Dmi8 inverted microscope

For fluorescence microscopy, exponentially growing cells were placed on slides containing a thin pad of 1% SeaKem LE agarose (Cambrex) with TPM buffer (10mM Tris-HCl pH 7.6, 1mM KH₂PO₄ pH 7.6, 8mM MgSO₄) and 0.2% CTT medium, and covered with a coverslip. After 30 min at 32°C, cells were visualized using a temperature-controlled Leica DMi8 inverted microscope and phase contrast and fluorescence images acquired using a Hamamatsu ORCA-flash V2 Digital CMOS camera. For time-lapse recordings, cells were imaged for 6 hrs using the same conditions. To induce expression of genes from the vanillate inducible promoter [51 (link)], cells were treated as described in the presence of 300 μM vanillate. The data sets used for fluorescence microscopy quantification are available in S9 Table.

All live imaging movies were acquired on a Leica DMi8 inverted microscope with a ×63 objective (oil immersion, NA = 1.4, Hamamatsu Orca Flash 2.0 camera) and at room temperature (22–25°C). To reduce autofluorescence during imaging, the culture medium was replaced with Live Imaging Solution (Thermo Fisher Scientific A14291DJ). Culture media was not replaced for imaging cells in nocodazole, DMSO, and osmo+ to enable measurement of MT dynamics in the chosen media. For EB1-GFP imaging, an image (exposure time 500 ms) was taken every 2 s for 70–150 frames depending on sample bleaching. When imaging both EB1-GFP and Jupiter-mCherry simultaneously, one image was taken every 3 s for 100 frames (exposure 500 ms). Lamp intensity was set to the lowest level that enabled visual identification of labels.

In this study, beads are sorted based on their fluorescence intensity or pattern in both red and green channels. Simultaneous dual color fluorescence microscopy was performed using a LEICA DMi8 inverted microscope connected to a Hamamatsu Orca-D² camera equipped with two charge-coupled devices (CCDs) enabling simultaneous microscopy in wavelengths of interest. The ChemMatrix beads used in this study tend to aggregate in solution, leading to clogging of the microfluidic platform and sorting errors (false positives/negatives). To prevent aggregation, beads were maintained in a diluted suspension in PBS buffer (150 beads per 80 ml) and gently stirred on an orbital shaker throughout the duration of the sorting cycle. On-chip valves were filled and degassed with a 50% glycerol solution with a similar refractive index as PDMS, which improves image quality in the vicinity of the valves. A custom-built pressure box equipped with pressure regulators was used to drive fluid flow in the tubing and device. Valve operations was controlled by a custom-developed MATLAB Graphical User Interface (GUI).