Initial imaging of embryos and micro-dissected urogenital tracts was performed on a Leica M205 FA microscope with an ORCA-Flash4.0 V2 digital CMOS camera (Hamamatsu) or a Zeiss M2Bio microscope with a Retiga 4000R-F-M-C camera (Q-Imaging) and accompanying software. Imaging of cryosections was initially performed on a Leica DMI6000 B microscope using SimplePCI version 6.6.0.16 imaging software (Hamamatsu). Confocal microscopy was performed on a Zeiss Scanning Microscope LSM510 using a 633 nm laser for imaging Cy5 (649–745 bandpass filter), 543 nm laser for imaging Cy3 (560–615 bandpass filter), and a 488 nm laser for imaging Venus (YFP, 505–550 bandpass filter) to visualize transgene expression and secondary antibody fluorophores. Images were captured with the Zeiss LSM Image Browser Software (free download, http://www.zeiss.com/microscopy/en_de/website/downloads/lsm-image-browser.html ). Images were then exported from the Image Browser software as .tiff files and assembled in Adobe Photoshop (2014 2.2 release, Adobe Systems Inc.).
Dmi6000b microscope
Manufactured by Hamamatsu Photonics
Sourced in Japan
The DMI6000B is an inverted microscope designed for a wide range of applications. It features a motorized focus drive, a high-performance illumination system, and a large sample stage. The DMI6000B is capable of various imaging techniques, including brightfield, darkfield, and phase contrast.
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Lab products found in correlation
Lsm image browser software, by Zeiss (3 mentions)
Simplepci version 6.6.0.16 imaging software, by Hamamatsu Photonics (3 mentions)
Orca c4742 80 12ag camera, by Hamamatsu Photonics (2 mentions)
Flash 4.0 camera, by Hamamatsu Photonics (2 mentions)
Metamorph, by Molecular Devices (2 mentions)
Sp5 2 scanning confocal microscope, by Leica (2 mentions)
M205fa microscope, by Hamamatsu Photonics (1 mentions)
Orca flash4.0 v2 digital cmos camera, by Hamamatsu Photonics (1 mentions)
Af6000 lx widefield multidimensional microscopy system, by Leica (1 mentions)
Volocity, by Quorum Technologies (1 mentions)
Igepal, by Merck Group (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Slowfade antifade reagent, by Thermo Fisher Scientific (1 mentions)
Mz 12.5 stereomicroscope, by Leica (1 mentions)
8 protocols using dmi6000b microscope
1
Microscopic Imaging of Embryos and Tissues
2
Quantifying Neuronal Cell Growth in Culture
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To study the growth of neuronal cells in the culture without the need of fixing the cells, DIC pictures were acquired at 1 and 3 DIV. 5 pictures (×40 objective) were taken per well with a fluorescence microscope (AF6000 LX Widefield Multidimensional Microscopy System, Leica, Heidelberger, Germany), epifluorescence and transmitted light, consisting of an inverted Leica DMI6000B microscope and a 9100-O2 CCD (Hamamatsu Photonics K.K., Japan) and the software LAS X. Cells with neuronal projections were identified as neuronal cells.
3
Immunofluorescence and 3D Imaging of Nucleoli
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Cells were washed with PBS, fixed in 4% PFA for 15 minutes and permeabilized with 0.5% PBS/Triton for 5 minutes. Incubation with primary antibody was performed for 1 h in PBS-5% BSA and cells were stained with AlexaFluor 488/568 conjugated anti-rabbit or -mouse secondary antibodies (Molecular Probes) and counterstained with DAPI. After mounting in 50% glycerol/50% 0.2 M Na-glycine, 0.3 M NaCl, 3D epifluorescent 3D image stacks were acquired using a Leica DMI6000B microscope equipped with an Orca C4742-80-12AG camera (Hamamatsu) and Volocity (Perkin-Elmer Improvision) and were subsequently deconvoluted (Iterative Restoration, Volocity). In a few cases 3D stacks were also obtained using a Leica SP5 II scanning confocal microscope. Nucleolar statistics were obtained from three independent immunofluorescence experiments in which ∼20 nuclei were analysed by a protocol established using the Volocity software. DAPI staining was used to define the nuclear volume and Fibrillarin staining to define the nucleoli and their individual volumes. 3D Immuno-FISH was performed as previously described [77] (link) using a Cy3 labeled fragment from the mouse rDNA, (positions 20138 to 23651 in Genbank Accession BK000964.3). Colocalization of FISH and protein signals were estimated using Volocity and given by the Pearson Global Correlation [78] .
4
Immunofluorescence Imaging of Nucleolar Proteins
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Cells were cultured on poly-lysine-treated coverslips and after experimental manipulations were fixed in 4% PFA for 10 min and permeabilized with 0.25% PBS/Triton for 15 min. After a blocking step in PBS-N [PBS, 0.1% IGEPAL (Sigma)] and 5% donkey serum, cover slips were incubated with primary antibodies in PBS-N and 5% donkey serum for ∼16 h at 4°C. RPI was generally detected using a combination of anti-A194 and A135 antibodies. Cells were incubated for ∼2 h at room temperature with the appropriate AlexaFluor or Dylight 405/488/568/647 conjugated secondary antibodies (Thermo Fisher/Jackson ImmunoResearch) and counterstained with DAPI or Hoechst 33342. After mounting in Prolong Diamond (Thermo Fisher), epifluorescent 3D image stacks were acquired using a Leica DMI6000B microscope equipped with an Orca C4742-80-12AG camera (Hamamatsu) and Volocity (Quorum Technologies) or using a Leica SP5 II scanning confocal microscope as indicated. 3D image analysis and quantitation were performed with the Volocity software using DAPI or Hoechst staining to define the nuclear volume and fibrillarin staining to define the active nucleolar rDNA volume. The fluorescence background for each image channel was estimated from the average 3D signal of the image stack outside of all nuclei. The Otsu intensity threshold (41 ) was used to determine fluorescence signal significance.
5
Immunofluorescence Microscopy of M. xanthus
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Immunofluorescence microscopy was performed essentially as described53 (link). Briefly, M. xanthus cells were resuspended to a calculated density of 7.0 × 109 cells per ml in PBS from TPM agar plates after 6 h of starvation. Cells were fixed with 1.6% paraformaldehyde and 0.008% glutaraldehyde for 20 min on freshly prepared poly l -lysine-treated 12-well diagnostic slides (Thermo Fischer Scientific). Cells were permeabilized with GTE buffer (50 mM glucose, 20 mM Tris, 10 mM EDTA, pH 7.5) for 10 min, washed and blocked for 30 min at RT. Cells were probed with relevant rabbit polyclonal antibodies at RT for 90 min (α-Oar, dil. 1:2000) and 30 min (α-p17/p25, dil. 1:200). Alexa Fluor 594 goat anti-rabbit IgG (Thermo Fischer Scientific, cat.nr LSA11037) (dil. 1:200) was added as secondary antibody for 1 h after washing away the primary antibody. Secondary antibody was washed away followed by addition of Slow Fade Anti Fade Reagent (Molecular Probes) to each well. For each strain, at least 100 cells were analyzed. Cells were observed using a Leica DMI6000B microscope with a Hamamatsu Flash 4.0 camera. Images were recorded with Leica MM AF software package and processed with Metamorph (Molecular Devices).
6
Microscopic Imaging of M. xanthus
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Five microliters of an exponentially growing M. xanthus culture was spotted on 1.5% agarose pads supplemented with TPM (10 mM Tris-HCl pH 7.6, 1 mM KPO4 pH 7.6, 8 mM MgSO4). Agar pads were incubated for 30 min at 32 °C in a humid chamber before microscopy. Cells were observed using a Leica DMI6000B microscope with a Hamamatsu Flash 4.0 camera. Images were recorded with Leica MM AF software package and processed with Metamorph (Molecular Devices). For each strain, at least 100 cells were analyzed.
7
Microscopy Imaging of Embryos and Bladders
8
Multimodal Microscopy for Embryo Imaging
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