Journal: STAR Protocols

Article Title: Single-Molecule Fluorescence Imaging in Living Saccharomyces cerevisiae Cells

doi: 10.1016/j.xpro.2020.100142

Figure Lengend Snippet: Examples of Outcomes of Live-Cell Imaging Experiments All examples are cells expressing PP7 coat protein fused with GFP Envy. (A) Typical field of view of cells during an experiment. Cells shown are expressing PP7 coat protein fused to GFP Envy for visualizing transcription from 14 × PP7 loops at 5′GAL10, grown in galactose-rich media, imaged in widefield fluorescence excitation mode. (B) Zoom in to several cells from the sample shown in (A), imaged at high power using short (50 ms) exposure time, showing individual RNAs in the cytoplasm. (C) Zoom in to several cells from the sample shown in (A) imaged at lower power (500× less than in (B)) using longer (150 ms) exposure time, showing RNAs at the transcription sites.

Article Snippet: Microwave with at least 800 W power Incubator at 30C with humidity control and/or air circulation Shaker (at least 150 rpm) or rotating wheel (such as Brunswick Scientific TC-7 Tissue Culture Roller Drum Rotator M1053-4004) Sonicator (Bioruptor Diagenode) Milli-Q machine Spectrophotometer with capability to measure OD at 600 nm in cuvettes with a light path of 10 mm Inverted widefield microscope (e.g., Zeiss AxioObserver) with the following parts, see schematic in : • Objective with at least 100× magnification and numerical aperture (NA) of at least 1.3; e.g., 100× (Zeiss 4-alpha Plan-Apochromat 100×/1.46 Oil, 420792-9800-000).

Techniques: Live Cell Imaging, Expressing, Fluorescence

Journal: STAR Protocols

Article Title: Single-Molecule Fluorescence Imaging in Living Saccharomyces cerevisiae Cells

doi: 10.1016/j.xpro.2020.100142

Figure Lengend Snippet: Other Applications of the Described Technology (A and B) (A) Cells expressing Sth1-GFP showing nuclear localization of the protein, using widefield fluorescence excitation mode. Imaging settings: widefield illumination, exposure time 150 ms, excitation with 10% power using ND 1.0, 100× magnification, maximum intensity projection of 9 z-slices with a difference of 0.5 μm between consecutive slices. (B) Cells expressing Gal4-HALO. Bright spots represent individual Gal4 protein molecules. Imaging settings: HILO excitation mode, TIRF angle 62.33°, exposure time 1,000 ms, 12 μW laser power, 160× magnification, single z-plane. (C) Cells imaged using the ParS system showing localization of the GAL10 DNA locus. Imaging settings: widefield illumination, exposure time 150 ms, excitation with 100% power using ND 2.0, 100× magnification, 9 z-slices with a difference of 0.5 μm between consecutive slices,

Article Snippet: Microwave with at least 800 W power Incubator at 30C with humidity control and/or air circulation Shaker (at least 150 rpm) or rotating wheel (such as Brunswick Scientific TC-7 Tissue Culture Roller Drum Rotator M1053-4004) Sonicator (Bioruptor Diagenode) Milli-Q machine Spectrophotometer with capability to measure OD at 600 nm in cuvettes with a light path of 10 mm Inverted widefield microscope (e.g., Zeiss AxioObserver) with the following parts, see schematic in : • Objective with at least 100× magnification and numerical aperture (NA) of at least 1.3; e.g., 100× (Zeiss 4-alpha Plan-Apochromat 100×/1.46 Oil, 420792-9800-000).

Techniques: Expressing, Fluorescence, Imaging

Journal: STAR Protocols

Article Title: Expansion microscopy-based imaging of nuclear structures in cultured cells

doi: 10.1016/j.xpro.2021.100630

Figure Lengend Snippet: Imaging expansion gels When using a lower magnification objective with a longer working distance, the orientation of the cells embedded in the gel with respect to the bottom of the well is not critical (A). The increase in size and observable detail is demonstrated here by imaging U2OS cells pre- and post-expansion using a Zeiss AxioObserver D1 widefield system with a 20 × /0.8 NA air objective. In addition to the Hoechst-stained DNA (blue), the cells express GFP-G3BP2, boosted by staining with anti-GFP and AlexaFluor488 secondary antibodies (green), which has accumulated in cytoplasmic stress granules due to sodium arsenite treatment. Nucleoli have been stained using anti-fibrillarin (1:50 dilution) and AlexaFluor555 secondary antibodies (red). When using a higher magnification objective with a much shorter working distance, the cells embedded in the gel can only be brought into focus if they are lying directly on the bottom of the well (B). More detail can be observed for smaller structures (in this case, nuclear stress foci at which GFP-tagged RepoMan accumulates in response to sodium arsenite treatment) following expansion when a 60 × /1.4 NA oil objective is used on a DeltaVision Core restoration deconvolution system. The white boxes mark the regions that have been enlarged to demonstrate the increased detail. Although this is already super-resolution imaging, we routinely combine ExM with Airyscan imaging on a Zeiss LSM880 laser confocal scanning system, which provides a further increase in resolution (C). Volume rendering of the 3D z-stacks using Imaris helps to visualize the nuclear substructure (D), confirming that nuclear stress foci (containing GFP-RepoMan, green) overlap and are surrounded by localized accumulations of the heterochromatic epigenetic histone marker H3K9me3 (1:50 dilution; red; E).

Article Snippet: The increase in size and observable detail is demonstrated here by imaging U2OS cells pre- and post-expansion using a Zeiss AxioObserver D1 widefield system with a 20 × /0.8 NA air objective.

Techniques: Imaging, Staining, Marker