M. smegmatis cells expressing RpoB-GFP were grown overnight from frozen 100 μL aliquots in 10 mL of fresh standard ADC medium. The bacteria were subcultured once and allowed to reach midlog phase (OD600 ∼0.5 to 0.7). The culture was then filtered to remove aggregates of bacteria and loaded into a custom polydimethylsiloxane (PDMS) microfluidic device as previously described (54 (link)). Fresh medium was delivered to cells using a microfluidic syringe pump. The microfluidics device was attached to a custom PDMS mixing device for delivery of drug for the duration of time described below and then placed on an automated microscope stage inside an environmental chamber that was maintained at 37 °C. The bacteria were imaged for a total of 26 h using a widefield DeltaVision PersonalDV (Applied Precision, Inc.) with a hardware-based autofocus. Antibacterial compounds were introduced to the M. smegmatis after a 10-h growth phase. Drug treatment lasted for 6 h and was followed by a 10-h recovery phase.
Deltavision personaldv
Manufactured by Cytiva
The DeltaVision PersonalDV is a high-performance microscope system designed for advanced fluorescence imaging. It features a wide range of optical capabilities and integrated software, enabling researchers to capture high-quality images and conduct detailed analysis of cellular structures and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 with airyscan system, by Zeiss (2 mentions)
Plan apo 60x 1.42 na, by Cytiva (2 mentions)
Uplansapo 100x 1.4 na, by Cytiva (2 mentions)
Hcx pl apo cs 63.0x 1 40 na oil lens, by Leica camera (2 mentions)
Fret ab wizard, by Leica camera (2 mentions)
Plan apochromat 63 1.40 oil dic m27 objective, by Zeiss (2 mentions)
Tcs sp5 confocal microscope, by Leica camera (2 mentions)
Uplanfl objective, by Olympus (1 mentions)
Softworx acquisition software, by Cytiva (1 mentions)
H 7000fa, by Hitachi (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Glass bottom 24 well plate, by MatTek (1 mentions)
9 protocols using deltavision personaldv
1
Real-time Microscopy of Antibiotic Effects
2
Immunostaining of SNAP-tagged H3.3 in MEFs
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MEFs were transfected with H3.3-SNAP and treated 24 h later as described in Supplemental Figure 3 (Ray-Gallet et al. 2011 (link)). After treatment, cells were processed for immunostaining with anti-PML or anti-SNAP antibodies and analyzed using the same acquisition parameters. Immunostaining was done after cell fixation with 3% paraformaldehyde and permeabilization with 0.1% Triton X-100/0.01% Tween 20/2% BSA. Images were captured at 100×/1.4 NA on a DeltaVision personalDV (Applied Precision) and analyzed using ImageJ 1.48v (National Institutes of Health).
3
Multimodal Microscopy Techniques for DNA Repair
4
Live-cell Imaging of Transfected BHK Cells
5
Multimodal Microscopy Techniques for DNA Repair
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Panel 1C was acquired with a ZEISS LSM 880 with Airyscan system equipped with a Plan-Apochromat 63×/1.40 Oil DIC M27 objective and using 488 and 561 nm wavelength lasers. Panels 2A,B S3A, S6A and S7B were acquired with a DeltaVision PersonalDV (Applied Precision) with either a Plan Apo 60x/1.42 NA or a UPlanSApo 100x/1.4 NA oil lenses. Panels 2F, 3B,H, S3B,C, S5 and S7A were acquired on a Leica TCS SP5 confocal microscope equipped with a HCX PL APO CS 63.0x/1.40 NA oil lens. FRET experiments were performed on HeLa cells using the Leica FRET AB wizard. Ku70(AF488)/Ku80(488 DyLight)/DNA-PKcs(488 DyLight) served as donors and LMNA (AF594 for Ku80 and DNA-PKcs FRET; AF555 for Ku70 FRET) as acceptor. Panels 3C,F were acquired with a OMX Structured Illumination Super-resolution Scope equipped with a PlanApo 60x/1.40 Oil DIC objective and using 488 and 568 nm wavelength lasers.
6
Imaging Live and Fixed Cells with DeltaVision DV System
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Cells were imaged with a DeltaVision Personal DV (Applied Precision) imaging system equipped with a 40×/0.75 numerical aperture Ph UPlanFL objective (Olympus) and a CoolSNAP HQ2 (Photometrics/Roper Scientific) camera. The system was controlled with softWoRx acquisition software (Applied Precision). Images for fixed-cell experiments (Figure 3, B, C, and E ) were acquired as z-stacks with 200-nm step sizes. For live-cell experiments, (Figures 1 , 2 , 3D , 4 , and 5 ), cells were maintained at 37°C using an environmental chamber (Precision Control, Seattle, WA), and a single z-plane was acquired every 3 or 5 min, depending on the experiment.
7
Fluorescent Ebola VLP Characterization
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Fluorescent VLPs were stained negatively with 2% Phosphotungstic acid (PTA), and the morphology of VLPs was examined by electron microscope (Hitachi, H-7000FA, Japan). The fluorescent VLPs were put into a 2 cm-plate with glass bottom and dried, then fixed by 4% paraformaldehyde at room temperature for 15 min. After washing with phosphate buffered saline (PBS), the fluorescent VLPs were cultured with mouse anti-GP monoclonal antibodies (produced in our lab) at 4 °C overnight. After reactions with the Alexa-555 labeled rabbit anti-mouse second antibody (Life Sciences), the VLPs were examined under the fluorescence microscope Delta Vision personal DV (Applied Precision) to identify the co-localization of immunofluorescence stained by anti-GP antibody (red) and inherent fluorescence of VLPs (Green) to confirm the composition of the fluorescent Ebola-VLPs.
8
Quantifying Adipocyte Lipid Content
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Cells were differentiated for 15 days on 12-mm diameter coverslips in 24-well plates. Cells were washed 3 times with PBS before fixation in 4% paraformaldehyde for 10 min. Cells were then incubated in Bodipy (1µg/ml, Invitrogen D3922) for 15 min and washed 3 times in PBS before mounting in DAKO Fluorescence Mounting Medium (S3023, Agilent). Images were acquired on an IX81 microscope (Olympus) fitted with epifluorescence, an 100× 1.4 NA objective mounted on a piezo drive, and a DeltaVision personalDV (Applied Precision, Ltd.) imaging station. The Bodipy surface per field was quantified on threshold-adjusted images using ImageJ software.
9
Time-lapse Imaging of Bacterial Cultures
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