Collagen fibril samples were imaged on a Bioscope catalyst (Bruker, USA) atomic force microscope mounted on an IX71 inverted optical microscope (Olympus, USA) and operating in Peak Force Quantitative Nanomechanical Mapping mode (Peak Force QNM). The cantilevers used were made of silicon nitride and had an n-doped Silicon pyramidal tip with a nominal radius of 2 nm and a half angle of 18° (SCANASYST fluid+, Bruker USA). Note that in this architecture, the pyramidal tip behaves as an insulated conductor that is not necessarily grounded. The spring constant was calibrated for each cantilever before imaging using the thermal noise method and ranged from 1 to 1.5 N m−1. 1 μm images were acquired with 512 pixels per line at a scan rate of 0.5 Hz, peak force setpoint of 5 nN, cantilever oscillating frequency of 1 kHz and corresponding vertical tip velocity of 0.6 mm s−1. Two channels were recorded for analysis, the height and the adhesion force between the tip and the substrate (Fig. 2c ). For all the images, the humidity in the room was between 15 and 20% RH.
Ix71 inverted optical microscope
Manufactured by Olympus
Sourced in United States, Australia
The IX71 inverted optical microscope is a versatile and reliable instrument designed for a range of scientific applications. It features an inverted design that allows for easy specimen manipulation and observation. The IX71 provides high-quality imaging capabilities, delivering clear and detailed views of your samples.
Automatically generated - may contain errors
Lab products found in correlation
Scanasyst fluid, by Bruker (1 mentions)
Bioscope catalyst, by Bruker (1 mentions)
Ixon ultra 897 camera, by Oxford Instruments (1 mentions)
Scanasyst fluid cantilever, by Bruker (1 mentions)
Andor revolution xd, by Olympus (1 mentions)
Leibovitz medium, by Merck Group (1 mentions)
Bl rc150vb, by Olympus (1 mentions)
5 protocols using ix71 inverted optical microscope
1
Nanomechanical Mapping of Collagen Fibrils
2
Neutralizing Antibody Assay for BEFV
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Mouse sera were tested for the presence of anti-BEFV-neutralising antibodies by VN assay. Briefly, the sera were heat-inactivated at 56 °C for 30 min, and then 50 µL of two-fold serial dilutions of each serum was mixed with 50 µL of 100 TCID50 of BEFV in the 96-well tissue culture plate and then incubated for 1 h at 37 °C in 5% CO2. After the incubation period, 50 µL of the Vero cell suspension containing 15000 cells was added to each well and the plate was incubated for 4 days at 37 °C in a humidified incubator with an atmosphere of 5% CO2. After the incubation, the cells were examined for BEFV-specific cytopathic effects (CPEs) using an Olympus IX71 inverted optical microscope (Olympus Australia, Mt. Waverley, Australia).
3
Correlative AFM and Fluorescence Imaging
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Correlated fluorescence and AFM images were acquired as described previously (3 (link), 33 (link)). Briefly, fluorescence images were acquired with an electron-multiplying charge-coupled device (EMCCD) iXon Ultra 897 camera (Andor) mounted on an IX71 inverted optical microscope (Olympus) equipped with an UAPON100XOTIRF 100× oil immersion objective (Olympus) with the ×2 magnifier in place. Illumination was provided by a monolithic laser combiner (Agilent) using the 488- or 561-nm laser output coupled to an optical fiber with appropriate filter sets: F36-526 for calcein-AM and F71-866 for mCherry-Wag31 or cytosolic red fluorescent protein. The AFM was mounted on top of the inverted microscope, and images were acquired with a customized Icon scan head (Bruker) using ScanAsyst fluid cantilevers (Bruker) with a nominal spring constant of 0.7 N m−1 in peak force tapping mode at a set point of <2 nN and typical scan rates of 0.5 Hz. The samples were maintained at 37°C in 7H9 growth medium heated by a custom-made coverslip heating holder controlled by a TC2-80-150 temperature controller (Bioscience Tools).
4
Spinning-Disk Confocal Microscopy for Live-Cell Imaging
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The fluorescent images were acquired by a spinning-disk confocal microscope Andor Revolution XD with 100 × N.A. 1.4 oil immersion or 60 × N.A. water immersion objective on the Olympus IX-71 inverted optical microscope. Generally, the z-stacks were acquired before and after the acquisition of an AFM image of the same cell. The spacing between the optical sections was chosen based on the Nyquist criterion (0.21 μm for the used setup). The imaging parameters (laser intensity and exposition time) were adjusted in preliminary experiments to decrease the acquisition time and still preserve high signal-to-noise ratio and low phototoxicity. The fluorescent images were analyzed by Fiji (NIH, Bethesda, MD).
5
Atomic Force Microscopy of Myotubes
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The experimental system was an Asylum Research MFP-3D atomic force microscope mounted on an Olympus ix-71 inverted optical microscope, used both for optical imaging and force spectroscopy.
For all measurements gold-coated silicon-nitride Bio-levers (BL-RC150VB, Olympus, Japan) were used, having a nominal spring constant of 30 pN/nm and a resonant frequency of 37 kHz in air, which drops to 6 kHz in liquid. The cantilevers were equipped with a V-shaped tip, having a half-opening angle of 45° and a radius around 30 nm. After 6 to 8 days of differentiation in vitro, the myotubes were transferred under the AFM head in serum free Leibovitz medium (Sigma), which enables maintaining the physiological conditions for long time in CO2 free atmosphere. The measurements were taken at 32 °C within 4 h after the cells were taken out from the incubator. According to our observations, the cells preserve their viability during this period. Prior each measurement the spring constant of cantilevers was determined using a combination of thermal noise and Sader methods, available within the driving software35 (link)–37 (link).
For all measurements gold-coated silicon-nitride Bio-levers (BL-RC150VB, Olympus, Japan) were used, having a nominal spring constant of 30 pN/nm and a resonant frequency of 37 kHz in air, which drops to 6 kHz in liquid. The cantilevers were equipped with a V-shaped tip, having a half-opening angle of 45° and a radius around 30 nm. After 6 to 8 days of differentiation in vitro, the myotubes were transferred under the AFM head in serum free Leibovitz medium (Sigma), which enables maintaining the physiological conditions for long time in CO2 free atmosphere. The measurements were taken at 32 °C within 4 h after the cells were taken out from the incubator. According to our observations, the cells preserve their viability during this period. Prior each measurement the spring constant of cantilevers was determined using a combination of thermal noise and Sader methods, available within the driving software35 (link)–37 (link).
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