Cells in time course experiments were washed and resuspended in SDC prior to being imaged on glass slides with glass coverslips. Experiments involving FUN1, DAPI, and Nop1-GFP were imaged on a Nikon Eclipse E600 microscope equipped with a Nikon 100×, 1.4 NA objective and a Photometrics CoolSnap HQ camera run by Metamorph software. Images were taken using 11 stack z-series with a 350 nm step size. The time course imaging of YJB12626 (Hhf1-GFP) was performed on an Olympus DeltaVision Microscope, equipped with an Olympus UPlanSApo 100×, 1.4 NA oil objective and a CoolSNAP ES2-ICX285 camera run by softworX software. Images were taken in a 21-step z-series with a 200 nm step size. Mutants and cells exposed to drugs other than FLC were imaged on a Nikon Eclipse E600 microscope equipped with a Nikon 100×, 1.4 NA objective and a Clara Interline CCD camera, or for heat shock experiments, a Neo 5.5 sCMOS camera (Andor, Belfast, Ireland).
Neo 5.5 scmos camera
Manufactured by Oxford Instruments
Sourced in United Kingdom, United States
The Neo 5.5 sCMOS camera is a scientific-grade camera designed for a variety of imaging applications. It features a 5.5-megapixel sensor with a high-speed, low-noise readout. The camera offers a range of advanced capabilities, including high frame rates, low readout noise, and wide dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
Ti e inverted microscope, by Nikon (6 mentions)
Nis elements software, by Nikon (2 mentions)
Du885 emccd, by Oxford Instruments (2 mentions)
Dichroic and emission filters, by IDEX Corporation (2 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
Deltavision microscope, by Olympus (1 mentions)
Na 1.40 oil immersion objective, by Nikon (1 mentions)
Volocity 6, by PerkinElmer (1 mentions)
Application suite x las x software, by Leica (1 mentions)
Tcs sp8 microscope, by Leica (1 mentions)
Plan apo vc, by Oxford Instruments (1 mentions)
Fm4 64, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Ti e inverted microscope, by Oxford Instruments (1 mentions)
Ti inverted microscope, by Nikon (1 mentions)
Cfi s plan fluor elwd 60, by Nikon (1 mentions)
Sp5x laser scanning confocal microscope, by Leica (1 mentions)
Axiocam mrc digital camera, by Zeiss (1 mentions)
Szx16 stereomicroscope, by Olympus (1 mentions)
Nis elements ar, by Nikon (1 mentions)
19 protocols using neo 5.5 scmos camera
1
Time-course Microscopy of Yeast Cells
2
Cryo-FLM and Cryo-EM Imaging of Bacterial Cells
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Cells of strain JPA1558 were lysed as described above and plunge-frozen on copper EM finder grids (Quantifoil Micro Tools), loaded into a cryo-FLM stage (FEI company) and imaged on a Nikon 90Ti inverted microscope (Nikon Instruments Inc., Melville, NY) using a 60x ELWD air objective lens and a Neo 5.5 sCMOS camera (Andor Technology, South Windsor, USA) using the NIS Elements software (Nikon Instruments Inc., Melville, NY). The grid was then transferred to the cryo-EM and tilt series were recorded from the same cells imaged by FLM previously. The grid was kept below −150°C at all times during the imaging process.
3
Live-Cell Fluorescence Microscopy Imaging
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Cells were imaged on a Nikon Eclipse Ti-E inverted fluorescence microscope with a 100X (NA 1.40) oil-immersion objective (Nikon Instruments Inc., Melville, NY, USA). Images were collected using an Andor DU885 EMCCD or Neo 5.5 sCMOS camera (Andor Technology, South Windsor, CT, USA). Cells were maintained at 37 °C during imaging with an active-control environmental chamber (HaisonTech, Taipei, Taiwan). Images were collected using µManager v. 1.446 .
4
Single-Particle Fluorescence Imaging and Analysis
5
High-throughput Microscopy of Cell Growth
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Stationary-phase cells or cells from cryostocks were diluted 1:10 into 0.85X PBS and then taken from 96-well plates and placed on 1% agarose pads with 0.85X PBS to control for osmolality. Phase-contrast images were acquired with a Ti-E inverted microscope (Nikon Instruments) using a 100X (NA 1.40) oil immersion objective and a Neo 5.5 sCMOS camera (Andor Technology). Images were acquired using μManager v.1.4 [54 ]. High-throughput imaging was accomplished using SLIP, as described previously [37 (link)]. Including sample preparation and calibration, SLIP enables acquisition of 49 images per well of a 96-well plate in ~30 min. Since replicate growth curves appeared similar across the entire library (Additional file 4 : Fig. S2), we imaged one replicate culture for each strain.
6
Confocal and Wide-field Imaging of Fixed Cells
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Confocal images of fixed cells were taken using the Confocal White Light Laser (WLL) Leica TCS SP8 Microscope. All the images were acquired as z‐stacks (0.5 μm step size) and taken with the HC Plan Apo 100×/1.40 OIL (CS2) objective. Image acquisition was carried out with the Leica Application Suite X (LAS X) software (Leica Microsystems).
Wide‐field images of fixed cells were acquired as z‐stacks (0.3 μm step size) using a Nikon Eclipse TE2000 Inverted Microscope with Neo 5.5 sCMOS camera (Andor) and Plan Apo VC 60× or 100×/1.40 OIL objectives. Following acquisition, images were imported into Fiji (2.0.0‐rc‐59/1.51k) or Volocity 6.0 (Perkin Elmer) to obtain maximum intensity projections of z‐stacks. Images were then imported into Photoshop (Adobe CC 2017) and adjusted to use full range of pixel intensities. Images from each biological replicate were acquired using the same settings and processed in the same manner. For image analysis, seeAppendix Supplementary Methods .
Wide‐field images of fixed cells were acquired as z‐stacks (0.3 μm step size) using a Nikon Eclipse TE2000 Inverted Microscope with Neo 5.5 sCMOS camera (Andor) and Plan Apo VC 60× or 100×/1.40 OIL objectives. Following acquisition, images were imported into Fiji (2.0.0‐rc‐59/1.51k) or Volocity 6.0 (Perkin Elmer) to obtain maximum intensity projections of z‐stacks. Images were then imported into Photoshop (Adobe CC 2017) and adjusted to use full range of pixel intensities. Images from each biological replicate were acquired using the same settings and processed in the same manner. For image analysis, see
7
Agarose Pad and Flow-Cell Imaging of Bacterial Cells
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For experiments conducted on agarose pads, samples were taken from test tubes and placed on 1% agarose pads every 15 min, and then imaged within 5 min. For membrane staining, a small aliquot of cells was incubated with FM 4-64 (Invitrogen) at a final concentration of 5 µg/mL for 5 min and spotted on agarose pads without washing. Flow-cell experiments were performed in ONIX B04A microfluidic chips (CellASIC). Cells were loaded into the imaging chamber, and media reservoirs were filled with fresh or spent LB medium. Phase-contrast images and epifluorescence images were acquired with a Nikon Ti-E inverted microscope (Nikon Instruments) using a 100X (NA 1.40) oil immersion objective and a Neo 5.5 sCMOS camera (Andor Technology). The microscope was outfitted with an active-control environmental chamber for temperature regulation (HaisonTech, Taipei, Taiwan). Images were acquired using µManager v.1.449 (link).
8
Quantifying Intracellular Fluorescence Signals
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All image acquisition was done using an inverted fluorescence microscope (Olympus IX83) with an oil immersion phase-contrast 60× objective seated inside an incubator chamber (InVivo Scientific) pre-warmed to 37 °C and MetaMorph software (Molecular Devices). HCT fluorescence was measured using a DAPI filter cube. Images were captured using a Neo 5.5 sCMOS camera (Andor).
Fluorescence intensity was obtained with MicrobeJ (5.13 l (4))74 (link), a plug-in for the ImageJ software. MicrobeJ can automatically segment cell boundaries from phase-contrast microscope images and apply the binary masks from the segmentation to measure fluorescence intensities (in mean gray value) inside and outside the cells. The latter (background) is subtracted from the former to determine intracellular fluorescence signals. Autofluorescence of cells was determined by measuring fluorescence intensity in the absence of HCT. Reported intensities are the results of subtracting autofluorescence from intracellular fluorescence signals.
Fluorescence intensity was obtained with MicrobeJ (5.13 l (4))74 (link), a plug-in for the ImageJ software. MicrobeJ can automatically segment cell boundaries from phase-contrast microscope images and apply the binary masks from the segmentation to measure fluorescence intensities (in mean gray value) inside and outside the cells. The latter (background) is subtracted from the former to determine intracellular fluorescence signals. Autofluorescence of cells was determined by measuring fluorescence intensity in the absence of HCT. Reported intensities are the results of subtracting autofluorescence from intracellular fluorescence signals.
9
Single-Particle Fluorescence Imaging and Analysis
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The inverted optical microscope was custom-built around a TIRF objective (100x, NA 1.49, Nikon Instruments, Japan) mounted on a Piezo LEGS motor (Piezomotor, Sweden) to focus the sample plane precisely. For fluorophore excitation a laser diode (Nichia, Japan) of 488 nm wavelength was used. Dichroic and emission filters, optimized for GFP, were obtained from Semrock (USA), and all optomechanical parts and lenses were manufactured by Thorlabs (USA). Fluorescence intensity was recorded using a Neo 5.5 sCMOS camera (Andor, UK) at an exposure time of 0.1 s.
For the fluorescence data analysis, the mean intensity of a 7 × 7 pixels2 ROI enclosing the particle of interest was considered. As shown in theFigure S3 , any photobleaching effects were corrected by exponentially fitting the average over time of 15 ROIs (corresponding to 15 intact VLPs surrounding the particle hit by the AFM tip) and setting it constant to its initial value by adding the correction function A × (1 − exp(−t/B)), where parameters A and B were the amplitude and the decay rate of the exponential fit, respectively.
For the fluorescence data analysis, the mean intensity of a 7 × 7 pixels2 ROI enclosing the particle of interest was considered. As shown in the
10
Imaging of VSMC Migration Dynamics
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VSMC were transfected using electroporation (see below) and plated onto FN-coated 35mm iBIDI glass bottom dish and incubated for 48h. Then cells were transferred to DMEM supplemented with 20% exosome-free FBS, 10mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin and images were acquired every 1min for 20min at 37°C using a Nikon Ti-E (inverted) microscope equipped with a Yokogawa spinning disk and a Neo 5.5 sCMOS camera (Andor) and 60x or 100x/1.40 NA Plan Apo λ oil objectives (Nikon) were used. Images were acquired using NIS Elements AR 4.2 software. Cells were maintained at 37°C, 5% CO2 throughout the experiment via a CO2 chamber and a temperature-regulated Perspex box which housed the microscope stage and turret.
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