Data was acquired on a home-built widefield microscope with 8-plane detection described in detail in 20 (link) .
Briefly, four illumination laser lines, a 120 mW 405 nm laser (iBeam smart, Toptica), an 800 mW 635 nm laser (MLL-III-635, Roithner Lasertechnik), and an 800 mW 532 nm laser (MLL-FN-532, Roithner Lasertechnik) are collimated, expanded and combined with dichroic filters and focused into the back focal plane of the objective (Olympus UPLSAPO 60XW 1.2 NA). The fluorescence light is filtered using a dichroic mirror and quad band emission filter (zt405/488/532/640/730rpc and ZET 405/488/532/640m Chroma).
The detection path is arranged as a sequence of four 2-f configurations to ensure image-object space telecentricity. An image splitting prism placed behind the last lens directs the light into eight images recorded by two synchronized sCMOS cameras (ORCA Flash 4.0, Hamamatsu; back projected pixel size of 111 nm). For translating the sample, the microscope is equipped with piezoLEGS stage (3-PT-60-F2,5/5) and Motion-Commander-Piezo controller (Nanos Instruments GmbH). 3a WGA labeled U2OS cells in HBSS 8000/1000 50 <0.17 561 nm Figure 3b See above 1000/1000 50 See above
Briefly, four illumination laser lines, a 120 mW 405 nm laser (iBeam smart, Toptica), an 800 mW 635 nm laser (MLL-III-635, Roithner Lasertechnik), and an 800 mW 532 nm laser (MLL-FN-532, Roithner Lasertechnik) are collimated, expanded and combined with dichroic filters and focused into the back focal plane of the objective (Olympus UPLSAPO 60XW 1.2 NA). The fluorescence light is filtered using a dichroic mirror and quad band emission filter (zt405/488/532/640/730rpc and ZET 405/488/532/640m Chroma).
The detection path is arranged as a sequence of four 2-f configurations to ensure image-object space telecentricity. An image splitting prism placed behind the last lens directs the light into eight images recorded by two synchronized sCMOS cameras (ORCA Flash 4.0, Hamamatsu; back projected pixel size of 111 nm). For translating the sample, the microscope is equipped with piezoLEGS stage (3-PT-60-F2,5/5) and Motion-Commander-Piezo controller (Nanos Instruments GmbH). 3a WGA labeled U2OS cells in HBSS 8000/1000 50 <0.17 561 nm Figure 3b See above 1000/1000 50 See above