Spinning disc confocal imaging of mitochondria (MT_rk or MT_gk), GFP-Miro2 fusions and ER (ER_rk) in live tobacco epidermal pavement cells was performed using a VisiScope Confocal Cell Explorer under the control of VisiView software (Visitron Systems, GmbH Germany), composed of an IX81 motorised inverted microscope (Olympus, Germany), a CSU-X1 Spinning Disc unit (Yokogawa, Japan), a PlanApo UPlanSApo × 100 (1.4 NA) oil objective (Olympus, Germany) with a Photometrics CoolSNAP HQ2 camera (Roper Scientific, Germany). To achieve dual fluorescent imaging, GFP was excited with a Sapphire 488 nm 70 mW laser and mCherry with a Cobolt Jive 561 nm 70 mW laser. All movies were taken using a temporal resolution of five frames s−1, 100 frames long with a spatial resolution of 0.129 µm pixel−1.
Photometrics coolsnap hq2 camera
Manufactured by Teledyne
Sourced in Germany
The Photometrics CoolSNAP HQ2 camera is a high-performance, cooled digital camera designed for scientific and industrial imaging applications. It features a high-resolution, low-noise imaging sensor that can capture detailed images with high sensitivity and dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer microscope, by Teledyne (2 mentions)
Softworx software, by Cytiva (2 mentions)
Visiscope confocal cell explorer, by Visitron (1 mentions)
Csu x1 spinning disc unit, by Yokogawa (1 mentions)
Visiview software, by Visitron (1 mentions)
Softworx, by Cytiva (1 mentions)
Deltavision core imaging system, by Cytiva (1 mentions)
Metamorph 7, by Molecular Devices (1 mentions)
Prism 7, by GraphPad (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
Zen software, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Deltavision elite, by Cytiva (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Planapo n objective, by Olympus (1 mentions)
9 protocols using photometrics coolsnap hq2 camera
1
Live Imaging of Mitochondria and ER in Plant Cells
2
Imaging Neuronal Dendrite Bundles in C. elegans
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Image stacks were collected on a DeltaVision Core imaging system (Applied Precision) with a UApo 40×/1.35 NA oil-immersion objective (72 hr animals) or a PlanApo 60×/1.42 NA oil-immersion objective (24 hr and 48 hr animals) and a Photometrics CoolSnap HQ2 camera (Roper Scientific). Animals were mounted on an agarose pad with 20–40 mM sodium azide and imaged in yellow (excitation [EX] 513 nm/emission [EM] 559 nm), red (EX 575 nm/EM 632 nm), blue (EX 438 nm/EM 470 nm), and/or green (EX 475 nm/EM 525 nm) channels. To avoid possible complications due to left-right asymmetry, animals were selected for dendrite bundle imaging such that their right-hand side faced the coverslip and only this bundle was imaged.
Deconvolution and analysis of images were performed with Softworx (Applied Precision) and ImageJ (NIH, Bethesda, MD). Maximum-intensity projections were obtained using contiguous optical sections.
Deconvolution and analysis of images were performed with Softworx (Applied Precision) and ImageJ (NIH, Bethesda, MD). Maximum-intensity projections were obtained using contiguous optical sections.
3
Visualizing Localization of Fluorescent Fusion Proteins in MRSA
4
Multichannel Microscopy Imaging Techniques
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Time-lapse movies were acquired using a DeltaVision Elite® microscope (Applied Precision, GE Healthcare, Issaquah, WA, USA) mounted on an inverted IX71 microscope (Olympus, Center Valley, PA, USA) connected to a Photometrics CoolSNAP HQ2 camera (Roper Scientific, Martinsried, Germany). The primary image processing software used was SoftWorX 6.0. This microscope was also used for the production of deconvolution-based 3D image reconstructions, renderings supported by BITPlan software (Willich-Schiefbahn, Germany). Wide-range phase-contrast images and high content/high throughput microscopy was conducted on 96-well plates using a Hermes® microscope (IDEA BioMedical Ltd., Rehovot, Israel) equipped with automated scanning optics, high-precision autofocus, and a closed environmental chamber.
5
Suberin's Antimicrobial Effects on S. aureus and E. coli
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S. aureus NCTC8325 and E. coli TOP 10 cells (5 × 105 cells·mL−1) in Mueller-Hinton broth (MHB) media were exposed to suberin concentrations ranging from 0.25 to 2 μg mL−1 (11 h, 37 °C, without agitation). Suberin was added to the media from a stock solution in DMSO to a final concentration of 2% v/v. Cellular morphology and viability were visualized using light and fluorescence microscopy (with the fluorescent dye propidium iodide), respectively, with an inverted Zeiss Axio Observer microscope equipped with a Photometrics CoolSNAP HQ2 camera (Roper Scientific). Controls of the medium, bacterial growth, and hydrolysate cork monomers (obtained by alkaline hydrolysis) were also carried out. Under the same conditions, the average hydrodynamic diameter (dh), the dispersity (PDI), and the zeta potential (Zp) of each sample were measured using dynamic light scattering (DLS) analysis (triplicates; each in technical triplicate) (see Supplementary Information).
6
Fluorescence-based Bacterial Gene Silencing
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Overnight cultures of strains expressing dcas9-sgfp (from S. pyogenes or S. aureus) with corresponding sgRNA targeting fp650-rodZ (BCBMS10-13; BCBLS05-07) and control strains (BCBMS06-09; BCBLS02-04, lacking dcas9) were back-diluted 1:500 in 10 mL of TSB, with 10 µg/mL of Cm, 50 µg/mL of both Neo and Kan, and 0.1 µM CdCl2 and incubated at 37°C, with aeration, to an OD600 of 0.8. A 1 mL aliquot of culture was pelleted and re-suspended in 30 µL of phosphate-buffered saline (PBS). A 5 µL sample of each dCas9-producing culture expressing a specific sgRNA was mixed with an equal volume of the corresponding control culture lacking dCas9 but expressing the same sgRNA (see experimental setup in Fig. 2B ). One microliter of the mixed culture was placed on a thin layer of 1.2% agarose in PBS and imaged using an Axio Observer.Z1 microscope equipped with a Photometrics CoolSNAP HQ2 camera (Roper Scientific), using phase contrast objective Plan Apo 100×/1.4 oil Ph3. Cells were imaged using the ZEN software (Zeiss). The median fluorescence of sGFP and eqFP650 in each cell was determined using eHooke (68 (link)).
7
Quantitative Kinetochore Fluorescence Imaging
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Fixed-cell images were acquired using a DeltaVision PersonalDV Imaging System (Applied Precision/GE) on a Photometrics CoolSnap HQ2 camera (Roper Scientific) and a 60x/1.42NA Planapochromat DIC oil immersion lens (Olympus). All immunofluorescence images were collected as z-stacks at 0.2 μm intervals. Kinetochore integrated pixel intensity values were measured on deconvolved images with SoftWorx software (Applied Precision) applying background correction. To measure centrosome volume, the cells were stained with PCM1 antibody and then imaged using Deltavision imaging system. The centrosome volume was determined by centrosomal fluorescence intensity from deconvolved 3D images using Velocity software. An arbitrary threshold was applied to remove the particles with nonspecific staining [34 (link)].
8
Bacterial Cell Surface Binding Assay
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To test the ability of the recombinant proteins to bind to the bacterial cell surface, M. smegmatis, M. tuberculosis H37Ra, M. vaccae and M. bovis BCG cells were grown until an optical density at 600 nm (OD600) of 0.8, harvested by centrifugation and washed in SDS 1% in PBS for 1 h, with shaking at room temperature. Cells were then harvested by centrifugation and washed twice with PBS and incubated with 0.3 mg/mL of Ms6LysBPGBD-EGFP or EGFP proteins, for 45 min with shaking at room temperature. Cells were again harvested by centrifugation, washed trice with PBS, and 3 µL were placed on a 1% agar PBS microscope slide. In a parallel assay, M. smegmatis cells were pretreated with 0.3 mg/mL of Ms6 LysB (instead of SDS) for 45 min, with shaking at room temperature, followed by the washing steps describe above. Samples were observed using a Zeiss Axio ObserverZ1 microscope (Zeiss, Oberkochen, Germany) equipped with a Photometrics CoolSNAP HQ2 camera (Roper Scientific, Acton, MA, USA) using Metamorph software, Meta Imaging series 7.5 (Molecular Devices, San José, CA, USA) and analyzed using ImageJ software [29 ].
9
Quantifying DNA Damage Response
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Cells on cover slips were fixed in ice-cold methanol/acetone for 10 min at −20 °C, permeabilized with 0.5% TritonX-100 in PBS for 10 min and incubated in blocking buffer (5% goat serum and 0.3% Tween 20 in PBS) for 30 min. Samples were then incubated with anti-H2AX-pS121 and CREST diluted in blocking buffer for 1 h at room temperature, followed by incubation with anti-rabbit IgG conjugated with Alexa Fluor 488 (Life Technologies) and anti-human IgG conjugated with Alexa Fluor 594 (Life Technologies) secondary antibodies diluted in blocking buffer (1:200) for 1 h at room temperature. Nuclei were counterstained with Hoechst 33342 (1:1,000). Images were acquired with 0.2 μm sections using a DeltaVision Elite (Applied Precision) comprising an Olympus IX71 wide-field inverted fluorescence microscope, with a × 60/1.42 PlanApo N objective (Olympus), an oil-immersion objective and a Photometrics CoolSnap HQ2 camera (Roper Scientific). Images were maximum intensity projections of deconvolved stacks obtained using SoftWoRx software (Applied Precision).
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