Since polarity defects are emphasised in longer cells, cdc25‐22 cells were grown at 28°C until mid‐log phase at which point they were shifted to 35.5°C for 6 h to arrest at G2/M and then imaged live on the Nikon wide field system. Eighteen z‐stacks (0.2 μm apart) were taken with a 100 ms exposure time for bright field. Each experiment analysed at least 280 cells, was repeated three times and excluded cells that were less than 14 μm long. The z‐stack containing the middle of the cell was used for analysis, whereby a line was drawn manually between the two cell ends in the middle of the cell using the line tool in ImageJ. The length of this line (L, length) was then compared to that of a straight line between the cell ends (E, Euclidean distance) using the measure function of ImageJ. The ratio between these values, converted to a percentage, gives a measure of the curvature of each cell. The distribution of cell lengths was unaltered between backgrounds.
Widefield system
Manufactured by Nikon
The Nikon Widefield System is a microscope-based imaging platform designed for capturing high-quality wide-field images of samples. It provides a stable and versatile platform for a range of applications, including live-cell imaging, histology, and material science.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Zen 2, by Zeiss (2 mentions)
Plan apochromat oil objective, by Zeiss (2 mentions)
Csu x1 m1 spinning disk, by Sutter Instruments (2 mentions)
Alpha plan apochromat oil objective, by Zeiss (2 mentions)
Glass bottom petri dish, by Ibidi (1 mentions)
Cmtpx red cell tracker, by Thermo Fisher Scientific (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
4 protocols using widefield system
1
Quantifying Cell Curvature in G2/M
2
Super-resolution Imaging of Cellular Organelles
3
Super-resolution Imaging of Cellular Organelles
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Cells were maintained at 37°C and 5% CO2 (see Supplementary Data Table 2 ). Live cell Airyscan superresolution microscopy used a Zeiss LSM 880 confocal microscope with Airyscan detector and 63× Plan-Apochromat oil objective, NA 1.4. Sequential excitation of fluorophores was used, with 488nm laser line for Snap-Cell Oregon Green and MitoSOX, and 633nm laser for Snap-Cell 647-SiR, with appropriate dual bandpass filters. The Airyscan detector was adjusted regularly, and images were Airyscan processed in 2D using the Zeiss Zen 2.3 software package (Carl Zeiss Microscopy).
For spinning disk confocal imaging, cells were imaged on a 3i Marianas platform with a Zeiss Axio Observer Z1, Optovar 1×, 1.6× and 2.5×, with a Yokogawa CSU-X1 M1 spinning disk, Sutter LB-10W fast 10-position filter wheel with filters: 445/45, 525/30, 617/73, and Andor Neo sCMOS camera using a 63× Alpha Plan-Apochromat oil objective, NA 1.46 (Carl Zeiss Microscopy), using Slidebook 5.5 acquisition software (3i) and lasers: Violet (solid state 405nm/100mW), Blue (solid state 488nm/150mW), and Lime (solid state 561nm/50mW).
An inverted Nikon widefield system with Andor Zyla 4.2+ camera, external filter wheel and SpectraX LED was used for widefield imaging. Images were acquired using a 60× water objective, NA 1.20, and NIS-Elements 4.5 software.
For spinning disk confocal imaging, cells were imaged on a 3i Marianas platform with a Zeiss Axio Observer Z1, Optovar 1×, 1.6× and 2.5×, with a Yokogawa CSU-X1 M1 spinning disk, Sutter LB-10W fast 10-position filter wheel with filters: 445/45, 525/30, 617/73, and Andor Neo sCMOS camera using a 63× Alpha Plan-Apochromat oil objective, NA 1.46 (Carl Zeiss Microscopy), using Slidebook 5.5 acquisition software (3i) and lasers: Violet (solid state 405nm/100mW), Blue (solid state 488nm/150mW), and Lime (solid state 561nm/50mW).
An inverted Nikon widefield system with Andor Zyla 4.2+ camera, external filter wheel and SpectraX LED was used for widefield imaging. Images were acquired using a 60× water objective, NA 1.20, and NIS-Elements 4.5 software.
4
Live-cell Imaging of Myoblast Co-culture
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Live cell images were acquired using the inverted Ti eclipse Nikon Widefield system equipped with a Plan Apo 20× (NA 0.75) objective, SpectraX LED excitation (395 nm, 440 nm, 470 nm, 508 nm, 561 nm, 640 nm), Quad filter for DAPI/GFP/RFP/Cy5, and Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels). The environmental conditions of 37 °C with 5% CO2 were maintained using the Oko-lab environmental control chamber. Image acquisition used the NIS Elements Software Version 5.21.00 .
Control and DAG1 KO myoblasts were stained with CellTrackerTM Red CMTPX (Invitrogen, Waltham, MA, USA) and CellTrackerTM Green CMFDA (Invitrogen, Waltham, MA, USA), respectively. Control and KO cells were then co-cultured in a glass bottom petri dish (Ibidi, Fitchburg, WI, USA). Each experiment was imaged over 15 h with each XY position imaged every 10 min. For each time point, a brightfield image was taken in addition to images in the red and green channels to identify the two cell types.
Control and DAG1 KO myoblasts were stained with CellTrackerTM Red CMTPX (Invitrogen, Waltham, MA, USA) and CellTrackerTM Green CMFDA (Invitrogen, Waltham, MA, USA), respectively. Control and KO cells were then co-cultured in a glass bottom petri dish (Ibidi, Fitchburg, WI, USA). Each experiment was imaged over 15 h with each XY position imaged every 10 min. For each time point, a brightfield image was taken in addition to images in the red and green channels to identify the two cell types.
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