Time-lapse microscopy of E. coli MG1655 (wild-type) and ΔaceK strains expressing YFP (Supplementary Table 4 ) was performed with minor modifications51 (link). In brief, bacteria were grown to mid-log phase (OD600 ∼0.3) in M9 liquid medium containing 2 g l−1 glucose and 2 μl of culture were spread on a semipermeable membrane, which was then inverted onto a glass coverslip micro-patterned with rings of SU-8 (65 μm diameter × 1.5 μm height) containing a central post (10 μm diameter × 1.5 μm height). The bacteria-free side of the membrane was overlaid with a PDMS-based microfluidic device and the assembly was clamped between a transparent acrylic cover and base adapter, as described51 (link). The inlet and outlet ports of the microfluidic device were connected to plastic tubing and bacteria were fed by continuous flow of M9 minimal medium containing 2 g l−1 glucose or acetate (flow rate, 25 μl min−1). Bacteria were maintained at 37 °C in a microscope-fitted environmental chamber and imaged using a DeltaVision personalDV microscope (Applied Precision) equipped with a 100 × oil-immersion objective. Images were recorded at 5-min intervals on phase-contrast and fluorescence channels (GFP, excitation filter 490/20/, emission filter 528/38) using a CoolSnap HQ2 camera. Images were processed using Softworx software (Applied Precision) and custom-made macros in ImageJ.
Deltavision personaldv microscope
Manufactured by Cytiva
Sourced in United States
The DeltaVision personalDV microscope is a high-performance imaging system designed for advanced cellular and molecular research. It features a versatile optical configuration, enabling user-friendly and reliable imaging of a wide range of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Softworx software, by Cytiva (3 mentions)
Softworx software version 5, by Cytiva (3 mentions)
Softworx, by Cytiva (2 mentions)
Coolsnap hq, by Olympus (2 mentions)
Uplansapo 1.40 oil objective, by Olympus (2 mentions)
Volocity 6, by PerkinElmer (1 mentions)
Prolong live antifade reagent, by Thermo Fisher Scientific (1 mentions)
35mm glass bottom μ dishes, by Ibidi (1 mentions)
Ultimate focus system, by Cytiva (1 mentions)
α myc, by Santa Cruz Biotechnology (1 mentions)
12 protocols using deltavision personaldv microscope
1
Time-lapse Microscopy of E. coli Mutants
2
Quantitative Imaging of DNA Repair Foci
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High-resolution imaging was performed by imaging z-stacks containing the whole-cell nucleus with a wide-field Deltavision PersonalDV microscope (Applied Precision, 1024 × 1024 CoolSNAP HQ or HQ2 camera, z-stack of 0.2 mm interval) equipped with a 60× UPlanSApo/1.40 oil objective (Olympus). Deconvolutions were then performed with SoftWoRx (Applied Precision) in a conservative mode. KU80 and GFP-XRCC4 foci were counted by using the find object tool in Volocity 6.3 (Perkin Elmer).
3
Wide-field Fluorescence Microscopy of TADA-Labeled Cells
4
Live-cell Imaging of T. cruzi Infection
5
High-Resolution Bacterial Cell Imaging
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Bacterial cells were mounted on 1% agar pads and imaged with a DeltaVision Personal DV microscope followed by deconvolution using SoftwoRx software (Applied Precision). Further processing was performed using FIJI software (67 (link)). Image brightness and contrast were adjusted for visibility, and the files were converted to 600 dots per inch (dpi). Representative cells are shown from multiple images of each strain.
6
EGFP-V12C Binding Activity in Bacteria
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Cell wall binding activity of EGFP-V12C was observed by fluorescence microscopy (Loessner et al., 2002 (link)). Briefly, bacteria were harvested with centrifugation (12 000 g, 5 min), then washed and re-suspended with 1 × PBS (pH 7.4) for two times and finally re-suspended in 1 × PBS to a concentration of about 1 × 107 CFU ml−1. After mixing 200 μl of the cell solution with 100 μl of 2 μM EGFP–V12C in 1 × PBS buffer, the mixture was incubated at 37°C for 30 min. As a control, the cells in another tube were mixed with EGFP and incubated at 37°C for 30 min. After the incubation, the cells were separated from the supernatants by centrifugation (12 000 g, 5 min), washed three times with PBST (1 × PBS with 0.5% tween-20) buffer and re-suspended in 1 × PBS before fluorescence microscopy. The fluorescence images were obtained using a Delta Vision Personal DV microscope (Applied Precision, USA) with a 60× magnification oil-immersion objective lens. The exposure time used was set at 0.2 s when capturing all the fluorescence images. Individual images were obtained using suitable filter settings, and colour channels were processed using the image processing software installed with the microscope. In the experiments, EGFP was incubated with the cells of the individual strains, respectively, as the negative control.
7
Time-lapse Optical Microscopy of Bacteria
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For time-lapse optical microscopy, bacteria were cultured in a custom-made microfluidic device with a continuous flow of 7H9 medium6 (link),7 (link). Bacteria were imaged with a DeltaVision personalDV microscope (Applied Precision) equipped with a 100× oil immersion objective and an environmental chamber maintained at 37 °C (30 °C for M. marinum). Images were recorded on phase-contrast and fluorescence channels (490/20-nm excitation filter and 528/38-nm emission filter for GFP, 575/25-nm excitation filter and 632/60-nm emission filter for mCherry) with a CoolSnap HQ2 camera.
8
High-Resolution Imaging of Cell Nuclei
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High-resolution pictures were acquired by imaging z-stacks containing the whole-cell nucleus with a wide-field Deltavision PersonalDV microscope (Applied Precision, 1,024 × 1,024 CoolSNAP HQ or HQ2 camera, z-stack of 0.2 μm interval) equipped with a × 100 UPlanSApo/1.40 oil objective (Olympus). Deconvolutions were then performed with SoftWoRx (Applied Precision) in conservative mode. On all pictures in the manuscript, the white scale bars correspond to 10 μm. For RPA70 and P-RPAS4/S8 staining, micrographs correspond to the projection of the maximum intensity of two adjacent slices, while for other staining they correspond to a single slice.
9
Meiotic chromosome analysis in Arabidopsis
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Chromosome spreads of Arabidopsis pollen mother cells was prepared using fixed buds and DAPI-stained, as described100 (link). Pachytene cells were immunostained for ASY1 and ZYP1, and diakinesis cells were immunostained for MLH1, using fixed buds, as described100 (link),101 . Leptotene-stage meiocytes were immunostained for ASY1 and RAD51 using fresh buds, as described102 (link). The following antibodies were used: α-ASY1 (rat, 1:200 or 1:500 dilution), α-ZYP1 (rabbit, 1:200 dilution), α-MLH1 (rabbit, 1:200 dilution) and α-RAD51 (rabbit, 1:300 dilution)100 (link),102 (link),103 (link). Microscopy was performed using a DeltaVision personal DV microscope (Applied precision/GE Healthcare) equipped with a CDD Coolsnap HQ2 camera (Photometrics). Image capture was carried out using SoftWoRx software version 5.5 (Applied precision/GE Healthcare). For ASY1 and RAD51 co-immunostaining of leptotene-stage nuclei, individual cell images were acquired as Z-stacks of 10 optical sections of 0.2 μM each, and the maximum intensity projection for each cell was decided using ImageJ. Number of MLH1 foci per meiotic cell and RAD51 foci per cell associated with the axis protein ASY1 were manually scored. Wilcoxon tests were used to assess significant differences between wild type and hcr1-1 MLH1 and RAD51 foci counts.
10
Meiotic chromosome analysis in Arabidopsis
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