Live-cell imaging was essentially performed as described (Barrales et al., 2016 (link)). In brief, cells were grown overnight in rich medium (YES) to the logarithmic phase (OD600 = 0.4–0.6). Before imaging, cells were attached to lectin (Sigma) coated glass-bottom dishes containing a microwell (MatTek). Cells were imaged using a Zeiss AxioObserver Z1 confocal spinning disk microscope with an EMM-CCD camera (Photometrics, Evolve 512) through a Zeiss Alpha Plan/Apo ×100/1.46 oil DIC M27 objective lens. Z-stacks were obtained at focus intervals of 0.4 μm. FiJi/ImageJ software was used to count the number of foci in the yeast cells.
Alpha plan apo 100 1.46 oil dic m27 objective
Manufactured by Zeiss
The Zeiss Alpha Plan/Apo ×100/1.46 oil DIC M27 objective is a high-performance microscope objective lens. It has a magnification of 100x and a numerical aperture of 1.46, designed for use with oil immersion. The objective is compatible with differential interference contrast (DIC) microscopy, and it features an M27 thread mount.
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Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Micro well, by MatTek (2 mentions)
Evolve 512, by Zeiss (2 mentions)
Axio observer z, by Zeiss (1 mentions)
Axio observer z1 inverted microscope, by Zeiss (1 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 100 1.4 oil ph3 objective, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Zen blue, by Zeiss (1 mentions)
Sp8x wll microscope, by Leica (1 mentions)
4 protocols using alpha plan apo 100 1.46 oil dic m27 objective
1
Live-Cell Imaging of Yeast Foci
2
Spore Morphometry of S. venezuelae Strains
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Lawns of the respective S. venezuelae strains were generated by spreading a single colony onto MYM agar. The plates were incubated for 3–4 days at 30°C until sporulation was completed. Spores were washed off the agar using 20% glycerol and a sterile cotton pad through which spores were harvested using a sterile syringe. A small aliquot of the spore suspension was mounted on a microscope slide on top of a thin agarose pad (1% agarose dissolved in water) and imaged by phase-contrast microscopy using a Zeiss Axio Observer Z.1 inverted microscope and a Zeiss Alpha Plan-Apo 100×/1.46 Oil DIC M27 objective. Spore lengths were determined using the software Fiji (Schindelin et al., 2012 (link)) except for spore length measurements in Figure 7B in which case the MicrobeJ plug-in for Fiji was used (Ducret et al., 2016 (link)).
3
Membrane Staining and Sporulation Visualization
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For membrane staining, Streptomyces hyphae were incubated with 0.5 mg/ml FM 4-64 Dye (N-(3-Triethylammoniumpropyl)24-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide) (Molecular Probes) for 15 min in the dark. Hyphae were then directly spotted onto a 1% agarose pad and visualized using a Zeiss Axio Observer Z1 microscope using an Alpha Plan-Apo 100×/1.46 Oil DIC M27 objective. Spores of SV55 were loaded into a BA04 microfluidic plate (ONIX, CellASIC) and allowed to germinate and to grow using constant perfusion of MYM containing 5.5 μg/ml FM4-64 at 30 °C. To promote sporulation, MYM/FM4-64 medium was replaced after 3 h of growth by “spent-MYM”/FM4-64. The “spent-MYM” was prepared as described previously29 . Hyphae were visualized using a Zeiss Axio Observer Z1 microscope equipped with a Plan Apochromat 100×/1.4 Oil Ph3 objective. Images were collected and analyzed using Zen Blue (Zeiss) or Fiji28 . We note that FM4-64 staining using the ONIX microfluidics systems is inefficient as the membrane dye appears to bind to the internal plate material. Thus, the majority of images shown in the manuscript were generated using cells immobilized on agarose pads.
4
Live-cell Imaging of Cellular Foci
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Live-cell imaging was essentially performed as described38 (link). In brief, cells were grown overnight on rich medium (YES) to the logarithmic phase. Prior to imaging, cells were attached with lectin (Sigma) to glass-bottom dishes with a micro well (MatTek). Cells were imaged on a Zeiss AxioObserver Z1 confocal spinning disk microscope with an EMM-CCD camera (Photometrics, Evolve 512) through a Zeiss Alpha Plan/Apo ×100/1.46 oil DIC M27 objective lens. Z-stacks were obtained at focus intervals of 0.4 μm. FiJi/ImageJ software was used to measure the distances between the foci and the periphery.
For the imaging of cells expressing CFP-Mmi1, the following setup was used: confocal microscopy was performed at the Core Facility Bioimaging of the Biomedical Center (LMU Munich) with an inverted Leica SP8 X WLL microscope, equipped with 405-nm laser, WLL2 laser (470–670 nm), and acusto-optical beam splitter. Images were acquired with a HC PL APO ×93/1.30 GLYC motCORR-STED WHITE objective, and Z-stacks were obtained at focus intervals of 0.25 μm. Images were deconvolved using the SVI Huygens suite and FiJi/ ImageJ software was used to measure the distances between the foci and the periphery.
For the imaging of cells expressing CFP-Mmi1, the following setup was used: confocal microscopy was performed at the Core Facility Bioimaging of the Biomedical Center (LMU Munich) with an inverted Leica SP8 X WLL microscope, equipped with 405-nm laser, WLL2 laser (470–670 nm), and acusto-optical beam splitter. Images were acquired with a HC PL APO ×93/1.30 GLYC motCORR-STED WHITE objective, and Z-stacks were obtained at focus intervals of 0.25 μm. Images were deconvolved using the SVI Huygens suite and FiJi/ ImageJ software was used to measure the distances between the foci and the periphery.
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