Pom1 clusters and node components were imaged using simultaneous dual-color total internal reflection fluorescence (TIRF) microscopy to limit excitation of fluorophores to those nearest to coverslip. Imaging was performed on a commercially available TIRF microscope (Micro Video Instruments) composed of a Nikon Eclipse Ti microscope base equipped with a 100x Nikon Apo TIRF NA 1.49 objective and a two-camera imaging adaptor (Tu-CAM, Andor Technlogy) containing a dichroic and polarization filters (Semrock FF580-FDi01−25 × 36, FF02-525/40-25, FF01-640/40-25) to split red and green signal between two aligned Andor iXon electron-multiplied CCD cameras (Andor Technology). Red/green beam alignment was performed prior to imaging using a TetraSpeck Fluorescent Microsphere size kit (Thermofisher).
Standard #1.5 glass coverslips were RCA cleaned before use to remove fluorescent debris. Cells were grown in EMM4S, and washed into fresh EMM4S immediately before imaging to remove auto-fluorescent debris resulting from overnight culture. Cells were imaged in EMM4S media on glass slides at ambient temperature. Individual slides were used for no more than five minutes to prevent cells from exhausting nutrients or oxygen. Agar pads were not used due to increased background fluorescence. Image analysis and processing was performed using ImageJ2 (NIH).
Standard #1.5 glass coverslips were RCA cleaned before use to remove fluorescent debris. Cells were grown in EMM4S, and washed into fresh EMM4S immediately before imaging to remove auto-fluorescent debris resulting from overnight culture. Cells were imaged in EMM4S media on glass slides at ambient temperature. Individual slides were used for no more than five minutes to prevent cells from exhausting nutrients or oxygen. Agar pads were not used due to increased background fluorescence. Image analysis and processing was performed using ImageJ2 (NIH).