Local activation was performed by using a Mightex Polygon digital micromirror device (DMD) to pattern blue light (488nm) stimulation from a Lumencor SpectraX light engine using Nikon Elements software. U2OS cells expressing the optogenetic telomere coalescence constructs FUSN-miRFP-TRF1, NLS-GFP-iLId-Fe and FUSN-mCherry-sspB were imaged using a specific local activation protocol, as follows. Pre-activation, imaging the mCherry (541 nm beam) and miRFP (640 nm beam) channels every 5 s for 15 s. Activation, wherein an elliptical region of interest (ROI) was used to locally activate two telomere foci to nucleate and grow FUSN Corelet droplets using the 485 nm DMD laser every 5 s for 6 min. A second activation sequence used a smaller, circular ROI aimed at the junction between two FUSN Corelet droplets every 5 s for 4 min to encourage them to fuse. Finally, the FUSN droplet was deactivated for 10 min by only imaging the mCherry and miRFP channels every 5 s, which allows the droplets to dissolve and pull together any attached telomeres. The second set of telomere coalescence constructs (iLId-miRFP-TRF1) uses a similar local activation protocol but only a single circular activation ROI for 3 min and a longer deactivation sequence (15-30 min).
Polygon
Manufactured by Mightex
The Polygon is a versatile lab equipment designed for a range of applications. It features a multi-sided polygon-shaped surface that can be used for various purposes. The core function of the Polygon is to provide a stable, customizable platform for experiments, sample preparation, or other lab-based activities. Its modular design allows for flexibility in configuration and arrangement to suit the user's specific needs.
Automatically generated - may contain errors
4 protocols using polygon
1
Optogenetic Telomere Coalescence Dynamics
2
Optogenetic Telomere Coalescence Dynamics
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Local activation was performed by using a Mightex Polygon digital micromirror device (DMD) to pattern blue light (488nm) stimulation from a Lumencor SpectraX light engine using Nikon Elements software. U2OS cells expressing the optogenetic telomere coalescence constructs FUSN-miRFP-TRF1, NLS-GFP-iLId-Fe and FUSN-mCherry-sspB were imaged using a specific local activation protocol, as follows. Pre-activation, imaging the mCherry (541 nm beam) and miRFP (640 nm beam) channels every 5 s for 15 s. Activation, wherein an elliptical region of interest (ROI) was used to locally activate two telomere foci to nucleate and grow FUSN Corelet droplets using the 485 nm DMD laser every 5 s for 6 min. A second activation sequence used a smaller, circular ROI aimed at the junction between two FUSN Corelet droplets every 5 s for 4 min to encourage them to fuse. Finally, the FUSN droplet was deactivated for 10 min by only imaging the mCherry and miRFP channels every 5 s, which allows the droplets to dissolve and pull together any attached telomeres. The second set of telomere coalescence constructs (iLId-miRFP-TRF1) uses a similar local activation protocol but only a single circular activation ROI for 3 min and a longer deactivation sequence (15-30 min).
3
Spinning-Disk Confocal Microscopy for Live-Cell Imaging
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All images were taken with a spinning-disk (Yokogawa CSU-X1) confocal microscope with a 100X oil immersion Apo TIRF objective (NA 1.49) and an Andor DU-897 EMCCD camera on a Nikon Eclipse Ti body. A 488nm laser for imaging GFP and global activation, and a 561 was used laser for imaging mCherry. The imaging chamber was maintained at 37°C and 5% CO2 (Okolab) with a 96 well plate adaptor. Local activation was performed by using a Mightex Polygon digital micromirror device (DMD) to pattern blue light (488nm) stimulation from a Lumencor SpectraX light engine. All image acquisition was performed using Nikon Elements Advance Research software.
4
Spinning-Disk Confocal Microscopy for Live-Cell Imaging
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All images were taken with a spinning-disk (Yokogawa CSU-X1) confocal microscope with a 100X oil immersion Apo TIRF objective (NA 1.49) and an Andor DU-897 EMCCD camera on a Nikon Eclipse Ti body. A 488nm laser for imaging GFP and global activation, and a 561 was used laser for imaging mCherry. The imaging chamber was maintained at 37°C and 5% CO2 (Okolab) with a 96 well plate adaptor. Local activation was performed by using a Mightex Polygon digital micromirror device (DMD) to pattern blue light (488nm) stimulation from a Lumencor SpectraX light engine. All image acquisition was performed using Nikon Elements Advance Research software.
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