Fluorescence was excited with a 488 laser (Cell CMR-LAS-488, Olympus) and monitored using an inverted TIRF-equipped microscope (IX83, Olympus) under a ×150/1.45NA objective. Near-TIRF was achieved by adjusting the incident angle to 63.7°, which is near the critical angle of 63.63°. Images were acquired every 50 ms (with an exposure time of 49.38 ms) using a cooled EMCCD camera (iXon life 888, ANDOR). In experiments in Figure 1H, I , acquisition was performed at a rate of 25 ms/frame. Other experimental details, including solutions, field stimulation, etc., were the same as above.
Ixon life 888
Manufactured by Oxford Instruments
Sourced in United Kingdom
The IXon Life 888 is a scientific-grade, back-illuminated EMCCD camera designed for low-light imaging applications. It features a 1024 x 1024 pixel sensor with a 13 μm pixel size and a frame rate of up to 56 frames per second. The camera utilizes electron-multiplying technology to provide high signal-to-noise performance, making it suitable for a range of scientific research and industrial imaging tasks.
Automatically generated - may contain errors
Lab products found in correlation
Cell cmr las 488, by Olympus (3 mentions)
Whole microscope incubator chamber, by Tokai Hit (3 mentions)
Apon60xo tirf, by Olympus (1 mentions)
Ix3 zdc, by Olympus (1 mentions)
Nis elements ar, by Nikon (1 mentions)
Uapon, by Olympus (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
Sola se u nir light engine, by Lumencor (1 mentions)
Ix83 inverted microscope, by Olympus (1 mentions)
10 protocols using ixon life 888
1
Single-Molecule TIRF Microscopy
2
Widefield Fluorescence Imaging in Zebrafish
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Widefield epi-FL images of GCaMP5G signals were simultaneously captured with a high speed EMCCD camera (iXon Life 888, Andor Technology, Belfast, United Kingdom) having a 105 mm F-mount objective (Nikon, Chiyoda, Tokyo, Japan). FL emission was recorded using a longpass filter with a cut-on wavelength of 500 nm, effectively filtering the 488 nm excitation wavelength also used for OAT imaging (Figure 1C ). The camera was manually adjusted so that the zebrafish larvae lie in focus and synchronized with the pulsed laser source. Much like for the volumetric OAT recordings, differential FL signal changes with respect to the baseline [ΔF/F0 = (F−F0)/F0] were calculated from the same regions in the hind and forebrain. The baseline value (F0) was also calculated by averaging the first ten time points at the beginning of the acquired FL sequence.
3
Single-Molecule Imaging with Wide-Field Microscopy
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The setup was a single-molecule-sensitive,
wide-field microscope equipped with a single MEMS micromirror, which
has been previously described in detail.22 (link) Use of a NA 1.49, 60× oil immersion objective (APON60XOTIRF,
Olympus) and ∼1.8× postmagnification (OptoSplit II, Cairn)
led to an effective camera pixel size of 122 nm. The fluorescence
light was filtered with a zt532/640rpc dichroic mirror (Chroma) and
multibandpass filter ZET532/640 (Chroma), and imaged on a EMCCD camera
(iXon Life 888, Andor). For all measurements, the central area of
the EMCCD camera with 512 × 512 pixels was selected, thus resulting
in a total FOV of (62.5 μm)2; the camera was recording
30,000 frames at 20 Hz frame rate, except for the experiments on buffer
acidification with 15,000 frames at 10 Hz. Buffer acidification and
cell measurements were performed with a refractive beam shaping device
to generate a flat-field illumination (piShaper 6_6_VIS, AdlOptica),
whereas all other experiments were performed with an active MEMS micromirror.
The excitation intensity for the full FOV was measured to 0.48 kW
cm–2 for single-molecule photoswitching using the
MEMS illumination and 0.72 kW cm–2 for cell measurements
using the piShaper.
wide-field microscope equipped with a single MEMS micromirror, which
has been previously described in detail.22 (link) Use of a NA 1.49, 60× oil immersion objective (APON60XOTIRF,
Olympus) and ∼1.8× postmagnification (OptoSplit II, Cairn)
led to an effective camera pixel size of 122 nm. The fluorescence
light was filtered with a zt532/640rpc dichroic mirror (Chroma) and
multibandpass filter ZET532/640 (Chroma), and imaged on a EMCCD camera
(iXon Life 888, Andor). For all measurements, the central area of
the EMCCD camera with 512 × 512 pixels was selected, thus resulting
in a total FOV of (62.5 μm)2; the camera was recording
30,000 frames at 20 Hz frame rate, except for the experiments on buffer
acidification with 15,000 frames at 10 Hz. Buffer acidification and
cell measurements were performed with a refractive beam shaping device
to generate a flat-field illumination (piShaper 6_6_VIS, AdlOptica),
whereas all other experiments were performed with an active MEMS micromirror.
The excitation intensity for the full FOV was measured to 0.48 kW
cm–2 for single-molecule photoswitching using the
MEMS illumination and 0.72 kW cm–2 for cell measurements
using the piShaper.
4
TIRF Microscopy for Excitation Dynamics
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All experiments were conducted at 37°C within a whole-microscope incubator chamber (TOKAI HIT). Fluorophores were excited with a 488 laser (Cell CMR-LAS-488, Olympus), and monitored using an inverted TIRF-equipped microscope (IX83, Olympus) under a 150x/1.45NA objective (UapoN). The Z-drift compensation system (IX3-ZDC) was used to ensure a constant position of the focal plane during imaging. Near-TIRF was achieved by adjusting the incident angle to 63.7°, which is near the critical angle of 63.63°. Images were acquired every 50 ms using a cooled EMCCD camera (iXon life 888, ANDOR). Field simulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.).
5
Whole-sample Confocal Imaging of Antibody-labeled Cells
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OPAB were washed with phosphate-buffered saline (PBS) and transferred to flat bottom 24-well plate. Samples were fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT) and permeabilized for 24 h on a rocker with permeabilization buffer (0.5% BSA, 1% Triton in PBS). All subsequent steps were performed in permeabilization buffer with overnight incubation. Samples were labeled with primary antibody, washed, and labeled with appropriate secondary antibodies. After washes, the samples were incubated with RapiClear 1.52 reagent (Sunjin Lab) overnight at RT.
Image acquisition was performed on a spinning-disk confocal microscope (Dragonfly, Oxford Instruments) equipped with an ultrasensitive 1024 × 1024 EMCCD camera (iXon Life 888, Andor) and four laser lines (405, 488, 561, and 637 nm). A 20×, NA 0.8 air objective (Nikon) was used for whole sample imaging and a 60×, NA 1.4 (Nikon) oil-immersion long-distance objective was used for in-depth imaging of selected areas. Images were processed using FiJi (ImageJ) and Bitplane Imaris x64 (Oxford Instruments) version 9.2 and 9.7.
Image acquisition was performed on a spinning-disk confocal microscope (Dragonfly, Oxford Instruments) equipped with an ultrasensitive 1024 × 1024 EMCCD camera (iXon Life 888, Andor) and four laser lines (405, 488, 561, and 637 nm). A 20×, NA 0.8 air objective (Nikon) was used for whole sample imaging and a 60×, NA 1.4 (Nikon) oil-immersion long-distance objective was used for in-depth imaging of selected areas. Images were processed using FiJi (ImageJ) and Bitplane Imaris x64 (Oxford Instruments) version 9.2 and 9.7.
6
Simultaneous Fluorescence and Electrophysiology Imaging
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Fluorescence was excited with a 475 nm LED (Olympus) and monitored using an inverted microscope (IX83, Olympus) equipped with a ×150 1.45 NA oil-immersion objective. A continuous acquisition was performed at 50 ms/frame for 120 s using a cooled EMCCD camera (iXon life 888, ANDOR). The Z-drift compensation system (IX3-ZDC, Olympus) was used to ensure a constant position of the focal plane during imaging. All experiments were conducted at 37°C within a whole-microscope incubator chamber (TOKAI HIT). Field simulation was performed at 1 Hz precisely synchronized with the beginning of an acquisition frame using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.). Samples were perfused with bath solution containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 15 mM glucose, 50 μM APV, 10 μM CNQX, adjusted to pH 7.4.
7
Time-lapse Imaging for BFM Tracking
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The time-lapse phase-contrast and fluorescence imaging for BFM tracking was performed at room temperature on an inverted fluorescence microscope (Nikon, Ti-E) containing the Perfect Focus platform using Nikon NIS-elements AR (version 4.51). Alexa Fluor 594 was excited by a 580-nm LED light source (58 W/cm2) (pE4000, CoolLED, UK) and was observed through a 650-nm emission filter. Alexa Fluor 488 was excited by a 490-nm LED light source (151 W/cm2) and observed through a 530-nm emission filter. Both phase-contrast and fluorescence images were collected by a 1.45 NA ×100 oil objective and captured by an EMCCD camera (Andor, iXon Life 888). Images were captured at 2.5 min, 5 min, or 6 min intervals for 2 h depending on the experimental purpose.
8
Near-TIRF Imaging of Synaptic Vesicle Release
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All near-total internal reflection fluorescence (near-TIRF) experiments were conducted at 37°C within a whole-microscope incubator chamber (TOKAI HIT). Individual release events were evoked by 1Hz field stimulation for 200s, unless noted otherwise. Fluorophores were excited with a 488 laser (Cell CMR-LAS-488, Olympus), and monitored using an inverted TIRF-equipped microscope (IX83, Olympus) under a 150x/1.45NA objective (UApo N). The Z-drift compensation system (IX3-ZDC) was used to ensure constant position of the focal plane during imaging. Near-TIRF with a penetration depth of <1 μm was achieved by adjusting the incident angle to 63.7°, which is near the critical angle of 63.6°. Images were acquired every 50 ms (with an exposure time of 49.38 ms) using a cooled EMCCD camera (iXon life 888, ANDOR). Field simulation was performed by using a pair of platinum electrodes and controlled by the software via Master-9 stimulus generator (A.M.P.I.). Samples were perfused with bath solution (125 mM NaCl, 2.5 mM KCl, 2mM CaCl2, 1mM MgCl2, 10 mM HEPES, 15mM Glucose, 50 μM APV, 10 μM CNQX adjusted to pH 7.4).
9
Multimodal Microscopy of Plant Specimens
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SmFISH and MCP-GFP images were taken using either a spinning disk confocal or a wide field microscope. For spinning disk microscopy, we used a Dragonfly (Oxford instrument) equipped with four laser lines (405, 488, 561, 637 nm) and an ultrasensitive EMCCD camera (iXon Life 888, Andor) mounted on a Nikon Eclipse Ti2 microscope body, using a 40x, NA 1.3 Plan Fluor oil objective or a 60x, NA 1.4 Plan Apochromat oil objective coupled with a supplementary lens of 2x, using z-stacks with a 0.5 μm or 0.4 μm step. For wide field imaging, we used a Zeiss Axioimager Z1 wide-field upright microscope equipped with a camera sCMOS ZYLA 4.2 MP (Andor), using a 100x, NA 1.4 Plan Apochromat oil objective.
For these z stacks, a step of 0.3 or 0.4 μm was used. Maximal image projections (MIP) were generated with ImageJ, and figures were realized using Adobe Photoshop and Illustrator. The mosaic of Figure 3A is accessible with a viewer run with Imjoy 52 . Note that plant fixation and smFISH both reduced the GFP signals.
For these z stacks, a step of 0.3 or 0.4 μm was used. Maximal image projections (MIP) were generated with ImageJ, and figures were realized using Adobe Photoshop and Illustrator. The mosaic of Figure 3A is accessible with a viewer run with Imjoy 52 . Note that plant fixation and smFISH both reduced the GFP signals.
10
Live-Cell Microscopy Imaging Workflow
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All images were taken using a wide-field fluorescence microscopy system based on an IX-83 inverted microscope (Olympus) equipped with a UAPON 150´/1.45 NA oil objective (Olympus), iXon Life 888 electron-multiplying charge-coupled device (EMCCD) camera (Andor), SOLA SE u-niR light engine (Lumencor), Chamlide TC top-stage incubator system (Live Cell Instrument), and ET-EGFP filter set (Chroma, ET470/40x, T495lpxr, ET525/50m). Live-cell imaging was performed at 37 ℃, and time-lapse z-stack images were taken at an interval of every 8 s over a period of 67 min. Z-stacks were imaged at an interval of 0.5 µm for a total height of 6 µm.
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