The bending rigidity of E. coli cells was measured using a microfluidic assay similar to that used in Refs. 20 (link),21 (link). E. coli MG1655 was transformed with a plasmid (pDB192) containing sulA under an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible promoter. Cells were grown overnight in 2 mL LB containing 30 μg/mL kanamycin and 50 μg/mL ampicillin. IPTG was added to the medium to induce sulA throughout cell growth in the microfluidic flow chamber. Deflection of cells under fluid flow was monitored on a Zeiss Axiovert 100 microscope (Zeiss) equipped with a 60X oil objective. Images were collected with an Andor iXon 3 EMCCD (Andor) using μManager v. 1.426 . Deflection of the cells was determined using a custom Igor Pro (WaveMetrics Inc.) image-analysis algorithm.
Ixon3 emccd
Manufactured by Oxford Instruments
Sourced in United Kingdom
The IXon3 EMCCD is a scientific camera designed for low-light imaging applications. It features an Electron Multiplying Charge-Coupled Device (EMCCD) sensor that can amplify signals, allowing the camera to detect very faint light levels. The IXon3 EMCCD is capable of high-speed, high-sensitivity imaging and is commonly used in various scientific fields, such as astronomy, bioluminescence, and spectroscopy.
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Lab products found in correlation
Axiovert 100 microscope, by Zeiss (2 mentions)
Igor pro, by Wavemetrics (2 mentions)
Nie upright microscope, by Hamamatsu Photonics (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Coolsnap hq ccd, by Zeiss (1 mentions)
Nlo780 confocal system, by Zeiss (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Volocity, by PerkinElmer (1 mentions)
Pdb450c, by Thorlabs (1 mentions)
Csu x1 spinning disk, by Oxford Instruments (1 mentions)
Lysotracker, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Apotirf oil immersion objective, by Nikon (1 mentions)
Mlc 400 b laser box, by Agilent Technologies (1 mentions)
10 protocols using ixon3 emccd
1
Microfluidic Bending Rigidity of E. coli
2
Open-Source Modular SIM Imaging
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We have designed a complete instrument interface with LabView, which brings together on-the-fly pattern control, illumination color control, light intensity control and closed loop temperature control of the LED heat sink (Supplementary Fig. 5 ). It also saves the detailed acquisition parameters and pattern sequence used during the respective image acquisition. At the moment we offer several software versions with different functionalities in order to provide an adequate option for user with diverse requirements. For users who want to modify or customize the software we provide the full LabView project including camera control (exposure time, camera gains, binning options) and data handling (pre-formatting the data to be compatible directly with the open-source SIMToolbox SIM processing algorithm). The first version includes a full camera control for the camera models Andor Zyla PLUS sCMOS (6.5 µm pixel size) and Andor iXon3 EMCCD (8 µm pixel size). The modular design of the software provides the possibility to include the control of other commercial cameras. For user who want to primarily use the software, we offer an application (exe) of the openSIM software which is not camera model specific and does not require costly software licenses.
3
Spectral Analysis of Cone Photoreceptors
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To define the spectral signature of chromophore in cone photoreceptors, freshly isolated retinas were transversally sectioned using a vibrating microtome as described above, a section was place between slide and coverslip in Permafluor mounting medium to expose cone inner segments to wavelengths ranging from 300 to 490 nm in 15 nm increments. The corresponding emission spectra were recorded using focused excitation, a high-λ-pass filter to remove the excitation, and confocal detection with an Andor Shamrock SR303i spectrometer and Andor Ixon 3 EM-CCD (Andor Technology, Belfast, Northern Ireland). The resulting excitation–emission matrix was normalized by the excitation intensity to compensate its spectral variations, although this increases noises below 400 nm, where the available excitation intensity is weaker.
4
Multimodal Microscopy Imaging Protocol
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All fluorescence imaging was performed using a Nikon Eclipse-Ti inverted microscope equipped with 1.49 NA ×100 TIRF (total internal reflection fluorescence) and 0.95 NA ×40 air objectives and an Andor iXon3 EMCCD (electron-multiplying charge-coupled device) camera. The dark-field images were acquired using a Nikon NiE upright microscope with a Hamamatsu Orca-Flash 2.8 sCMOS (scientific complementary metal oxide semiconductor) camera.
5
Immunofluorescence Imaging of Cellular Structures
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For immunofluorescence analyses, cells were fixed in 3% paraformaldehyde/3% sucrose and permeabilised with 0.2% Triton X-100. After staining, the coverslips were mounted in ProLong® Gold antifade reagent (Invitrogen) or directly in PBS (for TIRF microscopy). Wide-field fluorescence was observed through 40X/1.3 oil objectives on a Zeiss Axiovert 200M microscope equipped with a CoolSnap HQ CCD. Image acquisition was performed using the MetaMorph Imaging System. Confocal imaging was performed on a Zeiss NLO780 confocal system using a 63X/1.4 objective. Total Internal Reflection Fluorescence (TIRF) microscopy was performed on our prototype system including 100x/1.49O Nikon objective, Ixon3 EMCCD (Andor, Belfast, Northern Ireland) and a laser bench with 491 nm (Oxxius, Lannion, France) and 561 nm (Cobolt, Solna, Sweden).
Images were analysed using MetaMorph Imaging System and Volocity (PerkinElmer, Waltham, MA) software. Quantification of VEC staining thickness was performed with the Integrated Morphometry Analysis module in MetaMorph software.
Images were analysed using MetaMorph Imaging System and Volocity (PerkinElmer, Waltham, MA) software. Quantification of VEC staining thickness was performed with the Integrated Morphometry Analysis module in MetaMorph software.
6
Fiber-Based Position Detection in Optical Tweezers
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The fiber based position detection mechanism on the inclined fiber optical tweezers platform is designed based on the dual fiber optical tweezers, which can be found in our previous work38 (link),41 ,42 (link). Briefly, the fiber based position detection mechanism, as well as the inclined fiber optical tweezers, was set up on a microscope platform. Light from a fiber-coupled 974 nm laser diode (AC 1405-0400-0974-SM-500, Eques) was split into two lensed fibers (TLF SM1060, Nanonics Imaging) through a 3dB coupler (22-12798-50-23162, GouldFiber Optics), as shown in Fig. 1a . All the fibers in the system were single-mode at 974 nm. The two light beams emitted from the lensed fiber tips can three-dimensionally trap microscale particles in water close to the beam intersection. The back-scattered light by the bead was collected by the two lensed optical fibers and measured by two inputs (PD1 and PD2) of a balanced photodiode (PDB450C, THORLABS). The differential output from the photodiode enabled the measurements of bead positions with nanometer resolution. The forward-scattered light collected by an objective lens was recorded by either a PSD (DL100-7-PCBA3, First Sensor) or a high-speed camera (iXon 3 EMCCD, Andor).
7
Microfluidic Bending Rigidity of E. coli
8
ß2-AR Trafficking and Lysosomal Localization
9
Single-Particle Tracking PALM for Precise Temperature Control
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Single-particle tracking PALM (sptPALM) was performed on a Nikon Eclipse Ti-E N-STORM system equipped with a Nikon 100× Apo TIRF oil immersion objective (NA 1.49) and perfect focus system. Photoactivation and excitation were performed with the 405 nm, 488 nm, 561 nm, and 647 nm excitation lasers within the MLC400B laser box (Agilent technologies) under TIRF or HiLo illumination through a quad-band polychroic mirror (Nikon 97335). An Ixon3 EMCCD (Andor) was used for detection, resulting in an effective pixel size of 160 nanometer. The microscope was fitted with a temperature-controlled stage that was adapted to fit the Nikon microscope. A pump was used to continuously flow cold water through the heat sink with 120 mL/min, to buffer the residual heat from the Peltiers. The Peltiers were controlled via Meerstetter Engineering TEC controllers and software. Additionally, the objective was cooled using a fitted copper collar with a fluid channel in the center that allowed a continuous flow of ~100 mL/min of cooled water through a peristaltic pump from a cold water bath of approximately 2.5 °C (see also Fig. 1A and SI Appendix, Fig. S1 for the full experimental setup). This allowed control of the sample temperature with <0.1° centigrade precision.
10
Sonochemical and Luminescence Analysis
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SCL and SL analysis was undertaken to measure the intensity of sonochemical/ionising reactions. SCL was performed using a 1 mM luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and 0.1 M NaOH (pH 13) solution [20] (link). An ANDOR iXon3 EMCCD camera and software was used to capture and quantify luminescence emission. The camera operated at −70 °C and applied an EM gain level/exposure time (seconds) of 50/40 for SL and 4/4 for SCL.
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