bx81 microscope, by Olympus - Product details

1

Microscopic Imaging of Bacterial Cells

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We used either (i) an Olympus BX81 microscope equipped with a 100x/1.40 Oil UPLSAPO100XO objective and an Orca-ER camera (Hamamatsu) or (ii) a Delta Vision Elite (GE Healthcare, Applied Precision) Olympus IX71 microscope with a CoolSnap HQ2 CCD camera (Photometrics), a 100x/1.40 Oil PSF objective (U-PLAN S-APO 100x Oil, 0.12 WD) and equipped with a four color standard set Insight SSITM illumination module. Mgryph cells were spotted onto a 1% “Mgryph agarose pad” as per Toro-Nahuelpan et al (2016), whereas R. rubrum was spotted onto a 1% PBS (NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 10 mM, KH₂PO₄ 1.8 mM, pH 7.3) agarose pad. Imaging was performed at room temperature (25ºC, in the Olympus BX81 microscope) or at 30ºC (Delta Vision Elite microscope).
When stated, deconvolution was carried out from optical sections using the softWoRx software (version 6.1.1) and the Ratio (conservative) method (GE Healthcare, Applied Precision) or Deconvolution of Z-stacks taken with the Olympus BX81 microscope was done with the deconvolution plug in of the CellM software package (Olympus) using “no neighbor” filtering and appropriate channel settings such as emission wavelength and refractive index used.

Toro-Nahuelpan M., Giacomelli G., Raschdorf O., Borg S., Plitzko J.M., Bramkamp M., Schüler D, & Müller F.D. (2019). MamY is a membrane-bound protein that aligns magnetosomes and the motility axis of helical magnetotactic bacteria. Nature microbiology, 4(11), 1978-1989.

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Microscopic Imaging of Bacterial Cells

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We used either (i) an Olympus BX81 microscope equipped with a 100x/1.40 Oil UPLSAPO100XO objective and an Orca-ER camera (Hamamatsu) or (ii) a Delta Vision Elite (GE Healthcare, Applied Precision) Olympus IX71 microscope with a CoolSnap HQ2 CCD camera (Photometrics), a 100x/1.40 Oil PSF objective (U-PLAN S-APO 100x Oil, 0.12 WD) and equipped with a four color standard set Insight SSITM illumination module. Mgryph cells were spotted onto a 1% “Mgryph agarose pad” as per Toro-Nahuelpan et al (2016), whereas R. rubrum was spotted onto a 1% PBS (NaCl 137 mM, KCl 2.7 mM, Na₂HPO₄ 10 mM, KH₂PO₄ 1.8 mM, pH 7.3) agarose pad. Imaging was performed at room temperature (25ºC, in the Olympus BX81 microscope) or at 30ºC (Delta Vision Elite microscope).
When stated, deconvolution was carried out from optical sections using the softWoRx software (version 6.1.1) and the Ratio (conservative) method (GE Healthcare, Applied Precision) or Deconvolution of Z-stacks taken with the Olympus BX81 microscope was done with the deconvolution plug in of the CellM software package (Olympus) using “no neighbor” filtering and appropriate channel settings such as emission wavelength and refractive index used.

Toro-Nahuelpan M., Giacomelli G., Raschdorf O., Borg S., Plitzko J.M., Bramkamp M., Schüler D, & Müller F.D. (2019). MamY is a membrane-bound protein that aligns magnetosomes and the motility axis of helical magnetotactic bacteria. Nature microbiology, 4(11), 1978-1989.

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3

Subcellular Localization Microscopy of CheW₁-EGFP and MamC-GBP Fusions

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Strains with genomic CheW₁-EGFP fusions and additional MamC-GBP fusions were grown in 1 ml FSM in 24-well plates for 16 h at 30°C and 1% O₂ without agitation. For microscopy, cells were immobilized on agarose pads (phosphate-buffered saline [PBS] buffer supplemented with 1% agarose) and imaged with an Olympus BX81 microscope equipped with a 100 UPLSAPO100XO objective (numerical aperture of 1.40) and a Hamamatsu Orca AG camera. The Olympus xcellence pro software was used to capture and analyze images.
To analyze relative positions of fluorescent foci, we manually segmented each cell along its long axis into four equal sectors and scored the fluorescent foci within each sector. The strongest fluorescence signal(s) was scored as “++,” and weaker signals were scored as “+.” Since the orientation of imaged cells was random and in many cases the distribution of fluorescent foci was not perfectly symmetric, we rotated the cells where necessary so that the sectors with the highest cumulated score were sectors 1 and 2. We then calculated relative frequencies of fluorescent focus positions based on the ratio of cumulated scoring points of all analyzed cells per sector divided by the total number of scoring points in all cells.

Borg S., Popp F., Hofmann J., Leonhardt H., Rothbauer U, & Schüler D. (2015). An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria. mBio, 6(1), e02117-14.

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4

Immunofluorescence Analysis of Taste Receptors

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hPASMCs, grown on coverslips, were processed for immunofluorescence analysis as described previously [15] (link). The cells were treated with primary antibodies for T2R1, T2R38 and the isotype-specific IgG sera (diluted in blocking solution at 1∶300) for 1 h at room temperature and visualized with Alexa fluorophores. Representative cells were selected and visualized using Olympus BX81 microscope for the localization of indicated proteins.

Upadhyaya J.D., Singh N., Sikarwar A.S., Chakraborty R., Pydi S.P., Bhullar R.P., Dakshinamurti S, & Chelikani P. (2014). Dextromethorphan Mediated Bitter Taste Receptor Activation in the Pulmonary Circuit Causes Vasoconstriction. PLoS ONE, 9(10), e110373.

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5

Fluorescent Protein Fusion Microscopy

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To image fluorescent protein fusions, 3 µl of cell suspension were immobilized on MSR agarose pads (7 (link)) and covered with a coverslip. For fluorescent staining, 20 µl of cell suspension was mixed with either 10 µl of a Nile red solution (0.5 µg/ml in dimethyl sulfoxide [DMSO]) or 6 µl of MM4-64 (an FM4-64 derivative [16 µM in DMSO]) and/or 10 µl of a DAPI solution (50 µg/ml). Conventional epifluorescence microscopy was performed on an Olympus BX81 microscope equipped with a 100× UPLSAPO100×O objective (NA1.4) and an Orca-ER camera (Hamamatsu). Time-lapse imaging and fluorescence recovery after photobleaching (FRAP) were performed on a Deltavision Elite system (GE Healthcare) equipped with a U-Plan S-Apo 100× oil PSF objective (NA1.4) and a CoolSnap HQ2 charge-coupled device (CCD) camera as described previously (7 (link)). Additional time-lapse series were acquired on a Nikon Eclipse Ti2-E microscope equipped with a CFI SR Apo TIRF AC 100×H oil objective (NA1.49) and Retiga R1 CCD camera (QImaging). Further methodological details with respect to image deconvolution and the different systems used for epifluorescence microscopy, FRAP, and time-lapse imaging are given in Text S1.

Pfeiffer D., Toro-Nahuelpan M., Bramkamp M., Plitzko J.M, & Schüler D. (2019). The Polar Organizing Protein PopZ Is Fundamental for Proper Cell Division and Segregation of Cellular Content in Magnetospirillum gryphiswaldense. mBio, 10(2), e02716-18.

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Microscopy Techniques for M. gryphiswaldense

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For fluorescence microscopy, 3 µl samples of M. gryphiswaldense over-night cultures were immobilized on 1% (w/v) agarose pads with FSM medium salts. The samples were imaged with an BX81 microscope (Olympus, Japan) equipped with a 100×UPLSAPO100XO 1.4NA objective and an Orca-ER camera (Hamamatsu, Japan) and appropriate filer sets using Olympus Xcellence software. For transmission electron microscopy (TEM), unstained formaldehyde-fixed (0.075% w/v) M. gryphiswaldense cells were absorbed on carbon coated copper grids. Bright field TEM was performed on a Phillips (Netherlands) CM200 instrument using an accelerating voltage of 160 kV. Images were captured with an Eagle 4k CCD camera using EMMenu 4.0 (Tietz, Germany).

Raschdorf O., Bonn F., Zeytuni N., Zarivach R., Becher D, & Schüler D. (2018). A quantitative assessment of the membrane-integral sub-proteome of a bacterial magnetic organelle. Journal of proteomics, 172.

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7

Fluorescence Microscopy of Magnetosome Expression

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For fluorescence microscopy, the plasmid pBam-Tet-mamD-GFP was transferred into different MSR-1 strains by conjugation. Exconjugants were screened for successful transposition of the mini-Tn5 transposon by PCR and subjected to wide-field epi-fluorescence microscopy. Four μl samples of MSR-1 overnight cultures were immobilized on 1% (w/v) agarose pads with FSM medium salts. The samples were imaged with an Olympus BX81 microscope equipped with a 100×UPLSAPO100XO 1.4NA objective and an Orca-ER camera (Hamamatsu) and appropriate filer sets using Olympus Xcellence software. All samples were recorded in Z-stacks with 500 ms exposure time per image. Images were processed with the ImageJ Fiji package (Schindelin et al., 2012) .

Uebe R., Keren-Khadmy N., Zeytuni N., Katzmann E., Navon Y., Davidov G., Bitton R., Plitzko J.M., Schüler D, & Zarivach R. (2018). The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization. Molecular microbiology, 107(4).

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Bx81 microscope

Lab products found in correlation

7 protocols using bx81 microscope

Microscopic Imaging of Bacterial Cells

Microscopic Imaging of Bacterial Cells

Subcellular Localization Microscopy of CheW₁-EGFP and MamC-GBP Fusions

Immunofluorescence Analysis of Taste Receptors

Fluorescent Protein Fusion Microscopy

Microscopy Techniques for M. gryphiswaldense

Fluorescence Microscopy of Magnetosome Expression

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