With Airyscan, ZEISS introduced a new detector concept for confocal laser-scanning microscopy (LSM). Whereas traditional LSM designs use a combination of pinhole and single-point detectors, Airyscan is a 32-channel gallium arsenide phosphide photomultiplier tube (GaAsP-PMT) area detector that collects a pinhole-plane image at every scan position. Each detector element functions as a single, very small pinhole. Knowledge about the beam path and the spatial distribution of each detector channel enables very light-efficient imaging with improved resolution and signal-to-noise ratio.
Mitochondrial morphology is regulated by continuous fusion-and-fission events that are essential for maintaining normal function. Despite the prominence of mitochondrial function in energy generation and cell signaling, understanding of processes of fusion and fission dynamics has been hampered by the lack of high-resolution optical systems that accommodate live-cell imaging. We have examined different confocal modalities in terms of resolution and signal-to-noise ratio (SNR) in a point scanning confocal microscope with Airyscan super-resolution (AS-SR). Results indicated that Airyscan (AS) provided speed, super-resolution, and high SNR. This modality was then used for monitoring mitochondrial dynamics in live tumor cells modified to harbor green-fluorescent protein localized to mitochondria. We then compared regular AS and fast-Airyscan modalities in terms of gentleness on the live-cell samples. The fast mode provided unprecedented imaging speed that permits monitoring dynamics both in 2D and also in three-dimensional dataset with time lapses (4D). Alterations to the mitochondrial network in U87 glioblastoma cells occurred within seconds and the cells were not affected by modest inhibition of fission. The super-resolution permitted quantitative measurements of mitochondrial diameter with a precision that enabled detection of significant differences in mitochondrial morphology between cell lines. We have observed swelling of mitochondrial tubules in A549 lung cancer cells after 2 hr treatment with deoxynyboquinone, an ROS-generating pharmacologic drug. We also tested different 3D analytical parameters and how they can affect morphometric quantitation. The AS-SR imaging enabled high-speed imaging of mitochondrial dynamics without the compromise to cell morphology or viability that is common with conventional fluorescence imaging due to photo-oxidation.
Sterile-alpha-motif (SAM) domains are common protein interaction motifs observed in organisms as diverse as yeast and human. They play a role in protein homo- and hetero-interactions in processes ranging from signal transduction to RNA binding. In addition, mutations in SAM domain and SAM-mediated oligomers have been linked to several diseases. To date, the observation of heterogeneous SAM-mediated oligomers in vivo has been elusive, which represents a common challenge in dissecting cellular biochemistry in live-cell systems. In this study, we report the oligomerization and binding stoichiometry of high-order, multi-component complexes of (SAM) domain proteins Ste11 and Ste50 in live yeast cells using fluorescence fluctuation methods. Fluorescence cross-correlation spectroscopy (FCCS) and 1-dimensional photon counting histogram (1dPCH) confirm the SAM-mediated interaction and oligomerization of Ste11 and Ste50. Two-dimensional PCH (2dPCH), with endogenously expressed proteins tagged with GFP or mCherry, uniquely indicates that Ste11 and Ste50 form a heterogeneous complex in the yeast cytosol comprised of a dimer of Ste11 and a monomer of Ste50. In addition, Ste50 also exists as a high order oligomer that does not interact with Ste11, and the size of this oligomer decreases in response to signals that activate the MAP kinase cascade. Surprisingly, a SAM domain mutant of Ste50 disrupted not only the Ste50 oligomers but also Ste11 dimerization. These results establish an in vivo model of Ste50 and Ste11 homo- and hetero-oligomerization and highlight the usefulness of 2dPCH for quantitative dissection of complex molecular interactions in genetic model organisms such as yeast.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.