Sola se 365

An inverted Leica DMi8 equipped with a Retiga R6 camera and Lumencor SOLA SE 365 light engine, using a 5X objective, has been used to acquire Time-lapse images. The filter cubes used were TXRed (excitation filter 560/40 nm, emission filter 630/75 nm, dichroic mirror 585 nm) and GFP (excitation filter 470/40 nm, emission filter 525/50 nm, dichroic mirror 495 nm). A live fluorescent dye (CellTrace, red) was used to selectively pre-stain the cancer cells before cultures on-chip. To monitor apoptotic death, a live fluorescent reporter for caspase activity (CellEvent Caspase-3/7, green) was added to the on-chip culture medium. The red channel was then used to locate cells^{10 (link)} while the transposition on the green channel of the cancer cell position allowed to monitor green emission signal and, therefore, death events. Breast cancer cells (BT474 cell line, representative of HER2 + breast cancer subtype) were co-cultured in 3D biomimetic collagen gels, within microfluidic devices, with immune cells (PBMCs, peripheral blood mononuclear cells from healthy donors), with or without the addition of targeted immunotherapy, the trastuzumab (brand name Herceptin). With the aim to demonstrate the advantage of using DM tool in common practice, we extracted handcrafted features related to green emission and compared the case of standard practice with the use of DM tool.

Time-lapse images were acquired with an inverted Leica DMi8 equipped with a Retiga R6 camera and Lumencor SOLA SE 365 light engine, using a 5X objective. The filter cubes used were TXRed (excitation filter 560/40 nm, emission filter 630/75 nm, dichroic mirror 585 nm) and GFP (excitation filter 470/40 nm, emission filter 525/50 nm, dichroic mirror 495 nm). The exposure times were 20 ms for the bright field and 700 ms for the fluorescent channels. The video-microscope was equipped with a motorized stage for multi-positioning acquisition, a CO₂ and temperature-controlled (37°C) incubator chamber (S5A Fig). The presence of a saturating humidity in the microscope chamber is crucial for optimal cell viability, therefore distilled water was added in the plastic wells of the DAX-1 chips and humidified small sponges were added in the chip surroundings (S5B Fig). Since in the AIM-Biotech devices the gas-permeability is provided by the underside sealing layer, before inserting them on the microscope stage, we placed them on a standard microscope glass slides and we lifted them using magnet holders (1 mm thick), in order to create an air circulating space underneath the devices, for CO₂ and temperature control (S5C and S5D Fig). The acquisition of images in transmission and fluorescent channels was performed every hour for a total duration of 48 h to 72 h.

Roots or root tips from 10‐ to 11‐day‐old seedlings were placed on growth plates or, in ‘wave’ experiments, a root was placed across a gap in growth medium agar (Matthus et al. 2019c ; Fig. S1). Recovery was for 5 to 10 min. Solution (3 µl) was applied by pipette; control solution (full Pi or zero Pi liquid half MS) ± 1 mM eATP. Imaging was with a Stereomicroscope M205 FA (Leica Microsystems, Wetzlar, Germany), with a DFC365FX camera (Leica) and a Sola SE365 light source (Lumencor, Beaverton OR, USA); excitation 470/40 nm, emission every 5 s at 525/50 nm, gain of 2.0 and 30× magnification. ImageJ Fiji was used to process GCaMP3 GFP signal intensities, fitting regions of interest (Roi) with the ‘ROI Manager’ tool. Z‐axis profiles were plotted for each Roi, and background signal was subtracted. Data normalization was as described by Vincent et al. (2017 (link)): ΔF/F₀ = (F–F₀)/F₀, where F is the fluorescence signal and F₀ is the baseline fluorescence signal. Maximal response was ΔF_max/F₀. Intensiometric false‐colour videos of response to control solution or ATP were compiled from a representative time series.