Generally, cells were mounted on glass slides or agarose pads and observed under a Nikon E800 phase contrast/fluorescence microscope equipped with Texas Red and FITC filter sets and ×100 and ×60 oil objective lenses. Some microscopy was done with a Zeiss Axio Imager Z2 epifluorescence microscope equipped with a Hamamatsu Orca-Flash4.0 sCMOS camera, 46HE and 63HE filter sets, and a ×100 oil objective lens. Time-lapse microscopy was done with live cells mounted on an agarose pad using the Zeiss microscope equipped with automated autofocus. Time series were processed with ZEN imaging software (Zeiss). FRAP assays were performed with confocal illumination provided by an LMM5 laser launch (Spectral Applied Research). Confocal images were acquired on an Olympus IX-81 microscope equipped with a ×100 oil objective lens, a Yokogawa spinning disk CSU-X1, and an ORCA-Flash 4.0 sCMOS camera. The supply and integration of all imaging components were carried out by Biovision Technologies.
Axio imager z2 epifluorescence microscope
Manufactured by Hamamatsu Photonics
The Axio Imager Z2 is an epifluorescence microscope manufactured by Hamamatsu Photonics. It is designed for high-resolution imaging of fluorescently labeled samples. The microscope features motorized focusing, an automated stage, and multiple illumination options for various fluorescence applications.
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Lab products found in correlation
Orca flash 4.0 scmos camera, by Hamamatsu Photonics (4 mentions)
Plan apochromat 100x 1.46 oil ph3 objective, by Hamamatsu Photonics (2 mentions)
Filter sets 38he, by Zeiss (2 mentions)
E800 phase contrast fluorescence microscope, by Nikon (1 mentions)
Csu x1 spinning disk, by Yokogawa (1 mentions)
Zen imaging software, by Zeiss (1 mentions)
Ix81 microscope, by Yokogawa (1 mentions)
Rat monoclonal anti ha antibody clone 3f10, by Roche (1 mentions)
Zen software, by Zeiss (1 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (1 mentions)
6 protocols using axio imager z2 epifluorescence microscope
1
Live Cell Microscopy Techniques
2
Immunofluorescence Analysis of Apicoplast Markers
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For IFA, intracellular tachyzoites grown on coverslips containing HFF monolayers were fixed for 20 min with 4% (w/v) paraformaldehyde in PBS, permeabilised for 10 min with 0.3% Triton X-100 in PBS and blocked with 0.1% (w/v) BSA in PBS. Primary antibodies used for detection of the the apicoplast were mouse monoclonal anti-ATRX1 (1/1000, kind gift of Peter Bradley, UCLA, USA) and rabbit anti-TgCPN60 (1/2000) [50 (link)]. Rat monoclonal anti-HA antibody (clone 3F10, Roche) was used at 1/500 to detect HA-tagged TgPROP1 and TgPROP2 and mouse anti-Ty [47 (link)] to detect Ty-tagged TgPROP1. Staining of DNA was performed on fixed cells incubated for 5 min in a 1 μg/ml DAPI solution. All images were acquired at the “Montpellier Ressources imagerie” facility from a Zeiss AXIO Imager Z2 epifluorescence microscope equipped with a Camera ORCA-flash 4.0 camera (Hamamatsu) and driven by the ZEN software. Adjustments for brightness and contrast were applied uniformly on the entire image.
3
Immunofluorescence Assay of Toxoplasma Gondii
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For IFAs, intracellular tachyzoites grown on a monolayer of HFFs and incubated in the presence or absence of ATc for various periods of time were fixed for 20 min with 4% (wt/vol) paraformaldehyde in PBS, permeabilized for 10 min with 0.3% Triton X-100 in PBS, and blocked with 0.1% (wt/vol) BSA in PBS. The primary antibodies used for detection were anti-TgATRX1 (1/1,000), anti-mitochondrial F1β ATPase (1/1,000) (P. Bradley, unpublished data), anti-TgCPN60 (28 (link)) (1/2,000), anti-TgATG8 (26 (link)) (1/1,000), anti-AMA1 (56 (link)) (1/10,000), anti-TgIMC1 (36 (link)) (1/1,000), anti-GRA3 (55 (link)) (1/500), anti-RON4 (57 (link)) (1/500), and anti-centrin1 (1/500) (I. Cheeseman, unpublished data) antibodies. Staining of both nucleus and apicoplast DNAs was performed with fixed cells incubated for 5 min in a 1-µg/ml DAPI solution. All images were acquired at the Montpellier RIO Imaging facility from a Zeiss Axio Imager Z2 epifluorescence microscope equipped with an ORCA-flash 4.0 camera (Hamamatsu) and driven by the ZEN software (Zeiss). Adjustments for brightness and contrast were applied uniformly to the entire image. For quantification, at least three independent replicates were used.
4
Visualizing Bacterial Polar Protein Localization
5
Time-lapse Imaging of G. obscuriglobus
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G. obscuriglobus cells were grown for 4 days at 28 °C in PYGV liquid medium with 100 μg mL−1 of terbinafine or without. 1 mL of each culture was pelleted, washed once in phosphate-buffered saline, and resuspended in sterile ddH2O. This suspension was spotted onto a 1% agarose-PYGV pad on a glass slide. The pad contained 100 μg mL−1 terbinafine for the treated cells. The pad was covered with a glass coverslip and sealed with a mixture of equal amount of petroleum jelly, lanolin, and paraffin wax (VALAP), leaving enough empty space around the agarose pad to provide the cells with sufficient oxygen. A single field of cells was imaged every 30 min during 14 h on a Zeiss Axio Imager Z2 epifluorescence microscope equipped with a Hamamatsu Orca-Flash4.0 sCMOS camera and a Plan-Apochromat 100×/1.46 Oil Ph3 objective. The images were collected in the Zen Blue software and processed using ImageJ and the EBImage package in Program R26 (link).
6
Visualizing Bacterial Polar Protein Localization
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Cells were immobilized on a 1% agarose pads. Live-cell imaging was performed using a Zeiss Axio Imager Z2 epifluorescence microscope equipped with a Hamamatsu Orca-Flash4.0 sCMOS camera and a Plan-Apochromat 100x/1.46 Oil Ph3 objective. Zeiss Filter sets 38HE and 63HE were used to acquire fluorescent images of msfGFP and mCherry, respectively. Images were collected and processed with Zen Blue software. For image panels, fluorescent signal from the YhjH-mChy-PopZ fusion (red) and GFP (green), or the YhjH-mChy-PopZ fusion (red) and BODIPY signal (yellow) are overlayed on a phase contrast image (grayscale). Every fluorescence microscopy experiment was repeated multiple times (as indicated in the figure legend) under the conditions described, which provided similar results. The figures show data from a single trial.
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