Axio observer z1 frame

10 to 12 week old tumors were dissected and fixed in 10% neutral buffered formalin overnight at room temperature. They were then embedded in paraffin and sectioned in 5–10 um sections. In some cases, sections were stained with H and E, picosirius red, or trichrome stain prior to SHG imaging. Prior staining had no effect on SHG signal. Images were acquired on a Zeiss LSM 880 Airyscan confocal microscope using an inverted, motorized Zeiss Axio Observer Z1 frame. Two-photon images were collected at 880 nm, using non-descanned detectors set to 440 nm for SHG. Three to four z-stacks were acquired (step size 2 um) per tumor. The z-stacks were compressed and TACS signature was scored by three blinded reviewers as previously described (Corsa et al., 2016 (link)) (Provenzano et al., 2006 (link)).

Embryos were collected and mounted as previously described (Bao and Murray, 2011 (link)). Briefly, embryos were collected by cutting gravid hermaphrodites in a droplet (20 μL) of M9 buffer (3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl, 1 ml 1 M MgSO₄, per liter H₂O). Embryos at the 2 or 4 cell stages were transferred to a droplet (1.5 μL) of M9 containing 20 μm polystyrene beads on 24 × 50 mm coverglass. An 18 × 18 mm coverglass was laid on top and sealed using melted Vaseline. Images were acquired on a spinning disk confocal microscope (Quorum Technologies, Puslinch, Canada) comprising a Zeiss Axio Observer Z1 frame. An Olympus UPLSAPO 60x objective was used with a thread adapter (Thorlabs, Newton, NJ) to mount on the Zeiss body. The timing of major developmental events was used to check for phototoxicity during 3D time-lapse imaging.

Single-molecule localization microscopy was performed on a home-built widefield microscope based on an Axio Observer Z1 frame (Zeiss) equipped with an Ixon Ultra X-7759 EMCCD camera (Andor) with a pixel size of 109 × 109 nm² and a quad-band dichroic mirror (z 405/473/561/640, AHF). Lasers emitting at 473 nm (Gem 473, Laser Quantum) and 561 nm (Gem 561, Laser Quantum) were used in combination with an acousto-optic tunable filter (AOTFnC-400.650, A-A Opto-Electronic) and two achromatic lenses (focal lengths 10 mm and 100 mm, Thorlabs) to expand the laser beam. Images were acquired using a Zeiss α Plan Apo 63× NA 1.46 Oil Corr M27 TIRF objective. GFP was excited with the 473 nm laser (~200 µW) and the signal was collected through a 525 ± 45 nm emission filter. TMR derivatives were excited with the 561 nm laser (~20 mW) and the signal was collected using a combination of 561 nm long-pass and 607 ± 35 nm emission filters. Typically, 80,000 frames (30 ms exposure time) were acquired and used for reconstruction of the SMLM images. SMLM analysis was performed with our a-livePALM software^{52 (link)} (MATLAB 2019b); image drift was corrected as previously described^{13 (link)}.