder between #1 coverslips for the transmittance study. The hydrogel stubs were further cut with a 15 mm punch prior to being photographed. For ALEXA-tagged hydrogel films preparation the monomer solution was confined between two glass coverslips, following the exact same procedure as above and scaling down for 45 mg of NiPAm. Specifically, 2.5 ul of monomer solution was cast on a 25 mm circular #1.5 coverslip (Bioscience Tools) which has been thoroughly solvent cleaned.

N,N'-bis(di-tert-butoxycarbonyl)-1,3-diaminopropan-2-ol
The coverslip was capped with a freshly piranha cleaned circular 25 mm #1 coverslip (VWR) and the coverslip sandwich was mounted into a Presslock sample holder. The holder assembly was transferred with the aid of a coolbox into a thermostated water bath and after 1 h the sample holder was disassembled. The coverslips where separated using a razor, exposing the laminated hydrogel film on the #1.5 solvent cleaned coverslip. The film was carefully washed with copious amounts of DI water, left to dry in ambient conditions and stored in the dark. The absence of free dye was confirmed by the absence of background in confocal images as well as the final superresolution images.

E. Reactivity ratios of monomers.
The relative insertion kinetics were calculated by following a procedure established by Acciaro et al. 8 Briefly, the reaction rate of the monomers is given by the equation below: where i = NIPAm, BIS or BMA-ALX, and Pol * denotes the polymeric radicals in the reaction mixture. The relative concentration (C(t)/C 0 ) of the monomers is plotted as a function of reaction time and the relative insertion kinetics calculated using the relationship below:

F. Resolution calculation
The resolution for the specific microgel system reported in this manuscript was calculated using the method used in Ref. 10 and 11. In brief, molecules that appear on consecutive frames and within 50 nm (in 3D) of each other are treated as arising from persistent emission of the same fluorophore and linked together. Localizations associated with molecules that are persistent across > 9 frames were identified and aligned by their center of mass to generate the localization distributions shown in Figure S6, below. From this data, we estimated the resolution to be ~24 nm in x, ~27 nm in y and ~18 nm in z.