Luca r camera

Micropatterned coverslips were prepared as described by Azioune et al.^{69 (link)}: O₂ plasma-cleaned coverslips were incubated with 0.1 mg/ml of PLL-g-PEG (Surface Solutions, Switzerland) in 10 mM HEPES, pH 7.4 for 1 h. They were then exposed to deep UV through micropatterned quartz/chrome photomasks (Toppan, Round Rock, TX) for 5 min, and incubated with fibronectin in 100 mM NaHCO₃ (pH 8.5) for 1 h. Releasable micropatterns were prepared similarly, with PLL-PEG being replaced by azido-PLL-g-PEG (APP) at 100 µg/ml. Migration was released by addition of 20 µM BCN-RGD for 10 min. Before imaging, cells were dissociated using Versene (Life Technologies) and seeded for adhesion on the previously mentioned coverslips for at least 2 h. Experiments were performed at 37 °C in 5% CO₂ in a heating chamber (Pecon, Meyer Instruments, Houston, TX) placed on an inverted microscope model No. IX71 equipped with a ×60 objective with NA 1.45 (Olympus, Melville, NY) and a Luca R camera (Andor, Belfast, UK). The microscope was controlled with the Metamorph software (Molecular Devices, Eugene, OR). TIRF images were acquired using an azimuthal TIRF module (iLas2; Roper Scientific, Tucson, AZ). Optogenetics stimulations were performed every 30−40 s with a DMD in epi-mode (DLP Light Crafter, Texas Instruments) illuminated with a SPECTRA Light Engine (Lumencor, Beaverton, OR USA) at 440 ± 10 nm.

Cells were imaged with an inverted fluorescent microscope (Zeiss AxioObserver) equipped with high magnification objectives, QD filter sets (Semrock), and a Luca-R camera (Andor) suitable for detecting discrete QD fluorescence. Data acquisition consisted of acquiring multiple fields of view randomly for each well of the multi-well chamber. The microscope was controlled by Micromanager (https://www.micro-manager.org/)37 . For each field of view, z-stacks were acquired in appropriate QD fluorescent channels (inter-slice distances of 275–300 nm, total z-depth of 18 μm for K562 cells, and 10–12 μm for MOLM-14 cells and MNCs from patients) along with a DIC image at mid-stack. Image acquisition was performed manually or by automated high-throughput scanning. For automated high-throughput scanning, we used a custom-written Java plugin for Micromanager that allows scanning of user-selected wells in multiple channels.