Apo tirf 1.49 na objective

Manufactured by Nikon

Sourced in United States

The 100× Apo TIRF 1.49 NA objective is a high-numerical aperture objective designed for Total Internal Reflection Fluorescence (TIRF) microscopy. It features an Apochromatic (Apo) optical correction and a numerical aperture of 1.49, which allows for high-resolution imaging of samples near the coverslip surface.

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Lab products found in correlation

Clara ccd camera, by Oxford Instruments (4 mentions) Eclipse ti wide field inverted microscope, by Nikon (3 mentions) Nis elements imaging software, by Nikon (3 mentions) Clara charge coupled device camera, by Oxford Instruments (2 mentions) Janelia fluor 549 halotag ligand, by Promega (2 mentions) Snap cell 647 sir, by New England Biolabs (2 mentions) Csu xi confocal spinning disk head, by Nikon (2 mentions) Cellometer auto 1000 bright field cell counter, by Revvity (2 mentions) Imagem em ccd digital camera, by Hamamatsu Photonics (1 mentions) Metamorph 7.7 imaging software, by Molecular Devices (1 mentions) Eclipse e600fn microscope, by Nikon (1 mentions) Matlab 2018b, by MathWorks (1 mentions) Chamber slide, by Ibidi (1 mentions) A1 scanning confocal microscope, by Nikon (1 mentions) Deltavision elite system, by Cytiva (1 mentions) Nis element, by Nikon (1 mentions) Orca er camera, by Hamamatsu Photonics (1 mentions) Eclipse te2000 u, by Nikon (1 mentions) Emccd camera, by Nikon (1 mentions) Inverted fluorescence microscope, by Nikon (1 mentions)

12 protocols using apo tirf 1.49 na objective

Time-lapse Microscopy of Plasmid Dynamics

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Time lapses of strains containing the 10-kb lacO/LacI-GFP array or containing the dicentric plasmid were performed at room temperature (25°C) using an Eclipse Ti wide-field inverted microscope (Nikon) with a 100× Apo TIRF 1.49 NA objective (Nikon) and Clara charge-coupled device camera (Andor) using Nikon NIS Elements imaging software (Nikon). Time lapses of strains containing the 10-kb lacO/LacI-GFP array were 10 min in duration with 30 s intervals. At each interval a seven-step Z-stack of 300-nm step size was acquired in the GFP, RFP, and Trans channels. Time lapses of strains containing the dicentric plasmid were the same as above but with a duration of 20 min.
Population images of the dicentric plasmid strains and of strains containing the 1.2-kb lacO/LacI-GFP array were imaged at room temperature (25°C) using an Eclipse E600FN microscope (Nikon) with a 100× Plan Apo TIRF 1.45 NA objective (Nikon) and ImagEM EM-CCD digital camera (Hamamatsu) with a custom Lumencor LED illumination system (Lumencor) using MetaMorph 7.7 imaging software (Molecular Devices). Each acquisition was a seven-step Z-stack with a 300-nm step size in the GFP, RFP, and Trans channels.

Lawrimore J., Doshi A., Friedman B., Yeh E, & Bloom K. (2018). Geometric partitioning of cohesin and condensin is a consequence of chromatin loops. Molecular Biology of the Cell, 29(22), 2737-2750.

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Confocal Imaging of Cell Cultures

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Confocal images were acquired with an Apochromat 63× 1.4 NA objective lens (Carl Zeiss, Jena, Germany) on a Marinas spinning disk confocal imaging system (Intelligent Imaging Innovations, Denver, CO) using an EM charge-coupled device camera (Evolve; Photometrics, Tucson, AZ), or a 100× Apo TIRF 1.49 NA objective (Nikon Instruments, Tokyo, Japan) on a Yokogawa CSU-X1 spinning disk system using an EM charge-coupled device camera (Evolve; Photometrics). Cells were imaged in HEPES-buffered growth media. Confocal z-stacks were taken using 200 nm steps. Images were deconvoluted using Slidebook 6. Individual 16-bit tiff image files were exported, then processed using ImageJ.

Manor U., Bartholomew S., Golani G., Christenson E., Kozlov M., Higgs H., Spudich J, & Lippincott-Schwartz J. (2015). A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division. eLife, 4, e08828.

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Yeast Cell Imaging: Fluorescence Microscopy Protocol

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Fluorescent image stacks of unbudded yeast cells were acquired using a Eclipse Ti wide-field inverted microscope (Nikon) with a 100× Apo TIRF 1.49 NA objective (Nikon) and Clara charge-coupled device camera (Andor) using Nikon NIS Elements imaging software (Nikon). Each image stack contained 7 Z-planes with 200 nm step-size.
Image stacks of experimental images were cropped to 7 Z-plane image stacks of single cells using ImageJ and saved as TIFF files. The cropped Z-stacks were read into MATLAB 2018b (MathWorks), converted into maximum intensity projections, and the projections of hmo1Δ and fob1Δ were cropped to 55 × 55 pixels, to match the dimensions of WT projections, using MATLAB function padarray with replicate option specified to extend outer edge of pixel values to ensure the center of all cropped images was the brightest pixel. The intensity values of all projections were normalized by subtracting all intensity values by the minimum value and then dividing the resulting values by the maximum intensity value after subtraction. The normalized intensity values were stored with double point precision, preventing any loss in dynamic range.

Walker B., Taylor D., Lawrimore J., Hult C., Adalsteinsson D., Bloom K, & Forest M.G. (2019). Transient crosslinking kinetics optimize gene cluster interactions. PLoS Computational Biology, 15(8), e1007124.

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Visualizing Centrosomes in Mouse Cells

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Centrosomes were visualized by the centrin-GFP marker in PRG5-derived mouse embryonic fibroblasts grown in chamber slides (ibidi) and were imaged (1 µM, 10×, 40×/1.35 NA) using the DeltaVision elite system (Applied Precision). Centrosomes visualized by the centrin-GFP marker in tissue cryosections were imaged (at 0.2-μm Z-sections with a Nikon 100× APO TIRF 1.49 NA objective) using the Nikon A1 scanning confocal microscope operated with NIS-Elements (Nikon). Additional quantification was done using an Olympus bx43 microscope equipped with a X-Cite series 120Q fluorescence lamp (60× magnification) and using Thermo Scientific Menzel-Gläser Deckgläser Coverslips ø12 mm #1.

Shoshani O., Bakker B., de Haan L., Tijhuis A.E., Wang Y., Kim D.H., Maldonado M., Demarest M.A., Artates J., Zhengyu O., Mark A., Wardenaar R., Sasik R., Spierings D.C., Vitre B., Fisch K., Foijer F, & Cleveland D.W. (2021). Transient genomic instability drives tumorigenesis through accelerated clonal evolution. Genes & Development, 35(15-16), 1093-1108.

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Visualizing Chromosomal Dynamics in Budding Yeast

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Budding yeast strain KBY8065 (Mat a CEN15(1.8)-GFP[10kb] ade2-1, his3-11, trp1-1, ura3-1, leu2-3,112, can1-100, LacINLSGFP:HIS3, lacO::URA3, Spc29RFP:Hyg) was grown in liquid yeast extract peptone dextrose at 24°C. Cells were imaged in liquid yeast complete medium at 24°C. Time-lapse images were acquired on an Eclipse Ti wide-field inverted microscope (Nikon) with a 100× Apo TIRF 1.49 NA objective (Nikon) and a Clara CCD camera (Andor) using the Nikon NIS Elements imaging software. Time lapses were 10 min in duration with 30 s intervals. At each interval, a seven-step Z-stack of 400-nm step size was acquired in the GFP, RFP, and Trans channels.
Metaphase yeast cells (medium budded cells with two Spc29-RFP foci) were cropped by hand from the original time-lapse images. Both original and denoised time lapses were automatically tracked using a custom MATLAB program. The motion of the two sister lacO/LacI-GFP foci and the two sister Spc29-RFP foci were the motion of one focus relative to the other as in Chacón et al. (2014) (link), and the radius of confinement was calculated by a custom MATLAB program. For the images shown as illustrations, the heterogeneous background was subtracted with the rolling-ball method (10 pixel radius) in FIJI. Tracking results are from original and N2V-denoised images.

Kefer P., Iqbal F., Locatelli M., Lawrimore J., Zhang M., Bloom K., Bonin K., Vidi P.A, & Liu J. (2021). Performance of deep learning restoration methods for the extraction of particle dynamics in noisy microscopy image sequences. Molecular Biology of the Cell, 32(9), 903-914.

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Imaging Cellular Processes with Fluorescence

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Cells containing lacO/LacI-GFP (KBY8065) were imaged in YC-complete media containing 0.02% sodium azide, and 1 μM of deoxyglucose for 10 min every 30 s using a Nikon Eclipse Ti wide-field inverted microscope with a 100× Apo TIRF 1.49 NA objective (Nikon, Melville, NY) and Andor Clara CCD camera (Andor, South Windsor, CT) using Nikon NIS Elements imaging software (Nikon) at room temperature (25°C). At each interval, a 7 Z-plane section image stack with a 400-nm step size was taken.
Cells containing fluorescently labeled cohesin (KBY9471) were imaged in YC-complete media with 2% filter-sterile glucose using a an inverted, wide-field microscope (Eclipse TE2000-U; Nikon) with a 100× Plan Apo 1.4 NA digital interference contrast oil-immersion lens with an Orca ER camera (Hamamatsu Photonics, Bridgewater, NJ) with MetaMorph 6.1 software at room temperature (25°C). At each interval, a 5 Z-plane section image stack with a 300-nm step size was taken.

Lawrimore J., Aicher J.K., Hahn P., Fulp A., Kompa B., Vicci L., Falvo M., Taylor RM I.I, & Bloom K. (2016). ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin. Molecular Biology of the Cell, 27(1), 153-166.

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Visualizing TMC1 and TMC2 in Mouse Inner Ear

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Inner ear tissue from mice expressing TMC1-mCherry and/or TMC2-AcGFP was dissected and mounted between slide and covers slip and viewed in a Nikon inverted fluorescence microscope, outfitted with a spinning disk confocal scan head, 100× Apo TIRF 1.49 N.A. objective, and an EM-CCD camera. NIS-Elements imaging software was utilized for image acquisition and analysis.For immunofluorescence wild type mice and rat tissue was labeled using polyclonal antibodies against mouse TMC1 (PB277 and PB612) and mouse TMC2 (PB361). See Supplemental Information for further details.

Kurima K., Ebrahim S., Pan B., Sedlacek M., Sengupta P., Millis B.A., Cui R., Nakanishi H., Fujikawa T., Kawashima Y., Choi B.Y., Monahan K., Holt J.R., Griffith A.J, & Kachar B. (2015). TMC1 and TMC2 Localize at the Site of Mechanotransduction in Mammalian Inner Ear Hair Cell Stereocilia. Cell reports, 12(10), 1606-1617.

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Tracking Sister Chromatid Motions in Budding Yeast

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Budding yeast strain KBY8065 (Mat a CEN15(1.8)-GFP[10kb] ade2-1, his3-11, trp1-1, ura3-1, leu2-3,112, can1-100, LacINLSGFP:HIS3, lacO::URA3, Spc29RFP:Hyg) was grown in liquid yeast extract peptone dextrose at 24°C. Cells were imaged in liquid yeast complete medium at 24°C. Time-lapse images were acquired on an Eclipse Ti wide-field inverted microscope (Nikon) with a 100× Apo TIRF 1.49 NA objective (Nikon) and a Clara CCD camera (Andor) using the Nikon NIS Elements imaging software. Time lapses were 10 min in duration with 30 s intervals. At each interval, a seven-step Z-stack of 400-nm step size was acquired in the GFP, RFP, and Trans channels.
Metaphase yeast cells (medium budded cells with two Spc29-RFP foci) were cropped by hand from the original time-lapse images. Both original and denoised time lapses were automatically tracked using a custom MATLAB program. The motion of the two sister lacO/ LacI-GFP foci and the two sister Spc29-RFP foci were the motion of one focus relative to the other as in Chacón et al. (2014) , and the radius of confinement was calculated by a custom MATLAB program. For the images shown as illustrations, the heterogeneous background was subtracted with the rolling-ball method (10 pixel radius) in FIJI. Tracking results are from original and N2V-denoised images.

Kefer P., Iqbal F., Locatelli M., Lawrimore J., Zhang M., Bloom K., Bonin K., Vidi P., & Liu J. (2021). Performance of deep learning restoration methods for the extraction of particle dynamics in noisy microscopy image sequences.

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Chromatin dynamics during mitosis

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We examined the in vivo dynamics of chromatin during mitosis to determine the behavior of a region of the chromosome visualized through the binding of lac repressor fused to GFP (LacI-GFP) to lac operator (lacO). The lacO is a repeated array of operator DNA sequences (256 repeats, 10 kilobase prs.) integrated 6.8 kb from the centromere on chromosome XV. The spindle pole bodies (sites of microtubulare nucleation) are visualized through a fusion protein between a spindle pole component (Spc29) and RFP (red fluorescent protein). Cells were grown to logarithmic phase at 24°C in rich media. Images were acquired on a Nikon Eclipse Ti wide-field inverted microscope with a 100x Apo TIRF 1.49 NA objective (Nikon, Melville, New York, USA) and Andor Clara CCD camera (Andor, South Windsor, Connecticut, USA) using continuous laser illumination. Images were streamed at the net effective camera acquisition rate of 22 frames/sec. The CCD camera’s exposure time was set nominally to be the inverse of the net frame rate. Images were acquired at room temperature with Nikon NIS Elements imaging software (Nikon, Melville, New York, USA). The program “Speckle Tracker” and other methods outlined in Ref. [66 (link)] were used to estimate the centroid of GFP and RFP spots for position measurements.

Calderon C.P, & Bloom K. (2015). Inferring Latent States and Refining Force Estimates via Hierarchical Dirichlet Process Modeling in Single Particle Tracking Experiments. PLoS ONE, 10(9), e0137633.

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Detecting CD80:CTLA-4 Clusters on Cell Membrane

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For detecting CD80:CTLA-4 clusters on cell membrane in Figure 4H, Raji (CD80–mGFP⁺CD86⁻PD-L1–SNAP⁺) cells were incubated with 1 μg/mL SC647-labeled CTLA-4-GCN4 on ice for 35 min, with or without the presence of 20 μg/mL atezolizumab, followed by 2 washes with 1x PBS plus 2% FBS. Cells were then dropped on a poly-D-lysine treated 96-well plate for TIRF microscopy. Images were acquired at 37°C on a Nikon Eclipse Ti microscope equipped with a 100x ApoTIRF 1.49 NA objective, controlled by the Micro-Manager software. Microscopy images were then analyzed by ImageJ.

Zhao Y., Lee C.K., Lin C.H., Gassen R.B., Xu X., Huang Z., Xiao C., Bonorino C., Lu L.F., Bui J.D, & Hui E. (2019). PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways. Immunity, 51(6), 1059-1073.e9.

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