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Cfi apo tirf 100 oil objective

Manufactured by Nikon
Sourced in Germany

The CFI Apo TIRF ×100 oil objective is a high-numerical aperture objective designed for total internal reflection fluorescence (TIRF) microscopy. It provides a magnification of 100x and is optimized for use with oil immersion.

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4 protocols using cfi apo tirf 100 oil objective

1

dSTORM Imaging of Fixed and Live Cells

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Two-color dSTORM imaging was conducted both for fixed and live cells.
Fixed cells were suspended in a STORM imaging buffer51 (link),52 (link). Live cells were suspended in Imaging buffer (RPMI without phenol red, 10% FBS, 25 mM HEPES.The imaging was performed using a total internal reflection (TIRF) Nikon microscope with a CFI Apo TIRF ×100 oil objective (NA 1.49, WD 0.12 mm). Imaging in TIRF mode served to visualize molecules at the PM of spreading cells in close proximity to the coverslip (up to ~100–200 nm). Fluorophores were activated using a low intensity laser illumination at 405 nm (~0.5% of 30 mW in maximum), and sequentially imaged in a following frame using laser excitation at either 488 nm, or 647 nm (80–100% of 90 mW in maximum for 488 nm or 200 mW for 647 nm). Laser illumination at all wavelengths covered a circular area with a diameter of 80 μm at the sample. dSTORM acquisition sequence typically took ~2.5 min at 13.4 fps of an EMCCD Ixon+ camera. The pixel size was equivalent to 160 × 160 nm2 at the sample. Excitation and imaging were performed through a quad dichroic (C-NSTROM QUAD 405/488/561/647/FILTER; Nikon). For two-color SMLM imaging, we used immunostaining as specified for each experiment.
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2

Automated Multicolor Fluorescence Microscopy

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Imaging was performed on a custom build setup based on an automated Nikon Ti Eclipse microscope equipped with appropriate dichroic and filters (ET dapi/Fitc/cy3 or ZET405/488/561/640m dichroic, ZT405/488/561rpc or ZT405/488/561/640rpc rejection filter, ET610/75 or ET655LP bandpass, all AHF Analysentechnik, Germany), and a CFI Apo TIRF ×100 oil objective (NA 1.49, Nikon). The 488 nm, 561 nm, and 637 nm lasers (Coherent) was modulated via an acousto-optical tunable filter (AOTF) (Gooch and Housego, United States). Fluorescence was detected by an emCCD (iXON Ultra 888; Andor, United Kingdom). The z-focus was controlled by a commercial perfect focus system (Nikon, Germany). The sample was placed on a heating stage and kept at the constant temperature 25°C. Acquisitions were controlled by μManager (Edelstein et al., 2010 (link)).
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3

Live-cell and PALM Imaging of Protein Dynamics

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Imaging was performed on a TIRF microscope (Nikon) with a CFI Apo TIRF × 100 oil objective (NA 1.49, WD 0.12 mm). Imaging in TIRF mode served to visualize molecules at the PM of spreading cells in close proximity to the coverslip (up to ∼100 nm). Cells were first dropped on the coverslip in imaging buffer and given ∼10 min to spread before imaging commenced. Imaging was conducted for 20–30 cells per experiment at a rate of ∼3 min between cells, hence ∼60–90 min typically passed between the first and last cells imaged. Imaging was conducted at room temperature.
For live cell imaging, cells were illuminated by a 561 nm excitation laser at 80% power for 5,000 frames at 85 fps of an EMCCD Ixon+ camera. Fixed cells were further imaged by PALM. For that, they were illuminated using a 405 nm laser for photoactivation of the PAGFP using a changing intensity over the duration of the imaging sequence (typically, using 1–20% power). Illumination with a 488 nm laser at 100% power was used for PAGFP excitation.
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4

Automated super-resolution microscopy

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For all imaging experiments, a custom build set-up based on an automated Nikon Ti Eclipse microscopy body with suitable dichroic and emission filters (ET dapi/Fitc/cy3 dichroic, ZT405/488/561rpc rejection filter, ET525/50 for GFP and the green form of mEos2 or ET610/75 for mScarlet-I and the red photoconverted form of mEos2, all AHF Analysentechnik, Germany) and a CFI Apo TIRF 100 × oil objective (NA 1.49, Nikon) was used. A perfect focus system (Nikon, Germany) was used for z-axis control, except for ExPALM imaging, where the sample was imaged at 10–80 µm depth. All lasers (405 nm OBIS, 488 nm Sapphire LP and 561 nm OBIS, Coherent Inc. USA) were modulated by an acousto-optical tunable filter (AOTF; Gooch and Housego, USA). Fluorescence detection was performed by an emCCD camera (iXON Ultra 888, Andor, UK) at a pixel size of 129 nm. Image acquisition was controlled by a customized version of µManager [52 (link)].
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