Ixon ultra 888 camera, by Oxford Instruments - Product details

1

Correlated Fluorescence and AFM Imaging

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Correlated AFM and fluorescence images were acquired as described previously ^{37 (link)}. Briefly, fluorescence images were acquired with an EMCCD iXon Ultra 888 camera (Andor) mounted on an IX73 inverted optical microscope (Olympus) equipped with a 100X oil immersion objective (UAPON100XOTIRF, Olympus). Illumination was provided by an MLC monolithic laser combiner (Agilent) coupled to an optical fiber. For combined fluorescence imaging of FtsZ-GFP and FM4-64 the excitation filter and dichroic mirror of the EGFP filter cube F36-526 (AHF Analysetechnik, Germany) were used. The emission light was split by an optical system (DV2, Photometrics) equipped with a second dichroic mirror (T565lpxr) placed between the EMCCD camera and the microscope frame to separate the red and green channels each to one half of the EMCCD camera chip. Cleanup filters used were F37-528 (EGFP) for the green channel and F37-624 (TxRed) for the red channel. Additionally, a neutral density filter with OD 0.6 was placed on the red channel. Typically, 5 mW power of 488 nm laser light at the MLC400B output was used for illumination. Images were recorded with EM gain set to 300 and exposure time to 500 milliseconds. The AFM was mounted on top of the inverted microscope (IX73, Olympus) and the AFM laser was switched off before acquiring fluorescence images. Contrast and brightness were adjusted with ImageJ.

Odermatt P.D., Hannebelle M.T., Eskandarian H.A., Nievergelt A.P., McKinney J.D, & Fantner G.E. (2019). Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division. Nature physics, 16(1), 57-62.

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Fluorescence Microscopy Imaging Setup

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Images were acquired using an inverted Nikon Eclipse Ti fluorescence microscope with a Plan Apochromat lambda 60X /1.40 Oil objective or a Plan Fluor 4X/0.13 objective for fluorescent images or DIC images, respectively, a CSU-W1 confocal spinning disk unit, an iXon Ultra 888 camera (Andor Technology), MLC 400B laser unit (Agilent Technologies), and NIS Elements software (Nikon).

Farshidfar F., Rhrissorrakrai K., Levovitz C., Peng C., Knight J., Bacchiocchi A., Su J., Yin M., Sznol M., Ariyan S., Clune J., Olino K., Parida L., Nikolaus J., Zhang M., Zhao S., Wang Y., Huang G., Wan M., Li X., Cao J., Yan Q., Chen X., Newman A.M, & Halaban R. (2022). Integrative molecular and clinical profiling of acral melanoma links focal amplification of 22q11.21 to metastasis. Nature Communications, 13, 898.

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3

Correlated Fluorescence and AFM Imaging

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Correlated AFM and fluorescence images were acquired as described previously ^{37 (link)}. Briefly, fluorescence images were acquired with an EMCCD iXon Ultra 888 camera (Andor) mounted on an IX73 inverted optical microscope (Olympus) equipped with a 100X oil immersion objective (UAPON100XOTIRF, Olympus). Illumination was provided by an MLC monolithic laser combiner (Agilent) coupled to an optical fiber. For combined fluorescence imaging of FtsZ-GFP and FM4-64 the excitation filter and dichroic mirror of the EGFP filter cube F36-526 (AHF Analysetechnik, Germany) were used. The emission light was split by an optical system (DV2, Photometrics) equipped with a second dichroic mirror (T565lpxr) placed between the EMCCD camera and the microscope frame to separate the red and green channels each to one half of the EMCCD camera chip. Cleanup filters used were F37-528 (EGFP) for the green channel and F37-624 (TxRed) for the red channel. Additionally, a neutral density filter with OD 0.6 was placed on the red channel. Typically, 5 mW power of 488 nm laser light at the MLC400B output was used for illumination. Images were recorded with EM gain set to 300 and exposure time to 500 milliseconds. The AFM was mounted on top of the inverted microscope (IX73, Olympus) and the AFM laser was switched off before acquiring fluorescence images. Contrast and brightness were adjusted with ImageJ.

Odermatt P.D., Hannebelle M.T., Eskandarian H.A., Nievergelt A.P., McKinney J.D, & Fantner G.E. (2019). Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division. Nature physics, 16(1), 57-62.

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Single Particle Optical Spectroscopy Setup

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Single particle
measurements were performed with a home-built microscope (DIY Cerna
Components). Dilute solution of QDs in 2 wt % poly(methyl-methacrylate)
were spin coated on silicon substrates leading to minimum separation
of 4–5 μm between the dots as confirmed by wide field
fluorescence microscopy. The samples were kept in a helium closed-cycle
cryostat (attoDRY800). The excitation light from a pulsed diode laser
(EPL475; Edinburgh Instruments) at a repetition rate of 5 MHz/1 MHz
was focused through a cold objective (100×; 0.8 NA, Attocube),
which was also used for collecting the emission. The emission light
was passed through a dichroic mirror (T505lpxr, Chroma) and additional
long-pass filter (ET542LP) before focusing either onto Avalanche Photodiodes
(MPD, 100 μm SPAD) or a spectrograph (Kymera 328i) with EMCCD
(iXon Ultra 888 camera, Andor) through a wave-plate and PBS (Thorlabs) mounted on
a motorized rotating stage (PRM1Z8,Thorlabs). Time-stamping was performed
using multichannel Time Tagger 20 (Swabian Instruments). Spectral
time traces and fluorescence lifetimes were extracted from the time-tagged
data using home-written MATLAB code.

Panfil Y.E., Cui J., Koley S, & Banin U. (2022). Complete Mapping of Interacting Charging States in Single Coupled Colloidal Quantum Dot Molecules. ACS Nano, 16(4), 5566-5576.

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Time-lapse Imaging of p53-Venus MCF7 Cells

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Time-lapse microscopy was used for the expression measurements of mCherry-tagged MCF7 p53-Venus cell lines. Two days prior to the microscopy experiment, cells were grown on poly-D-lysine-coated glass-bottom plates (MatTek Corporation) in selection medium. Approximately 45 mins before the experiment, medium of all samples were changed to transparent RPMI medium supplemented with 2% fetal bovine serum (FBS), 100 U/mL penicillin G, 100 mg/mL streptomycin sulfate, and 250 ng/mL amphotericin B. Cells were imaged on a Nikon Eclipse TiE inverted fluorescence microscope with a 20x plan apo objective (NA 0.75) using an iXon Ultra-888 camera (Andor) with constant temperature, CO₂ concentration, and humidity maintained. Images were acquired every 20 min over a 24-hour period. The mCherry filter set contained filters of 540–580 nm for the excitation light, 585 nm for the dichroic beam splitter, and 593–668 nm for the emission light (Chroma). We analyzed images using NIS-Elements software (Nikon) and custom written ImageJ (NIH) and MATLAB software (Mathworks), which is available upon request.

Koh W.S., Porter J.R, & Batchelor E. (2019). Tuning of mRNA stability through altering 3′-UTR sequences generates distinct output expression in a synthetic circuit driven by p53 oscillations. Scientific Reports, 9, 5976.

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Ixon ultra 888 camera

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5 protocols using ixon ultra 888 camera

Correlated Fluorescence and AFM Imaging

Fluorescence Microscopy Imaging Setup

Correlated Fluorescence and AFM Imaging

Single Particle Optical Spectroscopy Setup

Time-lapse Imaging of p53-Venus MCF7 Cells

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