Hybrid Structured Illumination Expansion Microscopy Reveals Microbial Cytoskeleton Organization.

Reagents

Unconjugated donkey anti-rat (712-005-151), donkey anti-mouse (715-005-151) and rabbit anti-mouse (315-005-003) secondary antibodies, conjugated Alexa Fluor 488 donkey anti-rat antibody (712-545-150), and unconjugated streptavidin (016-000-084) were purchased from Jackson Immunoresearch (West Grove, PA, USA). Conjugated Alexa Fluor 568 goat anti-rat antibody was purchased from Thermo Fisher (Waltham, MA, USA). Primary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA) including mouse anti-acetylated tubulin (clone 6–11B-1, T7451) and rat anti-HA (clone 3F10, 11867423001). Bovine serum albumin (BSA) was purchased from Santa Cruz Biotechnology. NHS-functionalized (amine-reactive) dyes and biotin were obtained from (ATTO 488) Sigma-Aldrich or (Alexa 546, Alexa 568, Alexa 647, EZ-link NHS-PEG-4-Biotin) Thermo Fisher. Dyes were obtained in 1 mg aliquots from the suppliers, dissolved at a concentration of ~100 mg/mL in anhydrous DMSO, sub-aliquoted into anhydrous DMSO at 10 mg/mL, and stored at −20 °C. NAP-5 size-exclusion chromatography columns were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). Methacrylic acid N-hydroxy succinimidyl ester (MA-NHS), anhydrous dimethyl sulfoxide (DMSO), sodium bicarbonate, PIPES salt (for buffer), Ethylene Glycol Tetraacetic Acid (EGTA), magnesium chloride, Triton X-100 (TX100), poly-L-lysine solution (0.1% w/v), cold water fish skin gelatin, and sodium acrylate were obtained from Sigma-Aldrich. MA-NHS was dissolved in anhydrous DMSO at a concentration of 1 M and stored at −20 °C until used. Guanidine hydrochloride was purchased from MP Biomedicals (Irvine, CA, USA). PBS and TAE (Tris Acetate EDTA) 10×stock solutions, and Proteinase K (>600 U/mL, EO0491) were obtained from Thermo Fisher. Acrylamide (40%), N,N’-methylenebisacrylamide (bis-acrylamide, 2%), ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) were purchased from Bio-Rad (Hercules, CA, USA). Paraformaldehyde (32%) and glutaraldehyde (50%) were obtained from Electron Microscopy Sciences (Hatfield, PA, USA). TetraSpeck 0.1 µm fluorescent beads were purchased from Thermo Fisher.

Preparation of fluorophore-labeled antibodies and streptavidin

Fluorophore-conjugated antibodies or streptavidin were prepared as follows. To 80 µL of unconjugated protein (~1.3 mg/mL IgG, or 1 mg/mL streptavidin) was added 10 µL of aqueous 1 M sodium bicarbonate (pH ~8.3) and 1 µL of NHS-dye stock in DMSO. These reagents were allowed to react at room temperature (22 °C) for ~30 min. For singly labeled antibody, typically ~5–10 µg of NHS-dye was added, and for dually-labeled antibodies containing fluorophores and biotin, 10 µg of NHS-PEG-4-Biotin was added after the first reaction. During the reaction, a NAP-5 size-exclusion chromatography column, for purification of labeled antibody from free dye, was equilibrated by flowing ~10 mL of PBS through each column. The ~100 µL reaction was loaded onto the column followed by flowing through and discarding 600 µL of PBS and flowing through and keeping 400 µL eluate. The eluate was characterized by absorption spectroscopy by measuring the average concentration of dye and average concentration of antibody according to the instructions provided by the dye manufacturers. Care was taken to avoid adding more than ~5 % DMSO to the antibody solution to avoid disturbing the antibody in all antibody-labeling reactions. The obtained dye to protein ratios are listed in Supporting Information, Table 1 .

Preparation of poly-L-lysine coverglass

For plating of Giardia lamblia , 12 mm round #1.5 coverglass was purchased from Electron Microscopy Sciences, and soaked in 5 M KOH overnight. After copious rinsing with water, the glass was dried before 1 µl of poly-L-lysine solution was evenly spread on the surface and heated on a hotplate at 90 °C for 10 min. The slides were then rinsed with water and dried. For immobilization of hydrogel specimens for SIM imaging, 25.4 mm × 50 mm #1.5 coverglass (Fisher) were exposed to an air plasma for 60 s to render the glass hydrophilic, then 3 µl of poly-L-lysine was evenly spread on the surface and allowed to dry. Excess water from hydrogel specimens was gently removed using a paper towel or compressed air, and the gel was transferred onto the lysine coated glass to form a drift-free adhesion just before imaging. Refer to Supplemental Protocol for additional information about sample mounting. Giardia Culturing and Immunostaining Giardia lamblia strain WBC6 (ATCC 50803) with integrated expression of HA-tagged DAP86676 or HA-tagged DAP16263 (see below) were cultured in TYI-S-33 medium supplemented with 10% bovine serum, 0.05 mg/ml bovine bile, and 35 µg/ml puromycin in tightly capped 15 mL polystyrene culture tubes. At 70–90% confluency, the cells were iced for 10 min, gently mixed, and 2 mL of cells were added to each well of a 24-well plate containing a 12mm round coverglass coated with poly-L-lysine as described above. The cells were allowed to recover in an atmosphere-controlled incubator containing 95% N 2 at 37 °C for 1–2 h until they were densely adhered to the coverglass. For fixation, the cell media was quickly aspirated and 1 mL of warm 3.2% PFA in 1× PEM buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgCl 2 , pH 6.9) was immediately added to each well. The fixation was allowed to proceed for 20 min at 37 °C, then the samples were washed and stored in PBS containing 3 mM NaN 3 at 4 °C. For immunostaining, Giardia were incubated in PEMBALG (50 mM PIPES, 5 mM EGTA, 10 mM MgSO 4 , 1% BSA, 0.1 M lysine, 0.5% gelatin, 3 mM NaN 3 and 0.1% TX100) for 30 min, followed by overnight incubation with primary antibodies in PEMBALG. The sample was washed, incubated with secondary antibodies for 1 hr, washed, and if necessary, incubated with tertiary antibody, or labeled streptavidin for 1 hr. All washes involved a minimum of three PBS exchanges, with the last step being allowed to rest for 15 min. Antibody dilutions and additional details are summarized in Supporting Information, Table 1 . Vector Construction and Giardia Transformation HA-tagged DAPs for integrated expression in Giardia were constructed by amplifying 86676 (δ-Giardin) or 16263 from WBC6 genomic DNA and inserted into pKS_3HA_Pur (linearized by NotI and AflII) by Gibson Assembly. After construction, 86676_3HA_Pur or 16263_3HA_Pur were linearized for homologous recombination by PstI or NsiI, respectively and ~10 µg of plasmid was electroporated into 300 µl of Giardia in a 0.4 cm cuvette using a GenePulser XCell (BioRad) set to 375 V, 1000 µF and 750 ohms. After overnight recovery in growth media without antibiotics, selection began with 15 µg/ml puromycin and ramped up to 35 µg/ml over 10 days. See Supporting Information, Table 2 for primers. Polymerization, Digestion and Expansion After immunostaining, the sample was washed and incubated in a solution containing 25 mM solution of MA-NHS in PBS for 30 min. The sample was then washed and incubated in monomer solution (8.6% sodium acrylate, 20% acrylamide, 0.075% bis-acrylamide, 2 M NaCl, 1× PBS) for 5 min. Fresh stocks of 10% APS (w/w) and 10% TEMED (w/w) in water were diluted in monomer to a final concentration of 0.2% (w/w) to initiate polymerization. Excess incubation monomer was immediately wicked off the sample and it was inverted onto a 70 µL droplet of the initiated monomer and allowed to polymerize for 20 min. The polymerized sample on the coverglass was then digested overnight in 1 mL of digestion buffer (0.8 M guanidine hydrochloride, 1× TAE, 0.5% TX100, pH 8.3) containing ~6 U/mL of Proteinase K at 37 °C. The sample released from the coverglass after digestion and was transferred to a dish containing ~50 mL of water. The sample was allowed to expand for ~2 hr in deionized water, with water exchanges every ~30 min until complete. See Supplementary Protocol for additional details.

Fluorescence Microscopes 3D

Structured-Illumination microscopy was performed using a GE Healthcare DeltaVision OMX with an Olympus 60× 1.42 NA oil objective. Raw images were acquired from 20–100 ms per frame at 95 MHz CMOS readout. Two-channel data spanned 10–15 µm in post-expansion depth and required acquisition times of ~2 minutes, however the specimen signal was typically limited to the central 5 µm post-expansion dimensions), the 568 nm channel exhibited significant “ghosting” that could not be compensated for using index oil alone; we applied a depth-dependent axial deconvolution to the 568 nm channel using an axial point-spread function determined from the axial profile of the beads obtained by SIM. To computationally straighten curved features in this work, structures were first isolated using a binary mask to determine the direction of propagation in 2D or 3D, then resampled in 40 nm increments in the planes perpendicular using cubic interpolation.

Supplementary Material Movie 1 Disk Movie 2 Axo Supplementary Protocol Supporting Information