4D Confocal Imaging of Yeast Organelles

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.

Keywords: 4D microscopy; Confocal; Deconvolution; ImageJ; Photobleaching; Yeast.

Fig. 1

Illustration of the effect of image processing on 4D movies. Shown are the first frames from Video 1 and Video 2, which are 4D movies from the same data set before and after deconvolution, respectively. Yeast cells expressing GFP-Vrg4 as an early Golgi marker and Sec7-DsRed as a late Golgi marker were imaged by confocal microscopy, with cell images in the blue channel. Z-stacks of 24 optical sections each were collected every 2 sec. Where indicated, the red and green channels were deconvolved. The movie frames are average projections of the Z-stacks. In the deconvolved movie, maturation events can be observed when green Golgi cisternae turn red [5].