Immersion

Images were collected using a Keyence BZ-X710 fluorescent microscope configured with 3 fluorescent channels (FITC, Cy3, Cy5) and equipped with Nikon PlanFluor 40x NA 1.3 oil immersion lens. Imaging and washes were iteratively performed automatically using a specially developed fluidics setup (Figures S7A–S7C). Images were subject to deconvolution using Microvolution software (http://www.microvolution.com/). The staining patterns of 28 DNA-conjugated antibodies were acquired over 14 cycles of CODEX imaging and overlaid with 2 additional fluorescent antibodies, CD45-FITC and NKp46-Pacific Blue and a DRAQ5 nuclear stain (Figure 3A and low-resolution views in Video S2). Each tissue was imaged with a 40x oil immersion objective in a 7x9 tiled acquisition at 1386x1008 pixels per tile and 188 nm/pixel resolution and 11 z-planes per tile (axial resolution 900 nm). Images were subjected to deconvolution to remove out-of-focus light. After drift-compensation and stitching, we obtained a total of 9 images (one per tissue) with x = 9702 y = 9072 z = 11 dimensions, each consisting of 31 channels (30 antibodies and 1 nuclear stain).

Imaging as well as single molecule photon counting was performed on a modified Olympus IX71 inverted microscope. A 641 nm laser (Coherent, CUBE 640-100C) and a 405 nm laser (Coherent, CUBE 405-100C) was reflected by a multiband dichroic (89100 bs, Chroma) on the back aperture of a 100×1.3 NA oil objective (Olympus, UplanFL) or 60×1.2 NA water immersion objective (Olympus UPLSAPO 60XW) (for Figure 4d–f and Figure S7d–f) mounted on a piezo objective scanner (P-725 PIFOC, Physik Instrumente). The collected fluorescence was filtered using a band-pass emission filter (ET700/75, Chroma) and imaged onto an EMCCD camera (IxonEM+, Andor) with a 100 nm pixel size (167 nm for the 60×Objective) and using the conventional CCD amplifier at a frame rate of 25 fps. Laser intensity on the sample measured after the objective was ∼2–4 kW.cm⁻² with the 100× objective, and ∼1 kW.cm⁻² with the 60× objective. 10,000–20,000 frames were recorded for the photon-counting experiments, and 15,000–20,000 for the imaging experiments.
3D imaging (Figure 4) was performed by adding a cylindrical lens to the imaging path, using one arm of an Optosplit system (CAIRN). The cylindrical lens (f = 1000 mm, Throlabs LJ1516RM-A) was added at the position of the fluorescence filter, which is close to the Fourier plane.
3D STORM imaging (Figure 5) was performed on a SR-200 inverted microscope (Vutara, Salt Lake City, UT) based on the biplane approach [6] (link) using a 60×/1.42 NA oil objective (Olympus, UIS2 PLANAPO). Extra magnification was used to achieve a pixel size of 101 nm on an EMCCD camera (Photometrics). A 647 nm laser (Coherent) was used for excitation, with a power of ∼4.5 kW.cm⁻², and a 405 laser (Coherent, CUBE) for re-activation (few mW.cm⁻²). Data was recorded at 25 fps, and the acquisitions consisted of 20,000 raw images. Raw data was analyzed by the Vutara SRX software (v4.04). In brief, particles were identified by their brightness from the combined images taken in both planes simultaneously. If a particle was identified in multiple subsequent camera frames, data from these frames was summed up for the specific identified particle. Particles were then localized in three dimensions by fitting the raw data in a 16×16 pixel region of interest centered on each particle in each plane with a 3D reference obtained from recorded bead calibration data sets. Sample drift was corrected by cross-correlation of the localized particles according to [33] (link).

Immunofluorescence experiments for C. elegans were performed as previously described (Zhang et al., 2018 (link)). Images shown in Figure 4—figure supplement 2A were obtained from worms dissected and fixed in the absence of Tween-20 to retain soluble proteins. Primary antibodies were obtained from commercial sources or have been previously described, and were diluted as follows: Rabbit anti-RAD-51 (1:5000, Novus Biologicals, #29480002), Rabbit anti-pHIM-8/ZIMs (1:500, Kim et al., 2015 (link)), Goat anti-SYP-1 (1:300, Harper et al., 2011 (link)), Rabbit anti-SYP-2 (1::500, Colaiácovo et al., 2003 (link)), Chicken anti-HTP-3 (1:500, MacQueen et al., 2005 (link)), Mouse anti-HA (1:400, Thermo Fisher, #26183), Mouse anti-GFP (1:500, Millipore Sigma, #11814460001), Mouse anti-FLAG (1:500, Sigma, #F1804), anti-ALFA-At647N (1:500, Nanotag Biotechnologies, N1502-At647N). Secondary antibodies labeled with Alexa 488, Cy3, or Cy5 were purchased from Jackson ImmunoResearch (WestGrove, PA) and used at 1:500. All images were acquired as z-stacks through 8–12 µm depth at z-intervals of 0.2 µm using a DeltaVision Elite microscope (GE) with a ×100, 1.4 N.A. or ×60, 1.42 N.A. oil-immersion objective. Iterative 3D deconvolution, image projection, and colorization were carried out using the softWoRx package and Adobe Photoshop CC 2021.