Peter D. Dahlberg 1, Annina M. Sartor 1, Jiarui Wang 1,2, Saumya Saurabh 2, Lucy Shapiro 2, W.E. Moerner 1

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1 Peter D. Dahlberg 1, Annina M. Sartor 1, Jiarui Wang 1,2, Saumya Saurabh 2, Lucy Shapiro 2, W.E. Moerner 1 1 Department of Chemistry, Stanford University, Stanford, CA 94305, United States 2 Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, United States S1

2 Supporting Text Purification of PAmKate, PAmCherry and PA-GFP pbad/hisb-pamkate and pbad/hisb-pamcherry1 were a gift from Vladislav Verkhusha (Addgene plasmids # and 31931). prseta-pagfp was a gift from Jennifer Lippincott- Schwartz (Addgene plasmid # 11911). The expression plasmids were transformed into competent E. coli (NEB, C2566) and single colonies were inoculated into 5 ml cultures and grown overnight. The 5 ml grown cultures were then diluted into 1 L of LB broth and the cells were grown to an OD600 of 0.5 at 37 C. Protein expression was induced with either 1% (w/w) arabinose or 1 mm IPTG at 22 C for 4-6 hours. Finally, cells were pelleted at 4000 rpm for 20 minutes and washed once with cold PBS (ph 7.4) before being frozen at -80 C. For protein purification, pellets were resuspended in 10 ml of wash buffer (50 mm HEPES KOH ph 7.4, 500 mm KCl, Roche Protease Inhibitors 1 tablet / 50 ml lysis buffer, 25 mm imidazole, 200U benzonase nuclease (0.3 ul / 1 L starting culture)). Cells were lysed by sonication with 40x3 second pulses and 10 second intervals at 100% amplitude prior to centrifugation for 45 min at 10,000 g. The lysed cells were spun down at rpm for 20 minutes and the supernatant was incubated with Ni-NTA agarose beads (Thermo Fisher) for 2 hours at 4 C with agitation. After binding, beads were washed four times with 10mL of wash buffer. Finally, the protein was eluted in elution buffer (50 mm HEPES- KOH ph 7.4, 500 mm KCl, 250 mm imidazole, 10% glycerol). Eluted protein was then dialyzed three times against 1 L of dialysis buffer (50 mm HEPES-KOH ph 7.4, 200 mm KCl, 1 mm DTT, 10% glycerol). Dialyzed protein was purified on Superdex 75 Gel Filtration Column (S GL, GE Healthcare) by fast protein liquid chromatography. Purity was evaluated using SDS- PAGE and protein was quantified using a DU730 UV/Vis spectrophotometer based on Bradford assay. All purification steps were performed on ice or in a cold room. Generation of C. crescentus pxyl-pamkate-popz strains PAmKate and PopZ genes were PCR amplified with a GGGSGGGS linker and cloned into pxyfpc-2 that was digested by NdeI/NheI using Gibson Assembly. 1,2 The ligation was transformed into E. coli DH5α cells and selected on LB-kan plates. Sequence verified plasmid was then transformed into C. crescentus NA1000 cells via electroporation. Cryogenic super-resolution data acquisition The reduced bleaching rate and limited excitation intensities at cryogenic temperatures necessitated much longer image acquisition times than are typical in super-resolution imaging. This slow acquisition coupled with thermal gradients of 200 K resulted in drift both axially and laterally. During acquisition, the cryostage is cooled by the addition of small amounts of liquid nitrogen from an internal dewar. This results in a slight (~100 nm) breathing motion of the stage on the minute timescale, primarily along the x and y axes. Additionally, approximately every 30 min, the internal dewar is refilled via an external dewar. This results in ~ 5 μm of rapid axial drift (Figure S5). To monitor axial drift, an astigmatic point spread function (PSF) was generated using a 1 meter focal length cylindrical lens in the emission path. Axial drift is corrected for during acquisition by automatic monitoring of the astigmatic PSF of over-activated PAmKate-PopZ poles. Feedback to the axial stage (Applied Scientific Instrumentation LS-50) onto which the objective is mounted is provided by custom LabVIEW code to maintain in-focus imaging. S2

3 Cryogenic super-resolution analysis Raw data collected at 1 Hz was drift corrected using the over-activated PopZ poles as fiducials. These poles had roughly constant brightness throughout imaging and an average uncertainty per frame of ~14 nm as calculated following the analysis of Quan et al. 3 The fiducial x-y position in each frame was used to drift correct the raw data. Activation frames were removed and the remaining data was binned into 5 s frames. Following binning, the x-y uncertainty due to fiducial localization per binned frame is just 14/ 5 or ~6 nm. This uncertainty is less than the uncertainty of a single emitter in a single-binned frame. Since single PAmKate emitters were commonly active for minutes at cryogenic temperatures, this binning improved the signal to noise before ThunderSTORM localization analysis. The binned data was processed using ThunderSTORM version dev b1. The analysis filter was the default wavelet filter (B-spline scale 2 order 3). Particle detection used a connectivity of 8 and a threshold of 2 times the standard deviation of the high pass filter from the wavelet (2*std(Wave.F1)). Identified particles were fit by an elliptical Gaussian using a maximum likelihood routine. Localizations lasting for many binned frames can be grouped to take advantage of the additional photons at cryogenic temperatures. Both the error from fiducial localization and that from molecular localization are random in any given binned frame and therefore the overall uncertainty scales with 1/ N where N is the number of binned frames for which a single emitter is in an emissive state. Localizations were grouped if they were within 50 nm of each other, had at most four consecutive binned off-frames, and at least four binned on-frames. In the grouping process we treat each binned frame localization as an independent measure of the true molecular position. These multiple measurements of an individual molecule s position yield a statistical distribution whose standard error of the mean is represented in Figure 2B as the radii of the golden circles. Room temperature super-resolution acquisition and analysis Caulobacter crescentus cells expressing PAmKate-PopZ fusions were placed on agarose pads following the same induction conditions used for cryogenic super-resolution imaging (0.3% xylose for 3 hours). Imaging was performed with a custom epifluorescence microscope. Fluorescence emission from the sample was collected using an oil immersion, super-corrected objective (Olympus PLANON60xOSC, 60X/NA1.4) mounted in a Nikon Diaphot 200 microscope body with additional optics inserted before the Andor Ixon EMCCD camera for a 4f system that was not used in this work. Images were acquired at 20 Hz using 240 W/cm 2 of 561 nm readout excitation and 1 second pulses of 20 W/cm 2 of 405 nm activation. The acquired data was processed with ThunderSTORM in a similar manner as the cryogenic data, but the identified particles were fit with a standard Gaussian model rather than an elliptical Gaussian model since there was no astigmatism. The localizations were grouped if they were within 50 nm of each other and had at most one consecutive off-frame. S3

4 Supporting Table Photoactivatable Protein Efficiency At 77 K Compared To Room Temperature Error PAmKate 1.19 ±0.12 PAmCherry ±0.02 PAGFP 0.77 ±0.19 Table S1: Same data presented in Figure 1B. Activation efficiency of the three photoactivatable proteins at 77 K compared to room temperature shows no activation for PAmCherry within error, and activation efficiencies for PAmKate and PAGFP comparable to their efficiencies at room temperature. Supporting References 1. Thanbichler, M.; Iniesta, A. A.; Shapiro, L. A comprehensive set of plasmids for vanillateand xylose-inducible gene expression in Caulobacter crescentus. Nucleic Acids Res. 2007, 35, e Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A.,3rd; Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 2009, 6, Tingwei Q.; Shaoqun Z.; Zhenli H. Localization capability and limitation of electronmultiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging. J. Biomed. Opt. 2010, 15, S4

5 Supplementary Figures Figure S1. Distribution of single-emitter brightness based on the integrated signal from a Gaussian fit at 294 Kelvin and at 77 Kelvin for PAmKate (left) PAmCherry (middle) and PAGFP (right) isolated and embedded in 1% MOWIOL 4-88 (Polysciences Inc.). Figure S2. Workflow for the activation assay. (Top Left) BL21 E. coli expressing PAmKate plunge frozen on an EM grid. The frame shown was acquired during an activation pulse of 405 nm light. Autofluorescence seen from 405 nm illumination was used to determine cell outlines and construct a binary mask of cellular fluorescence signal. (Top right) The binary mask applied to data set; red S5

6 denotes zero. (Bottom) Intensity trace of masked data showing an increase in the density of active PAmKate proteins following activation. Figure S3 Larger field of view from the same data set shown in Figure 2. White light image of plunge-frozen C. crescentus cells imaged at 77 K, outlined in blue. Regularly spaced holes in the EM grid substrate coating are visible in the background. Overlaid in red is a 2D histogram of localizations generated from the super-resolution reconstruction. PAmKate-PopZ poles are highlighted with white arrows. The colorbar of the histogram is truncated at 20 to make pixels with few localizations visible. S6

7 Figure S4 Same as Figure 2A and B except the colorbar of the histogram has not been truncated. Figure S5. (A) Axial drift correction applied automatically during acquisition. Correction was determined from elliptical Gaussian fits of an astigmatic point spread function. Large changes at 0, 33, and 66 minutes result from a replenishing of the cryostage s Nitrogen dewar (B) Post acquisition determination of lateral drift. Oscillations are due to periodic supply of liquid Nitrogen to sample chamber. S7

8 Figure S6. (A) Super-resolution reconstruction of PAmKate-PopZ fusions imaged at room temperature, shown as a 2D histogram of binned molecule localizations and overlaid with white light image of the same region. (B) Zoom of boxed region in A. S8