HEK-293 cells were plated on poly-d -lysine coated chambers (ibidi, Germany) in DMEM supplemented with 10% (vol/vol) FBS (DMEM/FBS). After 24 h, cells were transfected with 300 ng of rat (r) NK1R-GFP per chamber and cultured for 48 h. To identify endosomal compartments, HEK-293 cells were infected with Rab5a-GFP (resident in early endosomes) or Rab7a-GFP (late endosomes) CellLight BacMam2.0 (Thermo Fisher Scientific) for 16 h. To examine localization of nanoparticles, cells were incubated in Leibovitzś L-15 medium with DIPMA-Cy5 nanoparticles (20 μg ml−1, 30 min, 37 °C) or vehicle, followed by addition of SP (10 nM). Cells were imaged at 30 and 60 min post-SP addition using a Leica SP8 confocal microscope equipped with HCX PL APO ×40 (NA 1.30) and HCX PL APO ×63 (NA 1.40) oil objectives. Images were analysed using Fiji36 (link) and deconvolved with Huygens Professional version 18.04 (Scientific Volume Imaging, http://svi.nl ), using the CMLE algorithm with a signal-to-noise ratio of 10 and 100 iterations. Co-localization was evaluated by determination of the Manders overlap coefficient25 .
Huygens professional version 18
Manufactured by Scientific Volume Imaging
Sourced in Netherlands
Huygens Professional version 18.04 is a software package for deconvolution and analysis of 3D and 4D microscopy data. It provides advanced algorithms for improving the resolution and contrast of microscopy images.
Automatically generated - may contain errors
Lab products found in correlation
Celllight bacmam 2, by Thermo Fisher Scientific (1 mentions)
Hcx pl apo 40, by Leica (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Ix83 fluoview1200, by Olympus (1 mentions)
Evolve 512 emccd camera, by Teledyne (1 mentions)
Csu x1 a1, by Yokogawa (1 mentions)
C shg1, by Nikon (1 mentions)
Inubg2e zilcs, by Tokai Hit (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
Application suite x software, by Leica (1 mentions)
Sp8 multiphoton microscope, by Leica (1 mentions)
5 protocols using huygens professional version 18
1
Nanoparticle Trafficking in Transfected HEK-293 Cells
2
Confocal Imaging and ELAVL4 Quantification
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Confocal images of Figures 1 , 2 , 3 , and 4 were acquired at the Olympus iX83 FluoView1200 laser scanning confocal microscope using a 60x NA1.35 oil objective and 405nm, 473nm and 559nm lasers. Filter setting for DAPI, Alexa Fluor 488 and Alexa Fluor 594 were used. For ELAVL4 signal quantification, the raw stack images were first deconvolved with Huygens Professional version 18.04 (Scientific Volume Imaging, the Netherlands, https://svi.nl ), using the CMLE algorithm, with SNR:15 and 50 iterations. Then 3D quantitative analysis was performed using Surfaces in Imaris 8.1.2 (Bitplane Scientific Software, USA), mean intensity and volume values of each speckle or stress granule were exported in Prism 7 for statistical analysis and graph display.
3
Spinning Disk Confocal Microscopy for 3D Imaging
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Spinning disk confocal microscopy was performed on a Nikon Eclipse Ti microscope equipped with a perfect focus system (Nikon), a spinning disk-based confocal scanner unit (CSU-X1-A1, Yokogawa, Japan), an Evolve 512 EMCCD camera (Roper Scientific, Trenton, NJ) attached to a 2.0X intermediate lens (Edmund Optics, Barrington, NJ), a super high pressure mercury lamp (C-SHG1, Nikon), a Roper scientific custom-ordered illuminator (Nikon, MEY10021) including 405 nm (100 mW, Vortran), 491 nm (100 mW, Cobolt), 561 nm (100 mW, Cobolt) and 647 nm (100 mW, Cobolt) excitation lasers, a set of BFP, GFP, RFP and FarRed emission filters (Chroma, Bellows Falls, VT) and a motorized stage MS-2000-XYZ with Piezo Top Plate (ASI). The microscope setup was controlled by MetaMorph 7.7.5 software. Images were acquired using Plan Fluor Apo VC 60x NA 1.4 oil objective. The 3D image reconstruction was carried out using Huygens Professional version 18.04 (Scientific Volume Imaging, the Netherlands). The temperature was controlled by a stage top incubator INUBG2E-ZILCS (Tokai Hit).
4
Quantifying Collagen in Tissue Samples
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Images were acquired on a Leica SP8 multi-photon microscope equipped with a Mai Tai DeepSee laser tuned to 930 nm. Second harmonic generation signal was collected between 455–475 nm using a HyD detector in photon counting mode and the autofluorescence was collected from 480–600 nm. Channel separation and stitching were performed within the Leica Application Suite X software (Leica Microsystems, Wetzlar, Germany) controlling the microscope, before being deconvolved with Huygens Professional version 18.04 (Scientific Volume Imaging, Hilversum, The Netherlands). The data was 3D median filtered to remove noise, the background subtracted, and the amount of collagen quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). A Fast Fourier Transform (FFT) filter was applied to the autofluorescence channel to remove stitching artifacts. Image projections are summed slices of the 3D z stack, and to enable visualisation of the total amount of collagen, spanning the entire dynamic range in a single image, a gamma correction of 0.2 was applied.
5
Deconvolution Processing of Microscopy Images
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Deconvolution processing was performed with Huygens Professional version 18.04 (Scientific Volume Imaging, Netherlands; http://svi.nl ). For that, a theoretical point-spread function was automatically computed based on the microscope and image acquisition parameters. The deconvolution process was numerically performed using the Classic Maximum Likelihood Estimation algorithm. In brief, this algorithm assumes that the photon noise is Poisson-distributed, and the likelihood of an estimate of the actual image given the computed point-spread function and the acquired image is iteratively optimized until either a quality factor or a maximum number of iterations is reached. In our deconvolutions, we used a quality factor equal to 0.001 and a maximum 50 iterations. The signal-to-noise ratio for each acquired image was computed based on three line profiles going through regions of background signals toward regions of positive, actual signals. Typically, signal-to-noise ratios were on the order of 7–20 for the different analyzed confocal images and 7 for the STED image.
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