For adipogenic differentiation, differentiated cells were fixed with four percent paraformaldehyde before staining with Hoechst (nuclei), Oil Red O (lipids) and anti-mouse Perilipin A/B with secondary antibody anti-rabbit AF488 (Figure 3 E, Sigma-Aldrich #SAB4600234). Fluorescence images were acquired at 20× magnification via Eclipse Ti inverted microscope (Nikon, Amsterdam, Netherlands). We acquired multiple independent wells per experiment. One well is represented (Figure 3 E). Image analysis was performed in ImageJ/Fiji, lipid droplets (red) and nuclei (blue) images were filtered using a Gaussian blur (sigma equal to 2) before an automatic thresholding. The automatic thresholding algorithm selections were chosen on the basis of visual inspection of output images. For myogenic differentiation, differentiated cells were fixed with four percent paraformaldehyde before staining with Hoechst 33342 (nuclei, Sigma-aldrich #14533) and MF20 (Myh1, hybridoma bank). Fluorescence images were acquired at 10× magnification via an Eclipse Ti inverted microscope (Nikon).
Eclipse ti inverted microscope
Manufactured by Nikon
Sourced in Japan, United States, United Kingdom, Italy, Germany, France, Singapore
The Eclipse Ti inverted microscope is a high-performance research-grade microscope designed for a variety of applications. It features a stable inverted optical system, providing consistent and reliable imaging capabilities. The Eclipse Ti is equipped with a range of advanced optical components, enabling clear and detailed observations.
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Lab products found in correlation
Nis element, by Nikon (15 mentions)
Nis elements software, by Nikon (14 mentions)
Nis elements ar software, by Nikon (14 mentions)
Nis elements ar 3, by Nikon (8 mentions)
Nis elements imaging software, by Nikon (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Perfect focus system, by Nikon (7 mentions)
Matrigel, by BD (7 mentions)
Ds qi2 camera, by Nikon (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Lsm 700, by Zeiss (6 mentions)
Ti ct e motorized condenser, by Nikon (6 mentions)
Volocity software, by PerkinElmer (5 mentions)
Hoechst 33342, by Merck Group (5 mentions)
Tirf objective, by Nikon (5 mentions)
Coolsnap hq2 firewire ccd camera, by Nikon (5 mentions)
Ixon ultra emccd camera, by Oxford Instruments (4 mentions)
Csu x1, by Yokogawa (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
513 protocols using eclipse ti inverted microscope
1
Adipogenic and Myogenic Differentiation Assay
2
Single-molecule dynamics of key signaling proteins
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An Eclipse Ti Inverted Microscope (Nikon) with a TIRF system and Evolve EMCCD Camera (Photometrics) were used for live-cell imaging. TIRF microscopy was performed with a 100× TIRF objective with a numerical aperture of 1.49 (Nikon) and an iChrome MLE-L multilaser engine as a laser source (Toptica Photonics). Immunofluorescent imaging was also acquired in an Eclipse Ti Inverted Microscope (Nikon) with CSU-X1 confocal spinning disk unit (Yokogawa).
Time-lapse single-molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2, and N-WASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5/µm2. Far-red channel (ex=647 nm, em>655 nm) were acquired before single-molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frames per s video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleach rate, acquired at the same microscopic setup.
Time-lapse single-molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2, and N-WASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5/µm2. Far-red channel (ex=647 nm, em>655 nm) were acquired before single-molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frames per s video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleach rate, acquired at the same microscopic setup.
3
Single-Molecule Imaging of Signaling Proteins
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An Eclipse Ti inverted microscope (Nikon) with a TIRF system and Evolve EMCCD camera (Photometrics)
was used for live cell imaging. TIRF microscopy was performed with a 100x TIRF objective with a numerical aperture of 1.49 (Nikon) and an iChrome MLE-L multilaser engine as a laser source (Toptica Photonics). Immunofluorescent imaging was also acquired in an Eclipse Ti inverted microscope (Nikon)
with CSU-X1 confocal spinning disk unit (Yokogawa).
Time-lapse single molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2 and NWASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5 / µm 2 . Far-red channel (ex =647 nm, em > 655 nm) were acquired before single molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frame per second video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleaching rate, acquired at the same microscopic set up.
was used for live cell imaging. TIRF microscopy was performed with a 100x TIRF objective with a numerical aperture of 1.49 (Nikon) and an iChrome MLE-L multilaser engine as a laser source (Toptica Photonics). Immunofluorescent imaging was also acquired in an Eclipse Ti inverted microscope (Nikon)
with CSU-X1 confocal spinning disk unit (Yokogawa).
Time-lapse single molecule imaging of Grb2-tdEos, SOS-tdEos, NCK-mEos3.2 and NWASP-mEos3.2 were performed by TIRF microscopy, in a way such as to optimize signal-to-noise and temporal resolution by coupling minimizing laser power and maximizing video rate. To increase tracking accuracy, the density of individual molecules was controlled by 405 nm laser illumination to be about ~0.5 / µm 2 . Far-red channel (ex =647 nm, em > 655 nm) were acquired before single molecule recording to localize mobile and immobile ephrinA1 corrals. The autofluorescence on the red channel was completely photobleached before photo-switching Eos by a 405 nm beam. After photo-switching, a small amount of Eos molecules were visualized and recorded by EMCCD with 20 frame per second video rate. Each movie contains 1000 frames for further analysis. Membrane localized CAAX-tdEos movies were used to calculate photobleaching rate, acquired at the same microscopic set up.
4
Quantifying Endocytosis of Nanoparticles in hMSCs
5
Fluorescence Microscopy Systems Comparison
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Fluorescence microscopy was carried out on one of the following systems: (1) an inverted AxioObserver.Z1 microscope with a × 100/1.3 oil EC Plan-Neofluar objective (Zeiss, Thornwood, NY), an Orca ER cooled CCD (charge-coupled device) camera (Hamamatsu, Bridgewater, NJ), a metal-halide lamp and an light-emitting diode (LED) Colibri system (Zeiss) including LED wavelengths at 365 and 470 nm; (2) a Nikon A1R MP laser scanning confocal attachment on a Nikon Ti Eclipse inverted microscope using a × 100/1.45 Plan Apo Lambda oil-immersion objective and standard lasers and filters (Nikon Instruments, Melville, NY); (3) a spinning-disk confocal microscope combining a CSU-X1 spinning disk attachment (Yokogawa Electronic Corporation, Tokyo, Japan) on a Nikon Ti Eclipse inverted microscope (Nikon) equipped with an electron multiplying CCD camera (Evolve, Photometrics, Tucson, AZ), 50 mW lasers at 488 and 561 nm, standard emission filters and a CFI Plan Apo × 100 1.45 numerical aperture oil objective (Nikon). System (1) was controlled by ZEN software (Zeiss). Systems (2) and (3) were controlled by NIS Elements Advanced Research software (Nikon). Details are given within the sections below describing each experiment.
6
Tendon-Bone Injury Histological Analysis
7
PASMC Membrane Potential Measurement
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Human PASMCs were incubated with the voltage-sensitive fluorescent dye, DiBAC4(3) (100 nM; Dojindo Laboratories), at room temperature for 30 min. DiBAC4(3) at the same concentration was added to the extracellular solution during measurements. Fluorescent signals were measured using the A1R confocal fluorescence imaging system equipped with an ECLIPSE Ti inverted microscope, a Plan Apo VC objective lens (20×/0.75), NIS-Elements imaging software (Nikon), and a solid-state 488-nm laser (Coherent, Santa Clara, CA, USA). PASMCs were illuminated at a 488-nm wavelength, and the fluorescent emissions (>520 nm) were obtained every 5 s. Membrane potential is presented as F/F140K, where F is the fluorescence intensity and F140K is the maximum fluorescence intensity in the 140-mM K+ HEPES-buffered solution (theoretically 0 mV). Standard HEPES-buffered solution was used as an extracellular solution (in mM): 137 NaCl, 5.9 KCl, 2.2 CaCl2, 1.2 MgCl2, 14 glucose, 10 HEPES, and pH 7.4 with NaOH. In the 140-mM K+ HEPES-buffered solution, the concentrations of NaCl and KCl in standard HEPES-buffered solution were changed to 2.9 and 140 mM, respectively.
8
Immunohistochemical Analysis of Muscle Tissues
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Muscle tissues were prepared for immunohistochemistry using standard protocols. Briefly, samples were fixed with 4% formaldehyde solution in PBS and embedded in paraffin; 10-µm-thick sections were prepared. After deparaffinization and rehydration, antigen unmasking was achieved by shortly boiling the slides, submersed in 1 mM EDTA (pH 8), using a microwave oven. After incubation for an additional 20 min at a sub-boiling temperature, slides were washed in distilled water and incubated in blocking buffer (0.1% Triton X-100 and 10% goat serum in PBS) for 1 h. Antibodies were diluted in staining buffer (0.1% bovine serum albumin in PBS). The primary antibodies were applied overnight at 4 °C, the secondary antibodies for 2 h at room temperature, with three washes in between incubations. Detailed information on the primary and secondary antibodies is given in Supplementary Table 2 . Sections were embedded in Mowiol and images were recorded at room temperature using a confocal laser scanning system (Nikon A1) equipped with an Eclipse Ti inverted microscope.
9
Multicolor TIRF Microscopy Protocol
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An Eclipse Ti inverted microscope (Nikon) with a TIRF system and iXon electron-multiplying charged-coupled device camera (Andor Technology) was used for imaging. TIRF microscopy was performed with a Nikon 100 × 1.49 numerical aperture oil-immersion TIRF objective, a TIRF illuminator, a Perfect Focus system, a motorized stage, and a U-N4S four-laser unit as a laser source (Nikon). The laser unit has the solid-state lasers for the 488 nm, 561 nm, and 640 nm channels and was controlled using a built-in acousto-optic tunable filter. The laser powers of 488 nm laser, 561 nm laser, and 640 nm laser were set to 5.2, 6.9, and 7.8 mW respectively measured with the field aperture fully opened. The 405/488/561/638 nm Quad TIRF filter set (Chroma Technology Corp.), along with supplementary emission filters of 525/50 m, 600/50 m, 700/75 m for 488 nm, 561 nm, 640 nm channels, respectively, were used. Images are acquired using the Nikon NIS-Elements software with the exposure time of 50 ms.
10
Imaging Embryos with Polystyrene Beads
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We mounted embryos as described previously (Robin et al., 2014 (link)) on glass slides under #1.5 coverslips in 3–5 µl of standard Egg Salts containing ∼100 uniformly sized polystyrene beads (18.7 ± 0.03 µm diameter; no. NT29N; Bangs Laboratories). The beads acted as spacers and allowed us to achieve uniform compression of the embryo surface across experiments (Robin et al., 2014 (link)).
We performed all imaging at 21–23°C on a Nikon ECLIPSE-Ti inverted microscope equipped with a Ti-ND6-PFS Perfect Focus Unit. A laser merge module (Spectral Applied Research) controlled fast, tunable delivery of 481-nm and 561-nm laser excitation from 50 mW solid-state lasers (Coherent Technology) to a motorized TIRF illuminator. We adjusted the laser illumination angle to achieve near-TIRF illumination (Tokunaga et al., 2008 (link)). We collected images using a Nikon CFI Apo 1.45 NA oil immersion TIRF objective combined with 1.5× intermediate magnification onto an Andor iXon3 897 EMCCD camera. All image acquisition was controlled by using Metamorph software. We used ImageJ to set minimum and maximum pixel values and perform gamma adjustments on the original 16-bit image data before converting images to 8-bit red, green, blue format or grayscale format for display. We performed these operations identically for all images that are compared directly.
We performed all imaging at 21–23°C on a Nikon ECLIPSE-Ti inverted microscope equipped with a Ti-ND6-PFS Perfect Focus Unit. A laser merge module (Spectral Applied Research) controlled fast, tunable delivery of 481-nm and 561-nm laser excitation from 50 mW solid-state lasers (Coherent Technology) to a motorized TIRF illuminator. We adjusted the laser illumination angle to achieve near-TIRF illumination (Tokunaga et al., 2008 (link)). We collected images using a Nikon CFI Apo 1.45 NA oil immersion TIRF objective combined with 1.5× intermediate magnification onto an Andor iXon3 897 EMCCD camera. All image acquisition was controlled by using Metamorph software. We used ImageJ to set minimum and maximum pixel values and perform gamma adjustments on the original 16-bit image data before converting images to 8-bit red, green, blue format or grayscale format for display. We performed these operations identically for all images that are compared directly.
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