β-lg amyloid fibril before and after gastric digestion were dyed with ThT and imaged using a Nikon N-STORM microscope (Nikon, UK Ltd.) using an SR Apochromat TIRF 100 ×1.49 N. A. oil immersion objective lens. Fluorescence was detected with an EM-CCD Camera iXon DU897 (Andor). The field of view imaged typically covered 512 × 512 camera pixels corresponding to an area of ~80 × 80 μm2 on the sample. An in-built focus-lock system was used to prevent axial drift of the sample during data acquisition. The laser excitation was 20% at 405 nm, with a maximum intensity measured at the tip of the optical fiber of 20 mW and an exposure time of 50 ms. The emission passed through a multi-edge dichroic filter with windows at 415–490 nm, 502–560 nm, and 660–800 nm. Images were acquired using the NIS Elements Nikon software.
N storm microscope
Manufactured by Nikon
Sourced in United Kingdom
The N-STORM microscope is a high-resolution imaging system designed for advanced biological research. It utilizes a specialized technique called Stochastic Optical Reconstruction Microscopy (STORM) to capture detailed images of cellular structures and processes at the nanometer scale. The core function of the N-STORM microscope is to provide researchers with the ability to visualize and study fine details within biological samples that are beyond the resolution capabilities of conventional light microscopes.
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Lab products found in correlation
N storm software, by Nikon (3 mentions)
Em ccd camera ixon du897, by Oxford Instruments (2 mentions)
Orca flash 4 v3, by Hamamatsu Photonics (2 mentions)
Andor ixon x3 du 897 em ccd camera, by Oxford Instruments (2 mentions)
Matlab, by MathWorks (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Catalase, by Merck Group (1 mentions)
Sr apochromat tirf 100 1.49 na, by Nikon (1 mentions)
Alpha plan apochromat 100 1.46 oil, by Zeiss (1 mentions)
Orca flash 4.0 scmos camera, by Hamamatsu Photonics (1 mentions)
200 proof ethanol, by Decon Labs (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
Sylgard 184 silicone elastomer kit, by Dow (1 mentions)
Ixon ultra 897, by Oxford Instruments (1 mentions)
Du 897u cs0 bv, by Oxford Instruments (1 mentions)
Nis element, by Nikon (1 mentions)
Emccd camera, by Oxford Instruments (1 mentions)
Tetraspeck microsphere, by Thermo Fisher Scientific (1 mentions)
1.49 na 100 oil immersion tirf objective, by Nikon (1 mentions)
Ixon ultra 897 emccd camera, by Oxford Instruments (1 mentions)
31 protocols using n storm microscope
1
Imaging of Amyloid Fibril Gastric Digestion
2
3D dSTORM Imaging of Virus-Bound Antibodies
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Three-color, 3D dSTORM imaging was carried out using the Nikon N-STORM microscope (Nikon Instruments Inc., Melville, NY). Using the 100X CFI Apo TIRF oil-immersion objective (1.49 NA), 256 by 256 XY scans, 0.165 μm/pixel size, were acquired, and 3D images were obtained using the astigmatism method of 3D localization [113 (link)]. The 647nm, 561nm, and 488nm laser lines were used to excite Alexa 647-conjugated OKT4, SNAP-ICAM-1 labeled with SNAP Surface Alexa 546, and Alexa 488-conjugated Mabs, respectively. The 405 laser was used to obtain TIRF images of the CLIP-Vpr Alexa 360 signal. The oxygen-scavenging imaging buffer was 14mg glucose oxidase and 50μL of 17mg/mL catalase (Sigma) in 200μL Component A (10mM Tris, 50mM NaCl); Component B (50mM Tris-HCl, 10mM NaCl, 10% glucose); and 1M cysteamine (MEA).
Single molecule fitting and Gaussian images were rendered using the N-STORM software NIS Elements (version 4.30.01). The localization precision for all three fluorophores was determined to be 20 nm using full width at half maximum (FWHM), with a 50 nm axial resolution. After high resolution images were obtained, ROIs were defined around single virions with ICAM-1 and Mab signals within 200nm. Virus-bound Mab signals and the number of localized signals were recorded.
Single molecule fitting and Gaussian images were rendered using the N-STORM software NIS Elements (version 4.30.01). The localization precision for all three fluorophores was determined to be 20 nm using full width at half maximum (FWHM), with a 50 nm axial resolution. After high resolution images were obtained, ROIs were defined around single virions with ICAM-1 and Mab signals within 200nm. Virus-bound Mab signals and the number of localized signals were recorded.
3
Nanodiamonds Imaged by STORM Microscopy
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Nanodiamonds were imaged using a Nikon N-STORM microscope (Nikon, UK Ltd) using an SR Apochromat TIRF 100 × 1.49 N. A., oil immersion objective lens. The illumination powers of light sources are reported as measured at the tip of the optical fiber. Fluorescence was detected with either an Orca Flash 4 v3 (Hamamatsu) or an EM-CCD Camera iXon DU897 (Andor). Imaging was performed in total internal reflection (TIRF) illumination mode to image close to the region above the coverslip. The field of view imaged typically covered 128 × 128 camera pixels corresponding to an area on the sample of ∼20 × 20 μm2. An in-built focus-lock system was used to prevent axial drift of the sample during data acquisition. The emission was collected and passed through a Laser QUAD filter set for TIRF applications, a multi edge dichroic filter with windows at 502–538 nm and 660–780 nm.
The laser excitation was at 561 nm, at a peak power density of 1.2 kW cm−2 and with an exposure time in the range of 10–30 m s.
The laser excitation was at 561 nm, at a peak power density of 1.2 kW cm−2 and with an exposure time in the range of 10–30 m s.
4
Live-cell TIRF microscopy for protein dynamics
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The two-color live-cell TIRF movies were acquired using a previously described custom-built microscope in the Princeton University Lewis-Sigler Imaging Core Facility (15 (link)). Three-color live-cell TIRF movies were acquired on a previously described Nikon N-STORM microscope in the Princeton University Molecular Biology Confocal Microscopy Facility (15 (link)), or a previously described custom-built ring-TIRF microscope in the Enquist laboratory (39 (link)). Images were prepared for publication using the following functions and plugins in Fiji/ImageJ: adjust brightness and contrast, Kalman filter (to reduce noise in time course microscopy images), and Z project (to make maximum-intensity projections). We calculated a maximum-difference projection, depicted in Fig. 1A , which emphasizes areas where the gM-pHluorin fluorescence intensity rapidly increases, as previously described (15 (link)).
5
DNA-PAINT imaging of neuronal proteins
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Fourteen days in vitro (DIV) neurons were fixed using 4% PFA in PEM (80 mM Pipes, 5 mM EGTA, and 2 mM MgCl2, pH 6.8) for 10 min. After blocking, they were incubated with primary antibodies overnight at 4 °C, then with DNA-PAINT secondary antibodies (Ultivue) for 1 h at room temperature. DNA-PAINT imaging was performed on an N-STORM microscope (Nikon Instruments). The two channels were imaged sequentially in imaging buffer (PBS, 500 mM NaCl, 5% dextran sulfate) using the 647 nm laser at 40–60% power for 40,000 frames at 20 Hz. The first channel imaged was Mical3 with 0.26 nM imager I2-650, then α2-spectrin with 0.125 nM imager I1-650 (imagers from Ultivue).
6
Single-Molecule Fluorescence Imaging Techniques
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Cells were spotted on a 2% agarose pad made with 0.22 µm-filtered M2G. Imaging was performed at room temperature. All images were acquired on either an N-STORM microscope (Nikon) or a custom-built setup assembled on a commercial Axio Observer D1 microscope stand (Carl Zeiss). The N-STORM microscope was equipped with a CFI Apo TIRF 100 × oil immersion objective (NA 1.49), lasers emitting at 405 nm and 561 nm (Agilent Technologies, Santa Clara, CA) and a built-in Perfect Focus system. Raw single molecule data were taken in a field of view of ∼43 × 43 µm2 (256 × 256 pixels) with an Andor iXon X3 DU 897 EM-CCD camera (Andor Technology, South Windsor, CT) at a frame rate of 28.5–29 frames per second (fps). Under this acquisition condition, diffusing molecules appeared blurred and were rejected from our analysis. The custom-built microscope set up (Huang et al., 2013 (link)) was equipped with a 100 × /1.46-NA oil-immersion objective (alpha Plan-Apochromat 100 × /1.46 oil, Carl Zeiss), lasers emitting at 405 nm (50 mW, CrystaLaser, Reno, Nevada) and 568 nm (Innova 300, ∼400 mW, Coherent, Santa Barbara, CA). Fluorescence was recorded with an ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics) at a frame rate of 100 fps and a field of view of 23 × 23 μm2.
7
Broadband Focusing Characterization of Graphene Oxide Lenses
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A Nikon N-STORM microscope with 700 nm plane wave illumination is used for the visible lens characterization. The cross-sectional distributions of the generated focal spots of the GO lenses are captured with a numerical aperture=1.4, × 100 objective into a charge-coupled device (CCD) camera. By gradually adjusting the distance between the objective and the GO lens in a step of 100 nm, we are able to obtain the optical intensity distributions at different axial positions, and the 3D images of the focal spot can be reconstructed. By normalizing the sensitivity and exposure time of the CCD camera, the lateral cross-sectional intensity distributions can be captured and the peak focusing intensities can be compared directly.
For the broadband focusing characterization, a super-continuum laser with illumination wavelengths from 450 to 1,500 nm is used as the light source. Two CCD cameras, operating at visible (Watec 902H3 SUPREME) and infrared (Xenics Xeva-1.7-320) regions, respectively, are used to capture the focal plane images of the GO lens at different wavelengths.
For the broadband focusing characterization, a super-continuum laser with illumination wavelengths from 450 to 1,500 nm is used as the light source. Two CCD cameras, operating at visible (Watec 902H3 SUPREME) and infrared (Xenics Xeva-1.7-320) regions, respectively, are used to capture the focal plane images of the GO lens at different wavelengths.
8
Live-Cell Imaging Coverslip Preparation
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Live-cell imaging dishes were prepared as follows: Schott Nexterion 1.5H 22-mm × 22-mm coverslips (170 µm ± 5 µm) were sonicated for 30 min in 1 M KOH and an additional 30 min in 200 proof ethanol (Decon Labs, 2701). The treated coverslips were attached to the bottoms of the 35-mm dishes containing a hole in the center using Sylgard 184 silicone elastomer kit (Dow Corning, 3097366-1004).
Cells were plated on these coverslips ∼17 h before imaging and labeled with HaloTag Janelia fluor 646 (JF646 was a gift from the Lavis laboratory) for 30 sec in 37°C complete medium at a concentration that produced ∼10 localizations per frame (Zhen et al. 2016 (link)). The concentrations used were 5 nM HaloTag-EZH2 and 25 nM HaloTag-SUZ12.
Cells were imaged in 2 mL of FluoroBrite DMEM (Thermo Fisher, A1896701) at 37°C and 5% CO2. All single-molecule imaging was performed under high-incline laser conditions (Tokunaga et al. 2008 (link)) on a Nikon N-Storm microscope described previously (Schmidt et al. 2016 (link)). All imaging was performed using HiLo illumination (Tokunaga et al. 2008 (link)). Diffusion imaging was performed at 97.5 fps, 25% AOTF, and continuous illumination, whereas lifetime analysis imaging was performed at 2 fps, 15% AOTF, and 31-msec exposures of intermittent illumination. n > 12 cells were analyzed for each biological replicate.
Cells were plated on these coverslips ∼17 h before imaging and labeled with HaloTag Janelia fluor 646 (JF646 was a gift from the Lavis laboratory) for 30 sec in 37°C complete medium at a concentration that produced ∼10 localizations per frame (Zhen et al. 2016 (link)). The concentrations used were 5 nM HaloTag-EZH2 and 25 nM HaloTag-SUZ12.
Cells were imaged in 2 mL of FluoroBrite DMEM (Thermo Fisher, A1896701) at 37°C and 5% CO2. All single-molecule imaging was performed under high-incline laser conditions (Tokunaga et al. 2008 (link)) on a Nikon N-Storm microscope described previously (Schmidt et al. 2016 (link)). All imaging was performed using HiLo illumination (Tokunaga et al. 2008 (link)). Diffusion imaging was performed at 97.5 fps, 25% AOTF, and continuous illumination, whereas lifetime analysis imaging was performed at 2 fps, 15% AOTF, and 31-msec exposures of intermittent illumination. n > 12 cells were analyzed for each biological replicate.
9
Single-Molecule Dynamics of B. subtilis PBP2B
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B. subtilis strain bGS28, in which the native Pbp2B has been replaced with an IPTG-inducible, Halo-tagged Pbp2B, was imaged as described in Bisson-Filho et al.12 (link). Briefly, cells were grown in CH media, Pbp2B was induced with 20 μM IPTG and labeled with 100 nM JF549 conjugated to HaloTag ligand, and cells were immobilized under an agarose pad for imaging. Cells were imaged in TIRF on a Nikon N-STORM microscope; time lapses were acquired with streaming 30 ms exposures for 1 min. Particle tracking was performed in TrackMate using the simple LAP tracker with the following settings: particle diameter was 300 nm, maximum linking distance was 300 nm, and no frame gaps were allowed. Tracks between 5 and 25 frames were analyzed further using custom MATLAB code12 (link). MSD vs. t was calculated for each track, and the diffusion coefficient was computed by the MSD equation noted above.
10
Super-Resolution Imaging of S. aureus
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SRRF imaging was carried out on a Nikon N-STORM microscope with a 100x objective (Plan-APOCHROMAT 100x/1.49 Oil, Nikon) and additional 1.5x magnification to collect fluorescence onto an EMCCD camera (iXon Ultra 897, Andor). Samples were prepared as follows. 10 µl of exponentially grown, fixed S. aureus SH1000 JGL232 (plsY-gfp) were resuspended in PBS and placed onto #1.5 thickness clean coverslips coated in poly-L-lysine and left for 20 min to settle. The coverslip was then washed once in milli-Q water and mounted in 100 mM mercaptoethylamine before imaging. For each SRRF image, 500 frames were acquired with a 488 nm laser operating at 100% power and 10 ms exposure time. The resultant time series were processed with the NanoJ-SRRF software package in Fiji.
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