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Sr apo tirf

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The SR Apo TIRF is a specialized imaging system designed for Nikon's microscopes. It utilizes total internal reflection fluorescence (TIRF) technology to capture high-resolution images of samples near the surface of a coverslip. The system features a high-numerical aperture objective lens and precise optics to provide optimal performance for TIRF microscopy applications.

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Lab products found in correlation

Orca flash4.0 digital cmos camera, by Hamamatsu Photonics (2 mentions) Igor pro 7, by Wavemetrics (1 mentions) Zet561 10, by Chroma Technology (1 mentions) Zt561rdc, by Chroma Technology (1 mentions) Et575lp, by Chroma Technology (1 mentions) Eclipse ti, by Nikon (1 mentions) Ti e inverted microscope, by Nikon (1 mentions) Metamorph imaging software, by Molecular Devices (1 mentions) Optosplit 2, by Cairn Research (1 mentions) Plan apo dic, by Nikon (1 mentions) Emccd 888e, by Oxford Instruments (1 mentions) Nis elements software, by Nikon (1 mentions) Ixon ultra 888, by Oxford Instruments (1 mentions) Orca flash 4, by Hamamatsu Photonics (1 mentions) Eclipse ti u, by Nikon (1 mentions) Nis elements imaging software, by Nikon (1 mentions) Lsm880 system, by Zeiss (1 mentions) N sim, by Nikon (1 mentions) Zen software, by Zeiss (1 mentions)

7 protocols using sr apo tirf

1

Fluorescence Recovery After Photobleaching (FRAP) Assay

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FRAP experiments were carried out by using the same laser scanning confocal microscope mentioned in the microscopy section at 37 °C. We used a 100× oil immersion objective lens (NA 1.49, Apo SR TIRF, Nikon). A circular region of the PM (r = 10 μm) was bleached with femtosecond laser pulses (800 nm, 2920 mW, 80 MHz, Chameleon Vision-S, Coherent, USA). Fluorescence images were simultaneously recorded at 2 frames per second by using a 488 nm laser line. To eliminate the effect of natural photobleaching, the time course fluorescence intensity of the center of the bleached region was normalized to the intensity of a nonbleached region (typically bottom left of the image). Plotted fluorescence curves were then fitted with the following equation, which is based on FRAP theory.37 (link)
ft=a+b·exp−2τDtI02τDt+I12τDt. Here, f is the normalized fluorescence intensity, a and b are constants, t is time, I0 and I1 are modified Bessel functions, and τD is the fluorescence recovery time. We determined the diffusion coefficient D using τD=w2/4D , where w is the radius of the bleached region. The fitting was carried out by using Igor Pro 7 software (WaveMetrics, USA). We obtained the mean values of D from more than four samples for each PM-cadDNA, PM-DNA, PM-cad, and Texas red-DHPE-labeled SOPC membrane condition.
Togo S., Sato K., Kawamura R., Kobayashi N., Noiri M., Nakabayashi S., Teramura Y, & Yoshikawa H.Y. (2020). Quantitative evaluation of the impact of artificial cell adhesion via DNA hybridization on E-cadherin-mediated cell adhesion. APL Bioengineering, 4(1), 016103.
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2

Super-resolution DNA-PAINT Imaging

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DNA‐PAINT imaging was carried out on an inverted Nikon Eclipse Ti microscope (Nikon Instruments) equipped with the Perfect Focus System using objective‐type total internal reflection fluorescence (TIRF) configuration (oil immersion Apo SR TIRF, NA 1.49 100× objective). A 200 mW 561 nm laser beam (Coherent Sapphire) was passed through a clean‐up filter (ZET561/10, Chroma Technology) and coupled into the microscope objective using a beam splitter (ZT561rdc, Chroma Technology). Fluorescence light was spectrally filtered with an emission filter (ET575lp, Chroma Technology) and imaged with a sCMOS camera (Andor Zyla 4.2) without further magnification, resulting in an effective pixel size of 130 nm after 2 × 2 binning. Images were acquired using a region of interest of 512 × 512 pixels. The camera read‐out rate was set to 540 MHz, and images were acquired with an integration time of 200 ms.
Geiger F., Acker J., Papa G., Wang X., Arter W.E., Saar K.L., Erkamp N.A., Qi R., Bravo J.P., Strauss S., Krainer G., Burrone O.R., Jungmann R., Knowles T.P., Engelke H, & Borodavka A. (2021). Liquid–liquid phase separation underpins the formation of replication factories in rotaviruses. The EMBO Journal, 40(21), e107711.
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3

FRET Microscopy Imaging of Fluorescent Proteins

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HEK293T transfected with fluorescent fusion proteins as mentioned above were cultured overnight in LabTek chambers coated with 10 μg/mL fibronectin in PBS for 2 h at 37°C. Image acquisition was performed on a Ti‐E inverted microscope (Nikon, Tokyo, Japan) equipped with a 100× objective (SR Apo TIRF, Nikon) and an Andor iXon Ultra‐897 EM‐CCD camera (Andor Technologies, Belfast, UK). The 488 nm (mNeonGreen) and 561 nm (mRuby3) laser line were used for fluorophore excitation in an objective‐based total internal reflection configuration and emitted light was split on the camera using a Optosplit II (Cairn Research, Faversham, UK) equipped with a 561 nm dichroic mirror, 525/50 (mNeonGreen) and 575LP (mRuby3) filters (Chroma, Bellow Falls, VT). MetaMorph imaging software (Molecular Devices, Downingtown, PA) was used to control the devices. Image analysis was performed using ImageJ (Version 1.51, National Institute of Health, Washington, DC; [54]).To determine FRET efficiencies, donor emission before (pre) and after acceptor photobleaching (post) as well as background signal was recorded and measured for a region centrally located in the laser beam. Complete ablation of the acceptor was confirmed, and FRET yields were calculated either pixelwise or for each individual region using the following formula:
FRET=donorpost−donorpredonorpost−background.
De Sousa Linhares A., Kellner F., Jutz S., Zlabinger G.J., Gabius H., Huppa J.B., Leitner J, & Steinberger P. (2020). TIM‐3 and CEACAM1 do not interact in cis and in trans. European Journal of Immunology, 50(8), 1126-1141.
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4

Super-Resolution Imaging of Cellular Structures

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Images were collected using a Nikon TiE (Nikon Instruments) inverted microscope stand equipped with a 100× PlanApo DIC, NA 1.4 objective. Images were captured using an Andor iXon EMCCD 888E camera with 0.3-μm Z steps. SIM images were acquired using a Nikon SIM (N-SIM) with a Nikon Ti2 (Nikon Instruments; LU-N3-SIM) microscope equipped with a 100× SR Apo TIRF, NA 1.49 objective. Images were captured using a Hamamatsu ORCA-Flash 4.0 Digital CMOS camera (C13440) with 0.1-μm Z step sizes. Reconstructions were generated with Nikon Elements. All images were collected at 25°C using NIS Elements software (Nikon). Raw SIM images were reconstructed using the image slice reconstruction algorithm (NIS Elements).
Stemm-Wolf A.J., O’Toole E.T., Sheridan R.M., Morgan J.T, & Pearson C.G. (2021). The SON RNA splicing factor is required for intracellular trafficking structures that promote centriole assembly and ciliogenesis. Molecular Biology of the Cell, 32(20), ar4.
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5

STORM and PWS Imaging on Inverted Microscope

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The STORM optical instrument was built on a commercial inverted microscope base (Eclipse Ti-U with the perfect focus system, Nikon). The microscope is coupled to two imaging modalities. For STORM imaging, a 637-nm laser (Obis, Coherent) is collimated through a 100× 1.49 numerical aperture (NA) objective (SR APO TIRF, Nikon) with an average power at the sample of 3 to 10 kW/cm3. Images were collected via a 100× objective and sent to an electron-multiplying CCD (iXon Ultra 888, Andor). At least 8000 frames with a 20-ms acquisition time were collected from each sample. For PWS imaging, samples were illuminated with low NA light (0.5), and images are collected using the same 100× objective and sent through a liquid crystal tunable filter (LCTF; CRI VariSpec) and then to an sCMOS camera (ORCA Flash 4.0, Hamamatsu). The LCTF allows for spectrally resolved imaging. Images are collected between 500 and 700 nm with 2-nm intervals.
Li Y., Eshein A., Virk R.K., Eid A., Wu W., Frederick J., VanDerway D., Gladstein S., Huang K., Shim A.R., Anthony N.M., Bauer G.M., Zhou X., Agrawal V., Pujadas E.M., Jain S., Esteve G., Chandler J.E., Nguyen T.Q., Bleher R., de Pablo J.J., Szleifer I., Dravid V.P., Almassalha L.M, & Backman V. (2021). Nanoscale chromatin imaging and analysis platform bridges 4D chromatin organization with molecular function. Science Advances, 7(1), eabe4310.
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6

Super-Resolution Imaging of Cellular Structures

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Superresolution imaging in Figure 2 was acquired using Nikon SIM (N-SIM) with a Nikon Ti2 (Nikon Instruments; LU-N3-SIM) microscope equipped with a 100× SR Apo TIRF, NA 1.49 objective. Images were captured using a Hamamatsu ORCA-Flash 4.0 Digital CMOS camera (C13440).
The fluorescence imaging utilized for Figures 1, 4, 5, and 7 is identical to that described in Dahl et al. (2015) (link). Briefly, images were acquired using a Nikon TiE (Nikon Instruments) inverted microscope stand equipped with a 100× PlanApo DIC, NA 1.4 objective. Images were captured using an Andor iXon EMCCD 888E camera or an Andor Xyla 4.2 CMOS camera (Andor Technologies). Images in Figure 3 were acquired using a Swept Field Confocal system (Prairie Technologies/Nikon Instruments) on a Nikon Ti inverted microscope stand equipped with a 100× Plan Apo λ, NA 1.45 objective. Images were captured with an Andor Clara CCD camera (Andor Technologies).
Nikon NIS Elements imaging software was used for image acquisition. Image acquisition times were constant within a given experiment and ranged from 50 to 400 ms, depending on the experiment. All images were acquired at ∼25°C. Images presented in most of the figures are maximum-intensity projections of the complete z-stacks. Exceptions include certain mitotic images that are constructed from selected z-planes to clearly distinguish kinetochores and lagging chromosomes.
Ganapathi Sankaran D., Stemm-Wolf A.J, & Pearson C.G. (2019). CEP135 isoform dysregulation promotes centrosome amplification in breast cancer cells. Molecular Biology of the Cell, 30(10), 1230-1244.
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7

Multimodal Imaging of Cellular Actin

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Images were collected via Airyscan or SIM imaging. Airyscan imaging was performed on a Zeiss LSM880 system, with a 63× 1.4-NA oil objective. Z collection was done with intervals at 0.18 µm. Raw images were processed using the 3D Airyscan processing function in Zen software (Carl Zeiss). SIM images were collected using a Nikon N-SIM and SR APO TIRF with a 100× 1.49-NA oil objective with 3D-SIM function. Images were processed with stack reconstruction using NIS-Elements AR v4.51 software 2016 (Nikon). Imaging parameters across conditions were kept identical for each set of experiments, to allow comparison of intensity between control and drug-treated conditions. All x–y (top view) images in the figures are maximum-intensity projections (MIPs) of the apical region of the cell, unless otherwise noted. 3D images were generated using Imaris v9.6 (Bitplane) with the surface function. To visualize myosin and Ecad signals at the border in the surface view, the actin signal at the borders was selected with surface gain size of 0.3–0.5 µm and then deducted from the total actin surface area. Photoshop CS6 (Adobe) was used to adjust brightness and contrast and adjust input levels so that the signal spanned the entire grayscale output.
Yu-Kemp H.C., Szymanski R.A., Cortes D.B., Gadda N.C., Lillich M.L., Maddox A.S, & Peifer M. (2021). Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions. The Journal of Cell Biology, 221(1), e202103074.
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