Live imaging of KO cultures grown in MFCs was conducted on DIV12. Lysotracker Red DND-99 (Thermo Fisher Scientific, L7528) was added to the somatodendritic compartment of the MFC to a final dilution of 1:10,000. Lysotracker-stained vesicles became visible in the axonal compartment a few minutes later. Imaging was conducted at 37°C and 5% CO2 on a Zeiss Axio Observer coupled to a spinning disk confocal system (CSU-W1-T3; Yokogawa) connected to an electron-multiplying CCD camera (ProEM+1024, Princeton Instruments). Images were taken every 1 s for 180 s with a 63×/1.46 NA oil immersion objective.
Proem 1024
Manufactured by Teledyne
The ProEM 1024 is a high-performance, scientific-grade CCD camera designed for advanced imaging applications. It features a 1024 x 1024 pixel sensor with a pixel size of 13 μm. The camera offers a wide dynamic range and low read noise, making it suitable for a variety of scientific and industrial imaging tasks.
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Lab products found in correlation
4 protocols using proem 1024
1
Live Imaging of Lysosomal Dynamics
2
Single-Molecule Fluorescence Microscopy Setup
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White light from a tungsten-halogen lamp passes through a condenser (IX-LWUCD, Olympus) and illuminates the sample on an inverted microscope (IX71, Olympus). The transmitted light passes through an infinity-corrected 60X water immersion lens with a 1.2 numerical aperture (UPLSAPO60XW, Olympus). The light exiting the side-port is relayed to an electron-multiplied charge-coupled device (EMCCD; ProEM 1024, Princeton Instruments) via a 4f system, where a bandpass filter with 650–670 nm passband (FB660-10, Thorlabs) at its Fourier plane (Supplementary Fig. 2 ).
3
sEV Analysis by Fluorescence Microscopy
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White light from a tungsten-halogen lamp passes through a condenser (IX-LWUCD, Olympus) and illuminates the sample on an inverted microscope (IX71, Olympus). The transmitted light passes through an infinity-corrected 60X water immersion lens with a 1.2 numerical aperture (UPLSAPO60XW, Olympus). The light exiting the side-port is relayed to an electron multiplied charge-coupled device (EMCCD; ProEM 1024, Princeton Instruments) via a 4f system, where a bandpass filter with 650–670 nm passband (FB660-10, Thorlabs) is at its fourier plane. sEVs PANORAMA measurements were done in PBS 1X.
4
Live-cell Fluorescence Recovery Analysis
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Live-cell video recordings were performed using an inverted microscope (Axio Observer, Zeiss) coupled to a spinning-disk confocal system (CSU-W1-T3, Yokogawa, pinhole aperture of 1 Airy unit) connected to wide-field electron-multiplying CCD camera (ProEM+ 1024, Princeton Instruments) and maintained at 37°C and 5% CO2. Images were taken at 1 Hz for 60 s using a 63× oil-immersion objective (1.46 NA). The raw fluorescence values from the unbleached area were subtracted from the values obtained in the photobleached areas. The values were then normalized to 0–100%, with 100% corresponding to the fluorescence value recorded just before photobleaching and 0% just after. A nonlinear fit with an unconstrained model “One site binding–Specific binding” from GraphPad Prism 6.0 was applied and indicated Bmax values for each experiment. To obtain a more accurate mean value, square roots of Bmax values were then represented.
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