Citation

  • Authors: Hu X. et al.
  • Year: 2020
  • Journal: J Cell Sci 133 jcs240796
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: BJ-5ta
    2. Name: MDCK
      Description: Canine kidney epithelial cells

Method

MDCK and BJ-5ta cells were reseeded overnight in 35 mm IWAKI glass bottom dishes before they were transfected with 1μg of total DNA [0.5 μg Rab5–GFP or Rab5–mApple, and 0.5 μg Rab7–mApple or Rab7–GFP]. The cells in the imaging dish were typically ∼60% confluent at the time of transfection, and were imaged at least a day after transfection.

Abstract

Despite progress made in confocal microscopy, even fast systems still have insufficient temporal resolution for detailed live-cell volume imaging, such as tracking rapid movement of membrane vesicles in three-dimensional space. Depending on the shortfall, this may result in undersampling and/or motion artifacts that ultimately limit the quality of the imaging data. By sacrificing detailed information in the Z-direction, we propose a new imaging modality that involves capturing fast 'projections' from the field of depth and shortens imaging time by approximately an order of magnitude as compared to standard volumetric confocal imaging. With faster imaging, radiation exposure to the sample is reduced, resulting in less fluorophore photobleaching and potential photodamage. The implementation minimally requires two synchronized control signals that drive a piezo stage and trigger the camera exposure. The device generating the signals has been tested on spinning disk confocal and instant structured-illumination-microscopy (iSIM) microscopes. Our calibration images show that the approach provides highly repeatable and stable imaging conditions that enable photometric measurements of the acquired data, in both standard live imaging and super-resolution modes.This article has an associated First Person interview with the first author of the paper.

Pubmed