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Emccd camera ixon2

Manufactured by Oxford Instruments

The iXon2 is a high-performance EMCCD (Electron Multiplying Charge Coupled Device) camera manufactured by Oxford Instruments. It is designed to capture and amplify low-light signals, enabling sensitive and high-resolution imaging in various scientific applications.

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2 protocols using emccd camera ixon2

1

Multimodal Imaging of Hydrogel Microstructures

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Confocal imaging experiments were conducted on an inverted Zeiss LSM 710 AxioObserver (Zeiss, Oberkochen, Germany). Images were acquired at room temperature using a 40× water objective (LD C-Apochromat 40×/1.1 NA W Corr M27, Zeiss) with the correction collar set for a No. 1 coverslip. Rhodamine-labeled PA gels were fabricated on No. 1 coverslips and imaged using a DPSS-561 laser at 0.25% power, using the MBS488/561/633 beam splitter and the Zen 2010 software (Zeiss). Z-stack images were acquired with a step size of 0.42 μm with line scanning at x = y=z = 0.42 μm pixel size.
Widefield epifluorescence images for microparticle imaging were obtained on an Olympus IX-71 inverted microscope with an Olympus LCPlanFI 40×/0.6 NA) objective and an EMCCD Camera iXon2 (Andor). For microparticle imaging, brightfield microscopy was first utilized to find the field of view including a microparticle in a microwell. Microparticles were then imaged with an exposure time of 50 ms using a Cy5 filter cube (Chroma, 49009) using a time series feature in MetaMorph (Molecular Devices). Images were collected every 1 s. For particle tracking, microparticles were imaged using an Olympus UPlanFi 10×/NA 0.3 objective at an exposure time of 500 ms and an EMCCD Camera iXon2 (Andor).
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2

Quantitative Analysis of Protein Distributions

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Brightfield images were obtained on an Olympus IX-71 inverted microscope with an Olympus UPlanFi 4× (numerical aperture (NA) 0.13) or 10× (NA 0.3) objectives and an EMCCD Camera iXon2 (Andor). Fluorescence images for ICC were obtained with the 10× objective and an X-Cite Exacte mercury arc lamp illumination source.
Immunoprobed gels were imaged on a microarray scanner (Genepix, Molecular Devices) under the 488, 535, and 635 excitation wavelength channels. Images were then analysed using an in-house MATLAB (R2017a) script (Fig. S2). The custom MATLAB algorithm segments peaks by first applying a threshold to identify protein peaks and centroids of each peak. A Gaussian is then fit laterally to each peak to generate a custom region of interest “lane” containing the entire peak (8σ). Intensity profiles of each peak were obtained by summing the intensities of each pixel along the separation axis and dividing by the number of pixels. The intensity profiles were fit to Gaussian curves with a minimum signal-to-noise ratio (SNR) of 3 and R2 threshold of 0.7. Protein mass of focused protein peaks was calculated by summing the area under the curve (AUC) of the fit Gaussian curve. Protein mass of protein retained on the fibronectin pattern was calculated by summing the background-subtracted intensities along the fibronectin pattern (integrated density, ID).
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