Total internal reflection fluorescence (TIRF) was used to determine the internalization of AT1R in transiently transfected cells by RFP‐fused AT1R. HEK293 cells were cultured on a 24‐mm‐diameter microscope coverglass (WHB‐6‐CS, WHB Scientific, China). After 48 hours of transfection, the cells were observed using a TIRF microscope (Olympus Corporation, Tokyo, Japan) equipped with an electron‐multiplying charge‐coupled device camera (Andor, Belfast, UK) and oil immersion objective (Olympus; magnification ×100, NA=1.49). A 101 to 103‐nm depth of field was chosen to observe AT1R fluorescence on the plasma membrane but not in cytoplasm. After stimulant administration, we monitored and pictured continuous quantifications of fluorescent receptors on the cell membrane for 2 000 s with use of MetaMorph software version 7.8.8.0 (Molecular Devices, Sunnyvale, CA).
Electron multiplying charge coupled device camera
Manufactured by Oxford Instruments
Sourced in Germany, Japan, United Kingdom
The Electron-multiplying charge-coupled device (EMCCD) camera is a specialized imaging device designed for low-light applications. It utilizes an electron-multiplying gain register to amplify the signal, enabling the detection of even single photon events. The camera's core function is to capture high-sensitivity images and video in challenging low-light conditions.
Automatically generated - may contain errors
Lab products found in correlation
Spinning disk confocal and tirf combined system, by Nikon (2 mentions)
Plan apo 1.49 na oil immersion objective, by Hamamatsu Photonics (2 mentions)
Flash 4, by Hamamatsu Photonics (2 mentions)
Oil immersion objective, by Olympus (1 mentions)
Tirf microscope, by Olympus (1 mentions)
Bafilomycin a1, by Thermo Fisher Scientific (1 mentions)
Bx61wi microscope, by Olympus (1 mentions)
Glass coverslip, by Warner Instruments (1 mentions)
Csu x1 spinning disk unit, by Oxford Instruments (1 mentions)
Dmi6000b microscope, by Yokogawa (1 mentions)
Eclipse ti inverted microscope, by Yokogawa (1 mentions)
10 protocols using electron multiplying charge coupled device camera
1
Visualization of AT1R Internalization
2
Imaging of Immune Cell Interactions
3
High-sensitivity Readout of Hippocampal Synaptic Properties
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For high-sensitivity readout of functional RRP properties, we used 10–17 day dissociated hippocampal cultures, prepared from P0 Sprague–Dawley rats23 (link)32 (link). Experiments were performed in HEPES-buffered extracellular solution with composition (in mM): 137 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 D -glucose, 5 HEPES, 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione, 50 μM AP-5. All experiments were performed at 23–25 °C. Synapses were labelled with field stimulation (10, 40 and 600 APs) in the presence of 10 μM FM1–43 and then washed extensively. 20 min after loading, time-lapse imaging was performed while a two-step FM-dye destaining protocol (40 APs then 600 APs at 20 Hz) was applied. Imaging used an Olympus BX61WI microscope (× 60 1.0 NA dipping objective, excitation: 480/20, emission: 520/35) attached to an Andor Ixon+ electron multiplying charge-coupled device camera (4 × 4 binning, 2 Hz acquisition) controlled by custom-written Micromanager routines. Some experiments used sypHy2x expression based on a viral construct (pAAV-pCAG-sypHy2x-WPRE-bGHpolA, a gift from T. Branco, Cambridge). Neurons were infected after 7 days in vitro and used for experiments after a further 9–10 days of expression. Imaging used 4 × 4 binning and 11 Hz acquisition rate. Cells were treated with 1 μM jasplakinolide (Calbiochem) or 0.5 μM bafilomycin A1 (Fisher Scientific).
4
Localization Microscopy of Gold Nanoparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Localization microscopy measurements were done for the optical investigation of the GNPs’ distribution as GNPs do not bleach under laser exposure but continue to blink even at maximum laser power [15 (link), 33 (link)]. The frequency of incident photons is set in order to establish the resonance condition matching the natural frequency of the surface electrons (plasmon resonance [34 ]). Probes were measured with 491 nm and 561 nm two diode-pumped, solid-state lasers at 200 mW laser power. The instrument was equipped with an oil objective lens 63x/NA 0.7….1.4 (Leica, Wetzlar, Germany), an electron-multiplying charge-coupled device camera (1376 x 1040 pixels; Andor Technology, South Windsor, CT) and band-pass filters (525/50 nm for 491 nm excitation and 609/ 54 nm for 561 nm excitation).
The cells were selected visually. Stacks of 2,000 frames were acquired at an integration time of 50 ms each. To get comparable conditions, the cell nuclei were selected in such a way that the image section was taken at the largest diameter. For quantitative image evaluation, in-house programs were applied (for details see Grüll et al. [35 ], Kaufmann et al. [36 ], Müller et al. [37 ], Stuhlmüller et al. [38 (link)]).
The cells were selected visually. Stacks of 2,000 frames were acquired at an integration time of 50 ms each. To get comparable conditions, the cell nuclei were selected in such a way that the image section was taken at the largest diameter. For quantitative image evaluation, in-house programs were applied (for details see Grüll et al. [35 ], Kaufmann et al. [36 ], Müller et al. [37 ], Stuhlmüller et al. [38 (link)]).
5
Synuclein Aggregate Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fusion proteins of α-synuclein with sGFP were expressed as described above and aggregates were enriched from the cell lysate using gradient centrifugation. Glass coverslips (#1.5 Warner Instruments) were cleaned with 70% (v/v) ethanol, dried and treated in a plasma cleaner (Harrick Plasma). Aggregates were absorbed from solution onto freshly cleaned coverslips. Coverslips were then rinsed with imaging buffer to remove unbound protein and imaged using an inverted TIRF microscope (TILL Photonics) with Zeiss α Plan-apochromat 100× oil objective (1.46 NA), 488 nm laser line and electron-multiplying charge-coupled device camera (Andor). Spots in the fluorescence image were detected as local maxima and fluorescence intensities were extracted by point-spread function fitting using image analysis software implemented using MATLAB.
6
Nanoparticle Size and Concentration Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NTA, using a NanoSight NS500 instrument (NanoSight Ltd. (Amesbury, UK)), determines the size and concentration of particles ranging from 50 nm to 1 µm in diameter. Samples were diluted 100- to 400-fold in PBS before measurement. The sample was illuminated by a laser beam; hence, any particle that scatters light would be detected, including protein aggregates. A dark-field microscope is used to determine the position of vesicles, which are continuously moving due to Brownian motion. For each vesicle, the movements are tracked and the mean squared velocity is calculated. As this depends on the particle diameter, an absolute size distribution of vesicles in suspension can be obtained [43]. The NanoSight NS500 (NanoSight Ltd.) equipped with an Electron Multiplying Charge Coupled Device camera (Andor Technology, Tokyo, Japan) and a 405-nm laser was used. Three or six video recordings of, respectively, 60 or 30 s were done with the following settings: camera level 14 to 15, screen gain 1.0 and detection threshold 5 to 10 using NTA v2.3.0.17 [33 ].
7
Nanoparticle Tracking Analysis for EV Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NTA is widely used in vesicle research for the characterization of the size and concentration of EVs (27 ). With NTA, the Brownian motion of individual particles in solution is tracked based on their light scattering. Variables include the camera level, detection threshold, viscosity, temperature, and the dilution of the sample. There are some attempts to standardize this method with regard to characterization of EVs (27 , 32 (link)). An NS500 (NanoSight Limited, London, UK) equipped with an electron multiplying charge coupled device camera (Andor Technology, Tokyo, Japan) and a 405 nm laser was used. During measurements, temperature was kept at 22°C. The viscosity of water at 22°C (0.95 mPa s) was used, as samples were diluted several fold in PBS buffer. Silica beads with a diameter of 100 nm and known concentration were used to adjust the focus height of the objective and to calibrate the concentration (27 ). NTA v2.3.0.17 software (NanoSight Limited) was used for data analysis. Before the measurement, samples were 500,000-fold diluted with PBS buffer. Ten videos of 30 seconds were captured per measurement at camera level 15. The detection threshold was set at pixel value 16.
8
Imaging Ventral Midbrain Neurons
9
Spinning-disk Confocal Microscopy Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All internalization and bead colocalization imaging performed for this study was done using a spinning-disk confocal microscope (Nikon Ti-Eclipse inverted microscope with a Yokogawa spinning disk). For bead internalization kinetic assays, images were acquired using a 40×/0.95 NA air objective. Live-image acquisition for internalization of corpses and beads was performed using the High Content Screening Site Generator plugin in µManager. All other images were acquired using a 100× 1.49 NA oil-immersion objective. Images were captured using an Andor iXon electron-multiplying charge-coupled device camera. The open source µManager software package was used to run the microscope and capture the images (Edelstein et al., 2010 (link)). All raw microscopy images were acquired as 16-bit TIFF stacks.
10
Spinning Disk Confocal and TIRF Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Imaging was performed on an inverted microscope (Nikon TiE,Tokyo, Japan) equipped with a Yokogawa spinning disk confocal and TIRF combined system (Spectral Diskovery, Ontario, Canada), a Nikon 100× Plan Apo 1.49 NA oil immersion objective and four laser lines (405, 488, 561, and 640 nm), a Hamamatsu Flash 4.0, and μManager software to run the microscope and 653 capture the images. Confocal images were captured using an Andor iXon electron-multiplying charge-coupled device camera. For TIRF imaging, a polarizing filter was placed in the excitation laser path to polarize the light perpendicular to the plane of incidence. The angle of illumination was controlled with either a standard Nikon TIRF motorized positioner or a mirror moved by a motorized actuator (CMA-25CCCL; Newport). Imaging experiments involving live cells were performed within a 37ºC chamber.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!