The imaging was performed on: (i) an inverted confocal spinning disk microscope CSU-W1 (Andor/Roper/Nikon) equipped with a sCMOS camera (Flash4 Hamamatsu), and a Borealis module from Andor for better field homogeneity, using either a 60x (NA1.4 OIL PL APO L) or 100x (NA1.45 OIL PL APO L); (ii) an inverted confocal spinning disk microscope from Zeiss (CSUW1, Roper/Zeiss) equipped with a sCMOS camera (Flash4 Hamamatsu), a Visitron module for better field homogeneity, using either 60x or 100x NA1.4 OIL DIC N2 PL APO VC objectives; (iii) an inverted spinning disk wide confocal microscope CSU-W1 (Roper/Nikon/GATACA) equipped with a sCMOS camera (BSI camera), a field illumination homogenizer, using either 60x or 100X (NA 1.4 OIL N2 PL APO VC) objectives. The Metamorph autofocus function was used for some time-lapse acquisitions.
Flash 4
Manufactured by Hamamatsu Photonics
Sourced in Japan, United States, Canada
The Flash 4.0 is a compact and versatile flash lamp system designed for laboratory and industrial applications. It features a high-intensity xenon flash lamp that can generate short, high-energy light pulses. The core function of the Flash 4.0 is to provide a controlled and repeatable source of pulsed illumination for various experimental and measurement purposes.
Automatically generated - may contain errors
Lab products found in correlation
Spectra x, by Lumencor (9 mentions)
Eclipse ti, by Nikon (8 mentions)
Lsm 700, by Zeiss (6 mentions)
Coolsnap kino, by Hamamatsu Photonics (4 mentions)
Tie perfect focus system microscope, by Hamamatsu Photonics (4 mentions)
Nis elements software, by Nikon (3 mentions)
Ti e epifluorescence microscope, by Nikon (3 mentions)
Perfect focus system, by Nikon (3 mentions)
Eclipse ti e, by Nikon (3 mentions)
Dmi6000, by Leica (3 mentions)
Axio observer 7, by Lumencor (2 mentions)
Na plan apo, by Hamamatsu Photonics (2 mentions)
Matlab, by MathWorks (2 mentions)
Spectra x led, by Hamamatsu Photonics (2 mentions)
Spectra x, by Hamamatsu Photonics (2 mentions)
Electron multiplying charge coupled device camera, by Oxford Instruments (2 mentions)
Spinning disk confocal and tirf combined system, by Nikon (2 mentions)
Plan apo 1.49 na oil immersion objective, by Hamamatsu Photonics (2 mentions)
N sim microscope, by Nikon (2 mentions)
Apochromat 100x objective na 1.49, by Hamamatsu Photonics (2 mentions)
72 protocols using flash 4
1
Imaging Protocols for Confocal Microscopy
2
Yeast Growth Monitoring via Microfluidics
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Cells were inoculated in SC, 0.1% glucose and grown to OD600 = 0.5 before transferring them to a CellASIC microfluidic perfusion chamber (Millipore). The growth medium was then replaced by preconditioned starvation medium obtained by filtering the medium of cells grown for 3 days in SC, 0.1% glucose. Fluorescence imaging was started 6 h following this medium change. For imaging a Nikon Ti-E epifluorescence microscope equipped with a 60× ApoTIRF oil-immersed objective (1.49 NA, Nikon), a 2048 × 2048 pixel (6.5 μm) sCMOS camera (Flash4, Hamamatsu) and an autofocus system (Perfect Focus System, Nikon) with either bright field or 470/40 excitation and 525/50 emission filters (Chroma). Images from one plane in the middle of the cell were taken every 15 min over the course of 24 h. Tracking and segmentation of cells was performed automatically using a custom script based on the CellX software (53 ) and quantified in R (R Development Core Team 2008) and Matlab (MathWorks).
3
Colon Cancer Cell Spheroid and Organoid Cultures
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Colon cancer cell lines were cultured in DMEM high glucose (4.5 g/l, Gibco, Thermo Scientific Inc., Waltham, MA) supplemented with 10 % FBS, 100 U/ml penicillin G, and 100 lg/ml streptomycin at 37 °C and 5% CO2.
Spheroid cultures were established as described [26 (link)] with 3000 cells per well. Images were acquired with an inverse microscope (Olympus IX83) equipped with a camera (Hamamatsu Flash4, Hamamatsu, Japan) and analyzed with the ImageJ software (NIH, National Institute of Health, Bethesda, Maryland, USA). Spheroid volume was determined by measuring projected areas, followed by radius and volume (μm³) determination.
Patient-derived organoids were cultured as previously described [64 (link)] in 80% Cultrex RGF BME Type 2 (R&D Systems #3533-005).
Spheroid cultures were established as described [26 (link)] with 3000 cells per well. Images were acquired with an inverse microscope (Olympus IX83) equipped with a camera (Hamamatsu Flash4, Hamamatsu, Japan) and analyzed with the ImageJ software (NIH, National Institute of Health, Bethesda, Maryland, USA). Spheroid volume was determined by measuring projected areas, followed by radius and volume (μm³) determination.
Patient-derived organoids were cultured as previously described [64 (link)] in 80% Cultrex RGF BME Type 2 (R&D Systems #3533-005).
4
Visualizing Viral Infection in PANC-1 Cells
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PANC-1 cells were seeded in a 6-well plate at 6 × 10^5 cells per well. Following overnight attachment, the cells were infected with CVB3, LACV, and SINV at an MOI of 1 or left uninfected for 72 h. Pictures of the cells were taken with a Levenhuk M500 Base camera with LevenhukLite software (Version ×64). Persistently infected PANC-1 cells were plated at the same density, and photos were taken as previously described. For immunofluorescence images, PANC-1 cells were seeded to coverslips and fixed 24 h later. Fixed cells were then stained for nuclei (DAPI) and dsRNA (J2, Invitrogen) as previously described (32 (link)). They were imaged on Zeiss Axio Observer 7 with Lumencor Spectra X LED light system and a Hamamatsu Flash 4 camera using Zen Blue software with a 40× objective.
5
Polarization Imaging of Bacterial Cell Sacculi
6
Immunofluorescence Analysis of ACE2 and CEACAM
7
Time-Lapse Imaging of Bacterial Dynamics
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Microfluidics experiments were performed on a Nikon Ti Eclipse inverted fluorescence microscope with a perfect focus system, oil immersion objective 100× NA1.4, motorised stage, sCMOS camera (Hamamatsu Flash 4) and LED excitation source (Lumencore Spectra X). The temperature chamber (Okolabs) allowed performing the experiments at 37°C. We recorded time‐lapse movies with a frame rate of 1/3 min using NIS‐Element software (Nikon) across three spectral channels (LED excitation wavelengths λ: 555, 508, 440 nm) to image mKate2, MutL‐mYpet and CFP reporters, respectively. One image is acquired from each channel consecutively using exposure times of 100 ms (λ = 555 nm), 300 ms (λ = 508 nm) and 75 ms (λ = 440 nm); LED intensity for all channels was 50% maximal output. One single triband dichroic and three separate emission filters were used to separate excitation and emission light. Up to 48 fields of view can be imaged across the microfluidic device within the 3‐min time‐lapse window. On average, 20 channels with bacteria were present per field of view such that about ~1,000 mother cell traces were recorded per experiment.
8
Live Cell Microscopy Protocol
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For light microscopy of living cells, in vitro haptomonad-like cells attached to a piece of a gridded glass coverslip were washed twice in DMEM, incubated in DMEM with Hoechst 33342 (1 µg/ml) for 5 min and then washed twice in DMEM. Coverslip pieces were mounted onto another glass coverslip and then onto a glass slide, with the cell attachment side facing up. Attached cells were imaged using a Zeiss ImagerZ2 microscope with 63×objective and a Hamamatsu Flash 4 camera.
9
Lymphatic Smooth Muscle Recombination Efficiency
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For assessment of smooth muscle recombination efficiency, cannulated and pressurized popliteal lymphatic vessels were equilibrated in calcium-free solution for 15 min to prevent movement during live imaging. Image stacks were acquired with a Yokagawa CSU-X Spinning Disc Confocal Microscope on an inverted Olympus IX81 with a Hamamatsu Flash4 camera and ×20 dry objective using Metamorph software. Each live vessel acquisition created a Z-stack of images in 1 micron increments from the lower surface of the vessel to its midpoint for both the membrane GFP (488 nm) and membrane tdTomato wavelengths (561 nm).
To assess recombination efficiency in the smooth muscle layer of popliteal lymphatic vessels, we assessed the percent area of the popliteal vessels covered by GFP+ lymphatic muscle cells. Maximum projections were made of the GFP and tdTomato image stacks using Image J. A mask of the GFP+ lymphatic muscle cells was created, despeckled, and then was used to assess the percent area of the tdTomato maximum projection encompassed by the mask using the Image J threshold and area measure function.
To assess recombination efficiency in the smooth muscle layer of popliteal lymphatic vessels, we assessed the percent area of the popliteal vessels covered by GFP+ lymphatic muscle cells. Maximum projections were made of the GFP and tdTomato image stacks using Image J. A mask of the GFP+ lymphatic muscle cells was created, despeckled, and then was used to assess the percent area of the tdTomato maximum projection encompassed by the mask using the Image J threshold and area measure function.
10
Measuring PrecA-GFPmut2 Expression Dynamics
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Mothermachine microfluidic experiments were performed as described4 (link) to measure PrecA-GFPmut2 expression dynamics during continuous unperturbed growth in M9 glucose medium at a single-cell level. Cells expressed fluorescent protein mKate2 constitutively and carried an flhD gene deletion to remove flagellum motility. Imaging was performed on a Nikon Ti Eclipse inverted fluorescence microscope equipped with perfect focus system, 100x NA1.45 oil immersion objective, sCMOS camera (Hamamatsu Flash 4), motorized stage, and 37°C temperature chamber (Okolabs). Fluorescence images were automatically collected using NIS-Elements software (Nikon) and an LED excitation source (Lumencor SpectraX). Time-lapse movies were recorded at 3-min intervals with 100 ms exposures for GFPmut2 and mKate2 using 50% LED excitation intensities. Movies were analysed using custom Matlab software to segment cells based on cytoplasmic mKate2 fluorescence and to construct single-cell lineages. PrecA-GFPmut2 expression traces represent the average pixel intensity within the area of a cell in each frame after subtracting the median background signal outside cells. Expression pulses were identified by applying a moving mean filter (30 frames window) and using findpeaks function in Matlab.
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