Wide-field microscopy was performed on a Zeiss Axio Observer Z1 microscope equipped with a Hamamatsu OrcaR2 camera and a Plan-Apochromat 100×/1.4 oil Ph3 objective (Zeiss). Fluorescence was visualized with the appropriate filter sets (Zeiss). An environmental chamber set to 30°C was used for time-lapse studies. Images were acquired with ZEN (Zeiss) and processed with Fiji (31 (link)). Final image assembly was conducted with Adobe Photoshop.
Plan apochromat 100 1.4 oil ph3 objective
Manufactured by Zeiss
The Plan-Apochromat 100×/1.4 oil Ph3 objective is a high-performance microscope objective manufactured by Zeiss. It is designed to provide excellent optical performance with a magnification of 100× and a numerical aperture of 1.4. The objective is optimized for use with oil immersion, and it includes a phase contrast (Ph3) functionality.
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Lab products found in correlation
Axiocam icc3, by Zeiss (2 mentions)
Hxp 120 c lightning unit, by Zeiss (2 mentions)
Sytox orange, by Thermo Fisher Scientific (2 mentions)
Axio imager m2 microscope, by Zeiss (2 mentions)
Orcar2 camera, by Zeiss (1 mentions)
Axio observer z1 microscope, by Hamamatsu Photonics (1 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Fm4 64 dye, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Zen blue, by Zeiss (1 mentions)
Alpha plan apo 100 1.46 oil dic m27 objective, by Zeiss (1 mentions)
Nile red, by Thermo Fisher Scientific (1 mentions)
Metamorph 7, by Molecular Devices (1 mentions)
Axio observer microscope, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
5 protocols using plan apochromat 100 1.4 oil ph3 objective
1
Wide-Field Microscopy for Time-Lapse Imaging
2
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.
3
Visualizing NLC-Bacteria Interactions
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Interaction of NLCs with bacteria was observed by wide-field fluorescence microscopy using a Zeiss Axio Observer microscope with a Plan-Apochromat 100×/1.4 oil Ph3 objective. Images were acquired with a Retiga R1 CCD camera (QImaging) using Metamorph 7.5 software (Molecular Devices, San Jose, CA, USA). Bacteria were visualized using membrane stain Nile Red (5 µg mL−1, Invitrogen) and DNA stain Hoechst 33342 (1 µg mL−1, Invitrogen). NLC particles were labelled with DiO. Bacteria were grown to mid-exponential phase (OD600nm 0.6–0.8), and labelled NLCs were added to 1 mL of culture and incubated for 30 min–3 h. Cells were then collected by centrifugation, washed with PBS and membranes and DNA labelled for 5 min at 37 °C. Cells were washed and placed on a thin layer of agarose on a microscopy slide for visualization.
4
Fluorescence Microscopy of C. glutamicum Strains
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Prior to analysis by fluorescence microscopy, the different C. glutamicum strains were grown overnight at 30°C and 170 rpm in BHI medium, diluted 1:50 to inoculate a main culture in CGXII medium with 2% (wt/vol) glucose, and incubated for 3–4 h under the same conditions. For membrane staining, cells were harvested by centrifugation (5 min, 4000 × g) and resuspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) containing 250 ng ml−1 Nile red followed by 10-min incubation in the dark. For DNA staining, 500 nM SYTOX Orange (Thermo Fisher Scientific, Waltham, MA, USA) was added, and the cultures were further incubated for 10 min in the same cultivation conditions. Afterwards, cells were collected by centrifugation at 5,000 × g, washed once, and resuspended in PBS. Afterwards, cells were immobilized on glass slides with agar pads [0.9% (wt/vol) NaCl, 1.5% (wt/vol) agarose] and analyzed using an Axio imager M2 microscope equipped with AxioCam ICc 3 and a Zeiss Plan-Apochromat 100×/1.4 oil Ph3 objective and an HXP 120 C lightning unit (Carl Zeiss). For detection of mVenus fluorescence, the filter 46 HE (λex 500/25 nm, λem 535/30 nm) and, in case of mCherry, Nile red, or SYTOX Orange, the filter 43 HE (λex 545/25 nm, λem 605/70 nm) were used. Image processing took place using the AxioVision SE64 Rel. software v. 4.8.2 (Carl Zeiss).
5
Fluorescence Microscopy of C. glutamicum Strains
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Prior to analysis by fluorescence microscopy, the different C. glutamicum strains were grown overnight at 30°C and 170 rpm in BHI medium, diluted 1:50 to inoculate a main culture in CGXII medium with 2% (wt/vol) glucose, and incubated for 3–4 h under the same conditions. For membrane staining, cells were harvested by centrifugation (5 min, 4000 × g) and resuspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) containing 250 ng ml−1 Nile red followed by 10-min incubation in the dark. For DNA staining, 500 nM SYTOX Orange (Thermo Fisher Scientific, Waltham, MA, USA) was added, and the cultures were further incubated for 10 min in the same cultivation conditions. Afterwards, cells were collected by centrifugation at 5,000 × g, washed once, and resuspended in PBS. Afterwards, cells were immobilized on glass slides with agar pads [0.9% (wt/vol) NaCl, 1.5% (wt/vol) agarose] and analyzed using an Axio imager M2 microscope equipped with AxioCam ICc 3 and a Zeiss Plan-Apochromat 100×/1.4 oil Ph3 objective and an HXP 120 C lightning unit (Carl Zeiss). For detection of mVenus fluorescence, the filter 46 HE (λex 500/25 nm, λem 535/30 nm) and, in case of mCherry, Nile red, or SYTOX Orange, the filter 43 HE (λex 545/25 nm, λem 605/70 nm) were used. Image processing took place using the AxioVision SE64 Rel. software v. 4.8.2 (Carl Zeiss).
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