Published: April 28th, 2019
This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.
The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.
Compartments of the endomembrane system are in constant flux, and their full characterization requires live cell imaging. Described here is a protocol that employs 4D (time-lapse 3D) confocal microscopy to visualize fluorescently labeled compartments in budding yeasts. The method was developed to track the dynamics of secretory compartments in Pichia pastoris and Saccharomyces cerevisiae1,2,3. This protocol focuses on S. cerevisiae, which has a nonstacked Golgi in which the individual cisternae are optically resolvable4. The ....
The example given here documents and quantifies the maturation of two yeast Golgi cisternae as visualized by dual-color 4D confocal microscopy3. A yeast cell contains on the order of 10-15 Golgi cisternae, each of which matures over a time course of approximately 2-4 min. Maturation can be visualized by tagging the early Golgi marker Vrg4 with GFP and by tagging the late Golgi marker Sec7 with a red fluorescent protein such as mCherry or mScarlet. An individual cis.......
4D confocal imaging of yeast organelles requires careful tuning of multiple parameters. The major concern is photobleaching and phototoxicity. A typical 4D movie involves collecting thousands of optical sections, so the laser illumination must be kept as low as possible. Tandem fluorescent protein tags can be used to boost the signal without increasing expression of the tagged protein16,17. Maximizing the scan speed helps to limit photodamage, and also allows Z-s.......
This work was supported by NIH grant R01 GM104010. Thanks for assistance with fluorescence microscopy to Vytas Bindokas and Christine Labno at the Integrated Microscopy Core Facility, which is supported by the NIH-funded Cancer Center Support Grant P30 CA014599.....
|35 mm glass bottom dishes No. 1.5
|Concanavalin A powder
|Leica SP8 confocal microscope
|Leica Application Suite X
|Huygens Essential software, version 17.04
|Scientific Volume Imaging
|Image processing and analysis software
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