Microscopy imaging was performed on a laser scanning confocal microscope (C1 si, Nikon) using a ×10 objective (CFI Plan Apochromat, Nikon) and a ×60 oil immersion objective (CFI Plan Apo VC, Nikon). The following lasers were used for excitation: a 405 nm continuous wave laser (Melles Griot 56ICS/S2695) for DAPI and Hoechst; a 488 nm continuous wave laser (Coherent Sapphire) for FD10, ATTO 488 and Alexa Fluor® 488; a 561 nm continuous wave laser (Melles Griot 85-YCA-010) for Alexa Fluor® 568 and a 640 nm continuous wave laser (Melles Griot 56ICS/S2695) for Alexa Fluor® 647. After photoporation, cells were incubated with 1 µg/mL Hoechst (Life Technology, Belgium) in CCM for 15 min at 37 °C. After the cells were washed twice with DPBS, time-lapse recordings were performed on a spinning disk confocal microscope (Nikon eclipse Ti-e inverted microscope, Nikon) equipped with an MLC 400 B laser box (Agilent technologies), a Yokogawa CSU-22 Spinning Disk scanner (Andor) and an iXon ultra EMCCD camera (Andor Technology, Belfast, UK). HeLa cells were imaged in a stage-top cell incubator (37°C with 5% CO2 supplied, Tokai Hit) for 1 h with a time interval of 3 min using a ×60 oil immersion objective lens (CFI Plan Apo VC 60 × oil, Nikon, Japan).
Cfi plan apochromat
Manufactured by Nikon
Sourced in Japan
The CFI Plan Apochromat is a high-performance microscope objective lens manufactured by Nikon. It is designed to provide excellent optical performance, including accurate color reproduction and a flat field of view. The lens is suitable for a variety of microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Cellmask, by Thermo Fisher Scientific (2 mentions)
Crossbeam 540 electron microscope, by Zeiss (2 mentions)
Coumarin 6, by Merck Group (2 mentions)
Jem 1400 plus transmission electron microscope, by JEOL (2 mentions)
Plan apo vc, by Nikon (2 mentions)
A1r confocal microscope, by Nikon (2 mentions)
Ixon ultra emccd camera, by Oxford Instruments (1 mentions)
Cfi plan apo vc, by Nikon (1 mentions)
Mlc 400 b laser box, by Agilent Technologies (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Nir apo, by Nikon (1 mentions)
Cryostat, by Leica (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Ixon ultra 897, by Oxford Instruments (1 mentions)
Ti e inverted fluorescence microscope, by Nikon (1 mentions)
Eclipse ti epifluorescence microscope, by Nikon (1 mentions)
14 protocols using cfi plan apochromat
1
Laser Scanning Confocal Microscopy of Cells
2
Intravital Imaging of Mouse Flank Skin
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Mice were anesthetized with 1.5% isoflurane gas and covered with a heating blanket to keep their body temperature at 37°C. Intravital images of the flank skin were captured with an inverted laser scanning microscope (A1R+; Nikon) equipped with CFI Plan Apochromat λ 10× or 20× objective lens. NIS elements software was used for acquisition and analysis of images.
3
Multimodal Imaging of Bioactive Nanofibers
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For TEM imaging, the nanofibers were directly electrospun on carbon-coated Cu grids (200-mesh). Following laser irradiation of the nanofibers, they were visualized by a JEM 1400 plus transmission electron microscope (JEOL, Tokyo, Japan) operated at 20-60 kV. For SEM imaging, samples were first coated with 5nm platinum using a Quorum Q150T ES sputter coater. Scanning electron microscope images were taken with a Zeiss Crossbeam 540 Electron Microscope using a SE2 detector at 20 kV.
For visualization by confocal microscopy, fluorescent PCL nanofibers were fabricated by electrospinning a PCL solution mixed with the fluorophores 3-(2-benzothiazolyl)-7-(diethylamino) coumarin (coumarin-6, #12779, Sigma-Aldrich). A confocal laser scanning microscope (C1si, Nikon, Japan) with 60X water lens (Plan Apo VC, Nikon) was used to image the fluorescent PCL nanofibers. HeLa and H1299 cells grown on PEN substrates were imaged by the C1si confocal with a 10X lens (CFI Plan Apochromat, Nikon). For confocal imaging of Jurkat cells, their plasma membrane was stained with 10 μg/mL deep red fluorescent CellMask (#C10046, ThermoFisher Scientific). A series of z-stack confocal images were acquired in two channels (green channel recorded for nanofibers and deep red channel for the cells) with the 60X water lens.
For visualization by confocal microscopy, fluorescent PCL nanofibers were fabricated by electrospinning a PCL solution mixed with the fluorophores 3-(2-benzothiazolyl)-7-(diethylamino) coumarin (coumarin-6, #12779, Sigma-Aldrich). A confocal laser scanning microscope (C1si, Nikon, Japan) with 60X water lens (Plan Apo VC, Nikon) was used to image the fluorescent PCL nanofibers. HeLa and H1299 cells grown on PEN substrates were imaged by the C1si confocal with a 10X lens (CFI Plan Apochromat, Nikon). For confocal imaging of Jurkat cells, their plasma membrane was stained with 10 μg/mL deep red fluorescent CellMask (#C10046, ThermoFisher Scientific). A series of z-stack confocal images were acquired in two channels (green channel recorded for nanofibers and deep red channel for the cells) with the 60X water lens.
4
Automated Quantification of Cell Loading and Viability
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The imaging processing quantification method was reported in our previous work1 (link). Briefly, after laser treatment, 3-5 confocal images were acquired with a confocal laser scanning microscope (C1si, Nikon, Japan) using a 10× lens (CFI Plan Apochromat, Nikon, Badhoevedorp, The Netherlands). Each image consists of green fluorescence (viability) and red fluorescence (loading efficiency) channels. A Matlab (The matworks, Natick, MA, USA) program was written for automated quantification of cell loading and cell viability. Untreated cells are used to define the threshold for positive cell loading, where the threshold value is defined as the 95% level of untreated cells. Similarly, cells are considered as alive when the green fluorescence intensity is higher than the 95% level of dead cells.
5
Cryosectioning and Immunofluorescence of Retina
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After fixation, the fixative below the explant filter is removed and replaced by 30% sucrose and incubated overnight at 4 °C. After snap freezing the samples in Tissue-Tek® O.C.T. (Sakura) with liquid nitrogen, 14 µm sections were cut with a cryostat (Leica). For reliability of our results, we applied the workflow drawn in Figure 2(C): 16 sections were analyzed per retina, resulting from 4 distinct retina locations. These retinal sections were permeabilized with 0.1% Triton for 5 min prior to a 1 hour incubation at room temperature with 5% goat serum in PBS as a blocking step. Next, sections were incubated overnight at 4 °C with 1:200 rabbit antibody against Collagen IV. Finally, after a 1 hour incubation at room temperature with goat anti-rabbit Alexafluor 647 conjugated secondary antibody and 10 µg/ml Hoechst the sections were mounted with Vectashield (Vector Laboratories) and prepared for imaging. The ‘cryosection imaging’ was done with a confocal microscope (C1-si, Nikon) using a 10× objective (CFI Plan Apochromat, Nikon) and a 60× water objective (NIR Apo, Nikon).
6
Quantifying T Cell Viability Post-Electroporation
7
STORM Imaging with Bead Experiments
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STORM and bead experiments were performed on a Nikon Ti-E inverted fluorescence microscope using an oil-immersion objective lens (Nikon CFI Plan Apochromat λ 100 × , NA 1.45) and the native 1.5x magnification on the microscope, without any modifications to the imaging path. Lasers emitting at 644, 561, and 488 nm were introduced to the back focal plane of the objective lens via a multi-line dichroic mirror (ZT405/488/561/640rpc-uf2, Chroma). A translation stage shifted the laser beams toward the edge of the objective lens so that they entered slightly below the critical angle, illuminating < 1 µm into the sample. Emission was filtered by a multi-notch filter (ZET405/488/561/640 m, Chroma) and recorded by an EM-CCD camera (iXon Ultra 897, Andor). Effective magnification and pixel size were ~ 150x and ~ 107 nm, respectively.
8
Automated Quantification of Cell Loading and Viability
9
Multimodal Imaging of Bioactive Nanofibers
10
Quantifying T Cell Viability Post-Electroporation
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After PEN photoporation or electroporation, using optimized delivery protocols (see main text), T cells were washed twice and seeded at 2 x 105 cells per well in a 96 well plate. After 4 hours T cells were stimulated with 5 ng/ml IL-2 and Immunocult human CD3/CD28 activator in complete IMDM. At the indicated timepoints T cells were washed and stained with Calcein AM and TO-PRO-3 iodide for 30 min in cell medium. Living cells were detected and quantified based on their green (Calcein AM positive, living cells) and red (TO-PRO-3 negative, dead cells) fluorescence levels using an A1R confocal microscope (Nikon, Badhoevedorp, The Netherlands) equipped with a perfect focus system and a X20 objective lens (CFI Plan Apochromat, Nikon, Badhoevedorp, The Netherlands). The software package ImageJ with the plugin of Analyze Particles was used for image processing.
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