SIM imaging was performed using an N-SIM microscope (Nikon Corporation, Tokyo, Japan), with SR Apochromat TIRF 100 × 1.49 NA oil immersion objective. Spherical aberration was reduced using the Ti2 automated correction collar at the beginning of the imaging session. Images were acquired using 405 nm, 488 and 561 nm lasers, with stacks of step size 0.12 µm (with top and bottom of samples determined visually), using 3D-SIM mode. Images were reconstructed with NIS Elements software (Nikon Corporation, Tokyo, Japan).
N sim microscope
Manufactured by Nikon
Sourced in Japan
The N-SIM microscope is a laser super-resolution microscope that uses structured illumination to achieve a higher resolution than conventional optical microscopes. It is capable of imaging live cells with high spatial resolution and minimal photodamage.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (8 mentions)
Nis elements software, by Nikon (7 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (6 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Nis element, by Nikon (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Flash 4, by Hamamatsu Photonics (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342 sc 200908, by Santa Cruz Biotechnology (2 mentions)
Hoechst 33342, by Santa Cruz Biotechnology (2 mentions)
Ixon 897 emccd camera, by Oxford Instruments (2 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions)
Du897, by Oxford Instruments (2 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Sr apochromat tirf 100 1.49 na, by Nikon (1 mentions)
Apochromat 100x objective na 1.49, by Hamamatsu Photonics (1 mentions)
Airyscan detector, by Zeiss (1 mentions)
Lsm 880 microscope, by Zeiss (1 mentions)
60 protocols using n sim microscope
1
Super-resolution Imaging of Samples
2
Super-resolution Microscopy Imaging
3
Live-cell Imaging of Cells Using SIM Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (20,000 cells) were seeded on a glass bottom 4 well plate (De Groot—76-D35C4-20). Imaging was started 24 h after the seeding of cells. Images were acquired depending upon the experiment. Cells were cultured, transfected and live cell was performed according to standard protocols19 (link),70 (link). For time-lapse imaging, we used a dual point-scanning Nikon A1R-si microscope equipped with a Piezo stage, using a 60 × PlanApo IR oil objective NA 1.4, 0.3 μm slices, and 0.2–2% laser power (from 65-mW 488-nm laser and 50-mW 561-nm laser) to acquire 3D movies. Images were acquired in resonant-scanning or Galvano-scanning mode. Each Z series was acquired with 0.5- to 1-μm step size and 10–35 steps. For super resolution Structured Illumination Microscopy (SIM) Cells were prepared as described above. Images were acquired using a Nikon nSIM microscope in 2D mode with a 488 nm and 561 nm lasers. A 100 × oil TIRF objective (NA 1.49) was used for the imaging. Prior to imaging the point-spread function was visualized with 100 nm fluorescence beads in order to adjust the correction ring of the objective to the coverslip thickness. The final image was reconstructed using NIS-Elements software (Version 4.1).
4
Immunofluorescence Microscopy of Trypanosoma brucei
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were settled onto poly-L-lysine treated slides, fixed in 4% formaldehyde and permeabilized with 0.3% Triton X-100. After blocking with 3% BSA in PBS, cells were stained with Rabbit anti-HA polyclonal (1:100, Beyotime, Shanghai, China) and rabbit anti-VP1 polyclonal (1:300) [32 (link)] primary antibodies to visualize HA-tagged protein and acidocalcisomes, respectively. As a secondary antibody, TRITC conjugated goat anti-rabbit IgG (H+L) (Invitrogen, Carlsbad, USA) was used. The mitochondrion in T. brucei was monitored by microscopy after incubation with 50 nM Mito-Tracker Red CMXRos (Life Technologies, Carlsbad, USA) in HMI-9 for 20 min at 37 °C, followed by washing with PBS. The cells were fixed on slides in 4% formaldehyde and washed in PBS with 0.1 M glycine. DNA contents were stained with DAKO fluorescent mounting medium containing 50 ng/ml DAPI. Images were documented using a Zeiss Axio Imager A2m epifluorescence microscope and a Nikon N-SIM microscope.
5
Imaging Virus Entry in RPE Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPE cells stably expressing epsin-EGFP or epsin mut-H0 EGFP and mCherry-Clc were seeded on 8-well chamber culture slide (#1.5, Nunc Lab-Tek) 12 to 16 h prior to each experiment. Cells were infected with labelled WSN-WT and WSN-UdM IAVs for 1 h. Immediately after, the cells were washed with PBS and fixed using 4% paraformaldehyde for 10 min on ice. 3D-SIM was performed using a Nikon N-SIM microscope equipped with an Apochromat 100X objective (NA 1.49) and a sCMOS camera (Flash 4.0; Hamamatsu Photonics, Shizuoka, Japan). Images were acquired in 3D SIM mode with nine images taken at each z depth of 200 nm of each color with linear translation of Moire pattern for SIM reconstruction. EGFP-epsin, mCherry-Clc and DiD-labeled IAVs were acquired at each z-step with 488 nm, 561 nm, and 640 nm excitation at exposure times of 100 ms, 100 ms and 50 ms respectively.
6
Super-resolution Microscopy Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Super-resolution images were acquired on a N-SIM microscope (Nikon Instruments, Inc.) equipped with an Apochromat 100 × /1.49 numerical aperture oil-immersion objective lens and solid-state lasers (488 nm, rated power ≥ 200 mW; 561 nm, rated power ≥ 150 mW; 647nm, rated power ≥ 100 mW). Images were captured with an electron-multiplying charge coupled device (EMCCD) camera (Andor; 512 × 512 px, 14 bit) with a gain value of 100.
Raw SIM images (containing nine images: three phases and three angles) were obtained and reconstructed with Nikon Elements software (Nikon Instruments, Inc.). The exposure time was set to 30 ms for each raw data capture (each single-color SIM frame was acquired over 270 ms in total). SIM frames were deliberately spaced at 1-s, 6-s, 10-s or 1-min intervals according to the purpose of each experiment. SIM images were analyzed with Nikon Elements and ImageJ software, and tested by using the ImageJ plugin SIMcheck18 (link) to assess the image quality. Detailed information regarding the imaging conditions used for SIM imaging is summarized in Supplementary Table2 .
Raw SIM images (containing nine images: three phases and three angles) were obtained and reconstructed with Nikon Elements software (Nikon Instruments, Inc.). The exposure time was set to 30 ms for each raw data capture (each single-color SIM frame was acquired over 270 ms in total). SIM frames were deliberately spaced at 1-s, 6-s, 10-s or 1-min intervals according to the purpose of each experiment. SIM images were analyzed with Nikon Elements and ImageJ software, and tested by using the ImageJ plugin SIMcheck18 (link) to assess the image quality. Detailed information regarding the imaging conditions used for SIM imaging is summarized in Supplementary Table
7
Imaging of LPL Mutants Under Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thirty thousand MV3 cells stably expressing eGFP, wt LPL eGFP, or LPL eGFP Cys-Ala mutants were added to either poly-D-lysine- or gelatin-coated 10 mm coverslips in 48-well plates and incubated for the indicated times. Thereafter, the cells were treated with the indicated concentrations of H2O2. The samples were fixed using 1.5% paraformaldehyde (PFA) for 10 min at RT. Then samples were washed and blocked in FW buffer for 30 min at RT63 (link). For immunostaining, the cells were stained as indicated with the primary antibodies against LPL (1:1,000), TRX1 (1 μg ml−1), MMP2 (1:250), and TRXR1 (1:200) in FWS buffer (PBS + 1% BSA + 0.1% saponin). Thereafter, secondary antibody staining were performed using the following antibodies and reagents for 30 min at RT: anti-rabbit Cy3 (1:800,), anti-rabbit AF405 (1:200), anti-mouse AF405 (1:200), anti-mouse Cy3 (1:800), DAPI (nuclei, 1:10,000), and SiR-actin (F-actin, 500 nM). 3D-SIM images or confocal images were obtained 1 day after sample preparation with a Nikon N-SIM microscope (×100 objective, NA 1.49).
8
Super-resolution Imaging of Mitochondrial Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Super-resolution images (Fig. 4c and Extended Data Fig. 3a ) were acquired with a Nikon N-SIM microscope. Nine z-stacks of 0.2 μm each were acquired using a SR Apo TIRF 100× 1.49 N.A. oil objective and a DU897 Ixon camera (Andor). Raw images were computationally reconstructed using the reconstruction slice system from NIS‐Elements software (Nikon). Mitochondrial diameter was calculated using the Fiji software.
Super-resolution images related to TOM20-PDH+TFAM+ MDVs (Extended Data Fig.11d ) were acquired with a Zeiss LSM 880 microscope with an Airyscan detector (Carl Zeiss Microscopy), using a Zeiss 63× oil lens, NA 1.4, as the primary objective. Four-colour excitation was performed with a blue diode laser for 405 nm, argon laser for 488 nm, He 543 laser for 561 nm and He 633 laser for 647 nm. The Airyscan detector was used in SR mode using all 32 pinholes, thus increasing the resolution to around 140 nm in x, y and z to capture the image. The image was reconstructed by pixel reassignment and deconvolution on the Zen Black platform (Carl Zeiss Microscopy). The final image was produced on ImageJ by background subtraction (rolling ball 50) and one run of the smooth filter.
Super-resolution images related to TOM20-PDH+TFAM+ MDVs (Extended Data Fig.
9
Immunofluorescence Staining of Focal Adhesions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed on 1.5mm glass coverslips with PHEMO buffer (68 mM, PIPES, 25 mM HEPES, 15 mM EGTA, 3 mM MgCl2, 10% DMSO) plus 3.7% formaldehyde, 0.05% glutaraldehyde and 0.5% Triton X-100 for 10 min at room temperature. After three 5 min washes with PBS, the coverslips were blocked with 10% normal goat serum for 1 h at room temperature. Primary antibodies were diluted in 5% normal goat serum and added to the coverslips overnight at 4 °C. After 3 PBS washes, the coverslips were incubated for 1 h at room temperature with a fluorophore conjugated secondary antibody diluted in 5% normal goat serum. After 3 PBS washes, the coverslips were incubated with 350 nM DAPI stain diluted in 1X PBS for 10 min at room temperature. The coverslips were mounted on glass slides using Prolong gold mounting medium (Invitrogen). The slides were imaged using Zeiss LSM 510 META confocal microscope using a 100X oil Plan-Apo objective (NA = 1.46). The images were processed using ZEN 2009 software. A Nikon N-SIM microscope housed within the Emory Integrated Cellular Imaging Core was utilized to acquire super-resolution images of vimentin directly entering FAs. Images were acquired using a 100× 1.49 NA oil objective. All images within an experiment were taken under identical settings. Intensity levels were adjusted equally on all samples within an experiment.
10
Immunofluorescence Staining of Focal Adhesions
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!