Cells were plated on poly-d -lysine–coated coverslips and transfected with Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer's recommendation. Cells were fixed in 4% formaldehyde (8 min) and permeabilized with 0.1% Triton X-100 in PBS with 5% BSA (10 min). After blocking with 5% BSA in PBS, primary antibodies were incubated overnight at 4 °C and stained with secondary antibodies for 1 h. Coverslips were mounted with SlowFade reagent (Life Technologies). Samples were imaged on a confocal microscope (A1R+, Nikon) equipped with GaAsP detectors and an oil objective (60×/NA1.40; Plan Apo) or on a STORM/TIRF microscope (Nikon) equipped with a laser (MLC400B, Agilent) charge-coupled device camera (iXon Ultra, Andor) and an oil objective (100×/NA1.49; ApoTIRF). The microscopes were controlled by Nikon Elements software. Image acquisition was performed at ambient temperature, and images were analyzed with ImageJ (National Institutes of Health) or Nikon Elements software.
Mlc400b
Manufactured by Agilent Technologies
The MLC400B is a laboratory instrument manufactured by Agilent Technologies. It is designed to perform liquid chromatography measurements. The core function of the MLC400B is to separate and analyze chemical compounds in liquid samples.
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Lab products found in correlation
Dmk 23u274, by Imaging Source (3 mentions)
Ixonem du 897 camera, by Nikon (2 mentions)
Eclipse ti e microscope, by Nikon (2 mentions)
Du897, by Oxford Instruments (2 mentions)
Perfect focus system, by Nikon (2 mentions)
Elements software, by Nikon (2 mentions)
Eclipse ti microscope, by Nikon (2 mentions)
Ixon ultra, by Oxford Instruments (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Plan apo, by Nikon (1 mentions)
Slowfade reagent, by Thermo Fisher Scientific (1 mentions)
Cfi apo tirf 100 1.49 na oil immersion objective, by Nikon (1 mentions)
Ultra du 897u cs0 bv, by Oxford Instruments (1 mentions)
Nis element, by Nikon (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Du 897e, by Oxford Instruments (1 mentions)
Clara cooled ccd camera, by Oxford Instruments (1 mentions)
Nis elements software, by Nikon (1 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Motorizedxy stage, by Nikon (1 mentions)
9 protocols using mlc400b
1
Fluorescent Labeling of Cellular Proteins
2
Thioflavin T Staining of Nanowire Fibrils
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A 600 µM Thioflavin T (ThT) stock solution was used to bring nanowire solutions to a final concentration of 100 µM ThT. Samples were incubated for 1 hour at room temperature before depositing 10 µl of solution on cleaned microscope slides and coverslips. Microscope slides (Fisherfinest Premium Catalogue No. 12-544-1) and coverslips were sonicated for 20 minutes each in 1 M KOH, Milli-Q water, and finally 70% ethanol before they were air-dried with nitrogen. Control samples that had only ThT or only nanowires did not show any fluorescent fibril structures (Fig. 6e ), confirming that the observed fluorescence is due to ThT binding to nanowires. Samples were excited with a 445-nm CW laser (Agilent Technologies MLC 400B) used at 30% of maximum power (<10 mW). TIRF microscopy images were acquired on an Eclipse TiE microscope (Nikon) equipped with an Andor iXonEM+ DU-897 camera. All images were processed and analysed using ImageJ software.
3
Thioflavin T Staining of Nanowire Fibrils
4
Super-Resolution Fluorescence Microscopy Setup
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Experiments were performed with a Nikon Ti Eclipse microscope with a Nikon APO 100 X objective (N.A. 1.49). The microscope stabilizes the sample in z-axis with the Perfect Focus System. An Agilent laser system MLC400B with four fiber-coupled lasers (405 nm, 488 nm, 561 nm and 640 nm) was used for illumination. Elements software from Nikon was used for data acquisition. A back illuminated EMCCD (Andor DU897) was used for recording. For 3-D imaging, a cylindrical lens (CVI Melles Griot, SCX-25.4-5000.0- C-425-675) of 10 m focal length was inserted below the back aperture of the objective. A motorized stage from ASI with a Piezo top plate (ASI PZ-2000FT) was used for x-y-z position control. A quad-band dichroic (Chroma, ZT405-488-561-640RPC) was used and band-pass emission filter 525/50, 600/50, 710/40, 641/75 was used for fluorescence imaging.
5
TIRF Microscopy Setup for Single-Molecule Imaging
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STORM data were acquired on Nikon Ti motorized inverted microscope with Perfect Focus System, equipped with Nikon total internal reflection fluorescence (TIRF) illuminator and Prior Proscan II motorized stage, filter wheels, and shutters. For illumination, we used the output of Agilent MLC400B monolithic laser combiner with 405-, 561-, and 647-nm laser lines, reflected by a quad-band dichroic beamsplitter (ZT405/488/561/647rpc; Chroma). Emitted fluorescence was collected by a CFI Apo TIRF 100×/1.49-NA oil-immersion objective (Nikon) and filtered by the dichroic beamsplitter, additional quad-band emission filter (ZET405/488/561/647m; Chroma), and an emission filter (ET600/50m or ET700/75m; Chroma) for CF568 and AF647 emissions, respectively. The filtered emission was projected onto electron-multiplying charge-coupled device camera (Ultra DU-897U-CS0-#BV; iXon). Additional 1.5× intermediate magnification was applied, which resulted in a pixel size of 104.3 nm. NIS-Elements software was used to control the hardware.
6
Super-resolution Fluorescence Microscopy Setup
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Imaging experiments were performed using Nikon inverted microscope (Ti-Eclipse) system. Four laser lines (MLC 400B, Agilent Technologies, equipped with 405, 488, 561, 640 nm) were combined through single mode optical fiber and focused on the back focal plane of the objective lens (APO 100×, NA 1.49, Nikon) which was used for illumination and collection of photons. Samples were loaded on xyz-translational stage (xy: motorized, z: piezo). Focus was maintained by Nikon perfect focus system (PFS). For 3D measurement a cylindrical lens (fcyl = 10 m) was put in the slide-in port between the objective lens and the tube lens to generate astigmatism.28 (link) Quad-band dichroic mirror (Chroma, ZT405-488-561-640RPC) was used for separating excitation and fluorescence emission, band pass filters were used for each spectrum (447/60, 525/50, 600/50, and 680/40). A back illuminated EMCCD camera (DU897, Andor Technology) was used for recording fluorescence signal. For drift correction, 760 nm LED was used for illuminating the sample from the top and transmitted IR signal was collected by separate IR sensitive CMOS camera (DMK 23U274, The Imaging Source) after reflected by 750 nm long pass dichroic mirror placed above the quad-band dichroic mirror. For image acquisition and device control Nikon NIS Element and IC capture were used.
7
Multi-modal Fluorescence Microscopy Techniques
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Fluorescence and super-resolution microscopy are performed on a Nikon Ti Eclipse microscope with 100X objective (CFI Apo TIRF 100x Oil, N.A. 1.49, Nikon). Excitation lasers (MLC400B, Agilent Technologies, 405 nm, 488 nm, 561 nm and 640 nm) were used for exciting different fluorescent probes. The images were recorded in an EMCCD (iXon DU-897E, Andor). A quad-band dichroic (ZT405-488-561-640RPC, Chroma) was used for reflectance of the excitation laser and transmission of fluorescence. The emission filter choices were 525/50 (DAPI, GFP, ATTO488), 600/50 (mCherry, Texas Red, PI), or 700/75 (Alexa647, ATTO647N). The live cell samples were imaged inside a 30°C temperature controlled chamber (InVivo Scientific) unless otherwise noted. The static conventional fluorescence images shown were averages of 10 frames taken at 50–100 ms, except for the kinesin experiment, where the spots were rapidly moving so a single frame image was shown. The cells were imaged in 10% FBS DMEM P/R free medium on a glass-bottom dish.
8
Multi-Modal Confocal Microscopy Imaging
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Microscopic imaging was performed on a spinning disk (Yokagawa CSU-X1; Andor Technology) confocal microscope using a 60 × 1.4 NA oil immersion or a 60 × 1.2 NA water immersion objective lens on a TiE microscope equipped with Perfect Focus System (Nikon) equipped with an electronic shutter (Sutter Instrument) for transmitted illumination, a linear encoded X and Y, motorized stage (ASI Technologies), and a multi-bandpass dichromatic mirror (Semrock) and bandpass filters (Chroma Technology Corp.) in an electronic filterwheel for selection of BFP, GFP, or RFP emission. 405-, 488-, 561-, and 640-nm laser illumination was provided by a high-powered (20 mW 405-nm; 50 mW 488-; 561 and 640-nm) monolithic laser combiner module (MLC 400B; Agilent Technologies) that were shuttered with electronic shutters and directed to a fiber-coupled output port with an Acousto optic tunable filter and to the confocal scan-head via a singlemode polarization-maintaining fiber coupled delivery system (Agilent Technologies). Images were acquired using a Clara cooled CCD camera (Andor) operated in the 14-bit mode. Microscope system automation was controlled with NIS elements software (Nikon Instruments).
9
Super-Resolution Imaging of Hippocampal Neurons
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Immunolabeled hippocampal neurons were imaged on a commercially available N-STORM system (Nikon) composed of an Eclipse Ti-E inverted microscope (Nikon) equipped with a Perfect Focus System (Ti-PSF, Nikon) and a motorized stage (Nikon). Total internal reflection fluorescence (TIRF) and highly inclined and laminated optical (HILO) sheet22 (link) configurations were adjusted using a motorized TIRF illuminator (Nikon) in combination with a oil-immersion objective (CFL Apo TIRF, NA 1.49, Nikon) resulting in a final pixel size of 158 nm. For imaging, an excitation wavelength of 647 nm was used, harbored in a laser combiner (MLC400B, Agilent). An optical fiber guided the laser beam to the microscope body and via a dichroic mirror (T660LPXR, Chroma) to the sample plane. Fluorescence emission were separated from excitation light via a bandpass filter (ET705/72m, Chroma) and detected by an iXon Ultra EMCCD camera (DU-897U-CS0-23 #BV, Andor). The software NIS-Elements Ar/C (Nikon) and 23 (link) were used to control the setup and the camera.
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