Widefield microscope

Images were collected on an Olympus FV1000 confocal microscope or a Nikon Widefield microscope. For each quantified value, at least 3 different animals were sampled on 10 consecutive 12 µm sections. Quantification was then manually performed using the Cell Counter plugin of ImageJ. Data shown are expressed as the mean±s.e.m. and are presented in Fig. S1D. Normality tests were applied on datasets and appropriate t-tests were used in order to assess the statistical significance of the data. Significance values are ***P<0.001, **P<0.01, *P<0.05.

Overnight cultures of P. aeruginosa grown in LB were used to set up liquid cultures in 5 mL LB using 100 µL of the overnight culture. The cultures were grown to 0.4–0.8 OD₆₀₀ and normalised to an 0.05 OD₆₀₀ in 1 mL of minimal medium (M9 succinate). Nanosensors were suspended at 5 mg mL⁻¹ in selected medium and filter sterilised using 0.22 µm PES filters. The BioFlux flow cell (BioFlux 200 48-well low shear plate, 0–20 dynes cm⁻²) was prepared according to the manufacturer’s instructions (Fluxion Biosciences, BioFlux System). For the seeding of the cells, 50 µL of the normalised cells was used with a seeding time of 45–60 min. After the seeding, 1 mL of the prepared medium with or without nanosensors was used as flow medium. The flow was set to 0.25 dyn cm⁻² and the system was run for 20–24 h. Time-lapse imaging was applied using a Nikon widefield microscope with brightfield and fluorescence channels (OG-FAM excitation = 460 nm, TAMRA excitation = 550 nm). Images were taken every 15 min for 16 h.⁸⁴

Measurements were taken on the Nikon spinning disc confocal microscope using CFP (445 nm excitation, 482/35 nm emission) and FRET (445 nm excitation, 540/30 nm emission) channels, acquiring images with a 40X (NA 0.95) air objective at 300 ms exposure, EM multiplier 300, 10 MHz camera readout speed, 20% laser power, and binning pixels 2 × 2. Measurements were also taken on the Nikon widefield microscope using CFP (434/16 nm excitation and 470/24 nm emission) and FRET (434/16 nm excitation and 535/20 nm emission) channels, acquiring images with a 20X (NA 0.75) or 40X (NA 0.95) air objective at 4–600 ms exposure, EM multiplier 300, 1 MHz camera readout speed, with a neutral density filter (ND8) restricting lamp light to 12.5% maximum.
Neuron cultures (DIV 10–14) were washed and put in RNIM at least ten minutes prior to imaging. Baseline measurements were obtained for 5–15 minutes. Calibrations were performed by adding 10 µM TPA for 2–5 minutes, followed by wash out with RNIM and addition of 10 µM ZnCl₂/0.5–2 µM pyrithione. Measurements were taken for several minutes after a maximum FRET ratio was reached. For KCl stimulation experiments, KNIM was mixed 1:1 with RNIM and measurements were taken for 400–600 seconds before washing out with RNIM and performing a calibration as above.

Tumor sections were imaged in an upright Nikon widefield microscope using a 10x objective (0.30 NA), and a gain of 1. Between 1–3 non-overlapping regions of interest (ROI) were obtained per sample, and in each ROI areas for segmentation were labeled as Nestin-positive (brown), background, or tissue (blue). The segmentation of each ROI was performed using the Trainable WEKA Segmentation from Fiji [43] (link). Between 1 to 4 regions of each label per ROI were manually selected and used to train the algorithm, followed by testing on the whole image. In each ROI, microvessel density (MVD) was quantified as the percentage of Nestin-positive area relative to total tissue area [41] (link). Additional details can be found in the Appendix.

Fluorometry was done on an inverted Nikon widefield microscope. A 63x oil immersion lens was used, in combination with a 34°C stage heater. The microscopy medium was kept at a pH of 7.2, using HBS (HEPES buffered saline). This buffer contained 10 mM glucose, 2 mM CaCl₂, 5 mM KCl, 140 mM NaCl, 1 mM MgCl₂ and 10 mM HEPES. The excitation of the Cyan Fluorescent Protein (CFP) was set at 425 nm. CFP and Yellow Fluorescent Protein (YFP) emissions were measured simultaneously using band-pass filters at 470±20 and 530±25 nm respectively. Ca²⁺ measurements were done using FRET biosensors and chemical dyes. The Ca²⁺ FRET sensor Twitch-2B was used. Also, the Ca²⁺ dyes Oregon Green 488 BAPTA-1-AM and Fura Red-AM were used. No quantitative differences could be detected between these methods, with respect to the TRPM7 Ca²⁺ signals. Before the experiments the FRET ratio was set at 1.0 and a baseline was recorded. After the Ca²⁺ experiments a calibration was done adding ionomycin (10 μM) and a high dose of Ca²⁺ (CaCl₂, 10 mM).