Sola light engine

Time-lapse live-cell imaging was performed on an ImageXpress Nano Automated Imaging System (Molecular Devices). A SOLA Light Engine (Lumencor) served as the excitation light source. Cells were plated on 24-well cell imaging plates (with black walls and glass bottom, Eppendorf) and treated with siRNAs and 100 nM nocodazole accordingly. Humidified 5% CO₂ was supplied to the environment chamber maintained at 37 °C.
Using Cre-lox recombination-mediated cassette exchange^{44 (link),54 (link),55 (link)}, we integrated the Tet-On expression cassette of either MAD1-mNG, MAD1^ΔL-mNG, MAD1^AL11-mNG, or MAD1^Lmut-mNG into the HeLa-A12 cell line. According to a previous study^{20 (link)}, the level of MAD1 and MAD2 must be balanced for a robust SAC. To make sure that the expression of exogenous, siMAD1-resistant MAD1(wild-type/mutant)-mNG in siMAD1-treated HeLa-A12 cells is close to the physiological level of endogenous MAD1 for all analyzed cells, we always imaged the heterozygous MAD1-mNG genome-edited HeLa-A12 cell line^{27 (link)} as the control in all our knockdown-rescue mitotic duration assays. Therefore, only cells with green fluorescence intensity (after correcting for the background and shading) close to two times the green fluorescence intensity in the heterozygous MAD1-mNG genome-edited HeLa-A12 cell line were analyzed in our knockdown-rescue mitotic duration assays.

For in vivo experiments, <5 mW of blue light (95–159 mW/mm² at the tip) was generated by 473-nm µLED (Prizmatix, US), and bilaterally delivered through two fiber optic patch cords (0.22 NA, 200 µm diameter, Prizmatix, US). Light delivery was controlled using a pulse generator (PulserPlus, Prizmatix, US) to deliver 5 ms light pulse trains at 10 Hz^{6 (link)} during behavior testing.
For ex vivo electrophysiology recordings, <5 mW of blue light was generated by a 473-nm LED (Sola Light Engine, Lumencor, US) and delivered through a 40× objective. Frequency (0.1 Hz) and pulse duration (1 ms) were controlled by LLE Sola-SE2 software (Lumencor, US).

S. pneumoniae cells were stored as exponential-phase frozen cultures. Frozen stock was inoculated 1:100 in C+Y medium and pregrown to an OD₆₀₀ of ∼0.1. Cells were diluted once again 1:100 in fresh C+Y (with antibiotic, if applicable) and grown to exponential phase to achieve balanced growth.
Cells were grown as described above to achieve balanced growth and subsequently concentrated and brought onto a multitest slide carrying a thin layer of 1.2% agarose in C+Y. Imaging was performed by fluorescence microscopy on a Leica DMi8 through a 100× phase-contrast lens objective (numerical aperture [NA] 1.40) with a SOLA Light Engine (Lumencor) light source. Light was filtered through external excitation filters, 470/40 nm (Chroma ET470/40x), for visualization of GFP. Light passed through a cube (Leica 11536022) with a GFP/RFP polychroic mirror (498/564 nm). External emission filters used were from Chroma ET520/40m. Images were captured using LasX software (Leica) and exported to ImageJ (48 (link)) for final preparation.
Cell outlines were detected using MicrobeJ (30 (link)). For all microscopy experiments, random image frames were used for analysis. The cell outline, object detection, and fluorescence intensity data were further processed using the R package BactMAP (31 (link)).

Imaging of DsRed or EGFP in mosquitoes, midguts and salivary glands was performed using a Nikon SMZ18 stereomicroscope with a Lumencor Sola Light engine, in brightfield and with appropriate fluorescence filters. Scale bars were implemented in reference to an objective micrometer (Edmund optics) that was imaged with the same magnification. Images were edited using Fiji [29 (link)].

Droplets were generated as previously described (Eyer et al., 2017 (link)), and the emulsion was directly injected into the 2D observation chamber. After chamber filling was complete, the chamber was gently closed and mounted onto an inverted fluorescence microscope (Ti Eclipse, Nikon). Two neodymium magnets (BZX082, K and J Magnetics) were placed on each side of the chamber during observation to hold the bead lines in place. Excitation light was provided by a LED source (SOLA light engine, Lumencor Inc). Fluorescence for the specific channels were recorded using appropriate band pass filters (GFP and TRITC filter sets, Nikon, and Cy5 filter set, Semrock) and camera settings (Orca Flash 4, Hamamatsu) at room temperature (25° C) and ambient oxygen concentration. Images were acquired using a 10x objective (NA 0.45). An array of 10 × 10 images were acquired for each experiment, every 7.5 min in all channels over 37.5 min (five measurements total).