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4D Confocal Imaging of Yeast Organelles

  • Kasey J. Day
  • Effrosyni Papanikou
  • Benjamin S. GlickEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1496)

Abstract

Yeast cells are well suited to visualizing organelles by 4D confocal microscopy. Typically, one or more cellular compartments are labeled with a fluorescent protein or dye, and a stack of confocal sections spanning the entire cell volume is captured every few seconds. Under appropriate conditions, organelle dynamics can be observed for many minutes with only limited photobleaching. Images are captured at a relatively low signal-to-noise ratio and are subsequently processed to generate movies that can be analyzed and quantified. Here, we describe methods for acquiring and processing 4D data using conventional scanning confocal microscopy.

Key words

Yeast Confocal 4D microscopy Photobleaching Deconvolution ImageJ 

Supplementary material

Video 1

4D movie of yeast Golgi compartments before deconvolution. See the legend to Fig. 1 (MOV 1139 kb)

Video 2

4D movie of yeast Golgi compartments after deconvolution. The data from Video 1 were deconvolved as described in the text. See the legend to Fig. 1 (MOV 315 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Kasey J. Day
    • 1
  • Effrosyni Papanikou
    • 1
  • Benjamin S. Glick
    • 1
    Email author
  1. 1.Department of Molecular Genetics and Cell BiologyUniversity of ChicagoChicagoUSA

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