Automating multimodal microscopy with NanoJ-Fluidics.

Cell lines COS7 (ATCC CRL-1651) and HeLa H2B-mCherry/mEGFP-α-Tubulin (HCTG, kind gift from Daniel W. Gerlich) stable cell line 26 , 27 cells were cultured in phenol-red free DMEM (Gibco) supplemented with 2 mM GlutaMAX (Gibco), 50 U/ml penicillin, 50 μg/ml streptomycin (Penstrep, Gibco) and 10% foetal bovine serum (FBS; Gibco). hTERT-RPE1 cells stably expressing zyxin-GFP 5 were cultured in DMEM F-12 GlutaMAX (Gibco), with 10% FBS, 3.4% sodium bicarbonate (Gibco), 1% Penstrep. All cells were grown at 37 °C in a 5% CO 2 humidified incubator. Cell lines have not been authenticated.

Plasmids

The plasmid expressing the calponin homology domain of utrophin fused to GFP (UtrCH-GFP) was a gift from William Bement 28 (Addgene plasmid #26737). Antibody conjugation Secondary antibodies were labelled with the corresponding DNA strands using the following protocol 29 : secondary antibodies were concentrated via Amicon 100 kDa spin filters to 2–6 mg/ml. A volume of 50–100 μg of antibody was labelled using a Maleimide-Peg2-succinimidyl ester (stocks of 10 mg/ml in DMF) for 90 min at 40× molar excess at 4 °C on a shaker. After the 90 min incubation, unreacted crosslinker was removed via a Zeba spin column. Thiolated DNA was reduced using DTT for 2 h at room temperature. DTT was separated from the reduced DNA via a Nap5 column and fractions containing DNA were concentrated via 3 kDa Amicon spin filters. The reduced DNA was then added to the antibody bearing a functional maleimide group in 25× molar excess and incubated overnight at 4 °C on a shaker in the dark. Antibody-DNA constructs were finally purified via 100 kDa Amicon spin filters. DNA-PAINT labelling: For Mitochondria: Goat anti-Mouse (ThermoFisher A28174,) with I1 (docking: 5′-TTATACATCTA-3′; imager: 5′-CTAGATGTAT-ATTO655-3′); For Vimentin: Goat anti-chicken (Abcam ab7113) with I2 (docking: 5′-TTAATTGAGTA-3′; imager: 5′- GTACTCAATT-Cy3B-3′); For Clathrin: Goat anti-Rabbit (ThermoFisher A27033 ). with I3 (docking: 5′-TTTCTTCATTA-3′; imager: 5′- GTAATGAAGA-Cy3B-3′); For alpha-tubulin: Donkey anti-Rat (ThermoFisher A18747) with I4 (docking: 5′-TTTATTAAGCT-3′; imager: 5′-CAGCTTAATA-ATTO655-3′). Sequences for DNA-PAINT strands were obtained from 3 . Both thiolated and fluorophore conjugated DNA strands were obtained from Metabion.

NanoJ-Fluidics framework

We provide the detailed instructions to easily build and use the system in a regular biology lab, as well as the software enabling its control and automation (Supplementary Note 1 ). NanoJ-Fluidics Lego syringe pump calibration NanoJ-Fluidics Lego syringe pump calibration was performed by injecting predefined volumes into a container sitting inside an analytical scale (Sartorius Cubis®, 0.1 mg ± 0.0003 mg). The procedure was repeated using different syringe pumps, and syringe sizes (see Supplementary Note 2 and 3 for discussion). The dispensed volume was weighed and an ordinary one-way ANOVA was used (GraphPad Prism version 7.00 for Mac, GraphPad Software, La Jolla California USA) to estimate the statistical difference between the different conditions used in Supplementary Fig. 4 (using a confidence interval of 99%). For the Overflow loading mode, a volume of 100 μL was measured with a Gilson P100 micropipette (by weighing it in an analytical scale, Sartorius Cubis®, 0.1 mg ± 0.0003 mg). The weighed volume was inserted into the tubing and then dispensed using the pump by infusing a 500 μL volume. By flushing out a larger volume than injected, this method ensures that the entire volume is dispensed. Live-to-fixed super-resolution imaging The NanoJ-Fluidics syringe pump array was installed on a Nikon N-STORM microscope equipped with 405, 488, 561 and 647 nm lasers (20, 50, 50 and 125 mW at the optical fibre output). One individual syringe pump module containing the fixative was kept within the incubator of the microscope at 37 °C. All steps after cell transfection were performed on the microscope, using NanoJ-Fluidics. COS7 cells (kind gift from M. Marsh) were seeded on ultraclean 30 25 mm diameter thickness 1.5 H coverslips (Marienfeld) at a density of 0.3–0.9 × 10 5 cells/cm 2 . One day after splitting, cells were transfected with a plasmid encoding the calponin homology domain of utrophin fused to GFP (UtrCH-GFP) using Lipofectamin 2000 (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Cells were imaged 1–2 days post transfection in culture medium using an Attofluor cell chamber (ThermoFisher), covered with the lid of a 35 mm dish (ThermoFisher), that was kept in place using black non-reflective aluminium tape (T205-1.0 AT205, THORLABs). Cells were fixed at 37 °C for 15 min with 4% paraformaldehyde in the cytoskeleton-preserving buffer “PIPES-EGTA-Magnesium” (PEM: 80 mM PIPES pH 6.8, 5 mM EGTA, 2 mM MgCl2) 31 . After fixation cells were permeabilised (PEM with 0.25% Triton-X) for 20 min, blocked with blocking buffer (5% Bovine Serum Albumin (BSA) in PEM) for 30 min, and stained with Phalloidin-AF647 (Molecular Probes, 4 units/mL) for 30 min.

Laser-illumination Highly Inclined and Laminated Optical sheet

(HILO) imaging of UtrCH-GFP in live COS7 cells was performed at 37 °C and 5% CO 2 on a Nikon N-STORM microscope. A 100x TIRF objective (Plan-APOCHROMAT 100 × /1.49 Oil, Nikon) with additional ×1.5 magnification was used to collect fluorescence onto an EMCCD camera (iXon Ultra 897, Andor), yielding a pixel size of 107 nm. For timelapse imaging, 100 raw frames (33 ms exposure) were acquired once every 10 min (with the illumination shutter closed between acquisitions) for 150 min with 488 nm laser illumination at 4% of maximum output.

STORM

HILO imaging of Alexa Fluor 647-phalloidin in fixed cells was performed on the same system. About 50,000 frames were acquired with 33 ms exposure and 642 nm laser illumination at maximum output power with 405 nm pumping when required (0.5–1% of maximum output when the blinking density was bellow 1 particle/μm 2 ). STORM imaging was performed in GLOX buffer (150 mM Tris, pH 8, 1% glycerol, 1% glucose, 10 mM NaCl, 1% β-mercaptoethanol, 0.5 mg/ml glucose oxidase, 40 μg/ml catalase) supplemented with Phalloidin-Alexa Fluor 647 (1 U/mL).

Multiplexed DNA-PAINT and STORM super-resolution microscopy

COS7 cells (obtained from ATCC) were seeded on 18 mm, 1.5 H glass coverslips (Menzel-Gläser). About 24 h after seeding, they were fixed using 4% PFA, 4% sucrose in PEM at 37 °C 31 . After blocking in phosphate buffer with 0.022% gelatin, 0.1% Triton-X100 for 1.5 h, cells were incubated with primary antibodies overnight at 4 °C: mouse monoclonal anti-TOM20 (BD Bioscience 612278), rabbit polyclonal anti-clathrin heavy chain (abcam ab21679), chicken polyclonal anti-vimentin (BioLegend 919101) and rat anti-alpha-tubulin (mix of clone YL1/2 abcam 6160 and clone YOL1/34 Millipore CBL270). After rinses, they were incubated with Exchange-PAINT secondary antibodies coupled to DNA sequences: goat anti-mouse I1, goat anti-chicken I2, goat anti-rabbit I3 and donkey anti-rat I4 for 1.5 h at RT (see Ab conjugation section for antibody and sequence details). After rinses, they were incubated in phalloidin-Atto488 (Sigma) at 12.5 μM for 90 min at RT and imaged within a few days 32 . For STORM/PAINT imaging, the NanoJ-Fluidics array was installed on an N-STORM microscope (Nikon) equipped with 405, 488, 561 and 647 nm lasers (25, 80, 80 and 125 mW at the optical fibre output). First, a STORM image of phalloidin-ATTO488 was acquired in buffer C (PBS 0.1 M pH7.2, 500 mM NaCl) by taking 30,000 frames at 30 ms/frame at 50% power of the 488 nm laser. After injection of the I1-ATTO655 (0.25 nM) and I2-CY3B (2 nM) imagers in buffer C, 60,000 frames were acquired by sequentially interleaving the two channels (60% power of the 647 nm laser and 30% power of the 561 nm laser) to image TOM20 and vimentin in a single acquisition, therefore providing 30,000 frames for each channel. After three rinses with buffer C, I3-Cy3B (1 nM) and I4-ATTO655 (0.5 nM) were injected in buffer C, and 60,000 frames were similarly acquired via frame interleaving (30% power of the 561 nm laser and 60% power of the 647 nm laser, 30,000 frames of each channel) to image clathrin and microtubules, respectively. Event detection and live-to-fix imaging hTERT-RPE1 cells stably expressing zyxin-GFP were incubated with 9 mM RO-3306 (Enzo Life Sciences ALX-270-463) to inhibit CDK1 activity for 15–20 h. Inhibition was released by replacing drug containing media by fresh media at the microscope immediately before imaging. Cells were imaged using a Nikon Eclipse Ti microscope (Nikon) equipped with a Neo-Zyla sCMOS camera (Andor), LED illumination (CoolLED) and a ×60 objective (Plan Apo 60 × /1.4 Oil, Nikon). Images were acquired every 3 min until enough cells had undergone mitotic rounding. At that point, determined by visual inspection, 16% warmed PFA (from a Lego syringe pump placed inside the incubation chamber) was added to cells in media to a final concentration of 4%, and incubated at room temperature for 20 min. They were then washed three times and 0.2% Triton was added for 5 min. 5% BSA in 1X PBS was used to block for 30 min at room temperature, before activated β1 Integrin (Abcam ab30394) primary antibody was added. After incubation and washing, Phalloidin-TRITC (Sigma-Aldrich) and anti-mouse AF647 antibody (Invitrogen) were added. All of these steps were performed automatically using the NanoJ-Fluidics platform, except the identification of the time point when to fix the cells. SMLM and SRRF image reconstruction For Fig. 4 images were reconstructed using NanoJ-SRRF 12 (magnification: 4, temporal analysis settings: TRPPM for live-cell data and TRM for fixed-cell data). Drift was estimated using the inbuilt function in NanoJ-SRRF and correction applied during SRRF analysis. For Fig. 5 localisations were detected using the N-STORM software (Nikon), and exported as a text file before being filtered (number of photons between 700 and 50,000; number of detections (after linking across frames)