Deepsee

The custom-built two-photon imaging setup was based on an Olympus BX51WI microscope equipped with a LUMPLFLN 60 × 1.0 NA objective, controlled by the open-source software package ScanImage (Pologruto et al., 2003 (link)) which was modified to allow user-defined arbitrary line scans at 500 Hz. Two Ti:Sapphire lasers (MaiTai DeepSee, Spectra-Physics) controlled by electro-optic modulators (350–80, Conoptics) were used to excite cerulean (810 nm) and GCaMP6s (980 nm). To activate ChR2(ET/TC)-expressing cells outside the field of view of the objective, we used a fiber-coupled LED (200 µm fiber, NA 0.37, Mightex Systems) to deliver light pulses to CA3. During the blue light pulses, sub-stage PMTs (H7422P-40SEL, Hamamatsu) were protected by a shutter (NS45B, Uniblitz).

Two-photon Ca2+ imaging was performed on days 1 and 6 using a Bergamo microscope (Thorlabs Inc., Newton, NJ, USA) controlled by ThorImage OCT software (ThorImageLS, v3). The visual cortex was illuminated with a Ti:Sapphire fs-pulsed laser (Mai Tai Deep-See, Spectraphysics) tuned to 920 nm. The laser was focused onto L2/3 of binocular V1 through a 16x water-immersion objective lens (0.8NA, Nikon). Ca2+ transients were obtained from neuronal populations at a resolution of 512 × 512 pixels (sampling rate ~30 Hz). The obtained images were motion-corrected using CaImAn (Giovannucci et al., 2019 (link)). Segmentation, neuropil subtractions, and deconvolution were performed using Suite2p (Pachitariu et al., 2016 ).

For raster image correlation spectroscopy (RICS), a series of 100 images of a dispersion of the PCDTBT NPs in water were acquired with a pixel dwell time of 16.38 µs using an LSM 880 (Zeiss) confocal laser scanning microscope on an inverted Axio observer motorized frame. A 256 × 256 pixel sized image, with a pixel size of 83 nm was acquired. The microscope was fitted with a Plan-Apochromat 20×/0.8 objective. A femtosecond pulsed titanium-sapphire laser (MaiTai DeepSee, Spectra-Physics) tuned to 810 nm was used as the excitation source for TPEM. The fluorescence emission was then channeled through a 690 nm low pass filter. The BiG-2 (Zeiss) non-descanned detector was used to capture the images. Arbitrary-region RICS (ARICS) analysis was applied to determine the diffusion coefficient [41 (link)]. Aggregates (if any) were masked out using an absolute intensity threshold, while the remaining areas of the image were left unprocessed. The analysis and fit were performed using the PAM software package using the Microtime Image Analysis and MIAFit modules [42 (link)].

CAFs-cancer cells cocultures were imaged using a two-photon laser-scanning microscope (Zeiss LSM880NLO) in single photon mode, using a 40×/1.30 OIL DICII PL APO (UV) VIS-IR objective, and laser lanes 488 and 561. CAF rings were ablated using a Ti:Sapphire laser (Mai Tai DeepSee, Spectra Physics) set at 800 nm and laser power of 10% Image acquisition was started 10 s before the ablation, every 2 s and for a total time of 50 s.
To perform laser ablations in vivo, living tumor tissue slices from control or myosinIIA KO mice were prepared as described previously^{10 ,40 (link)}, and placed into a 35 mm glass-bottom culture dish. A slice anchor (SHD-26GH/10; Harvard Apparatus) was placed on top of the tissue slices to minimize sample drift and covered with a drop (100 µl) of DMEM-GlutaMAX (Glibco), supplemented with 1% (v/v) antibiotic-antimycotic (Gibco), 2.5% (v/v) fetal bovine serum, 1% (v/v) Insulin-Transferrin-Selenium (ITS, ThermoFisher Scientific) and 10 ng/mL EGF (Peprotech). The ablation setup was adapted from the in vitro experiments, with laser power set at 20%. Image acquisition was started 10 s before the ablation, every 2 s and for a total time of 50 s. Para-Nitroblebbistatin (50 µM) (Optopharma) was added to 4–5 slices per mouse, incubated at 37 °C, 5% CO₂ for 2.5 h, and then ablations were performed as described.