The details on TIRF microscopy were described previously [57 (link)]. Briefly, using an inverted fluorescent microscope (IX-81; Olympus) with a 100X/1.45NA objective (Olympus), cells were excited using a combination of green (488 nm) and red (561 nm) lasers, passed through a LF405/488/561/635 dichroic mirror, and filtered emitted light was projected side-by-side on an electron multiplying charge-coupled device (EM-CCD) camera (DU 897; Andor). Using Andor IQ2 software, images of transfected cells were obtained at 5 Hz and 100 ms exposure times. Each day fluorescent beads (Invitrogen) were imaged in the green and red channels and superimposed by mapping corresponding fluorescent bead positions in both channels. The green and red images were subsequently transformed and aligned as described before [56 (link), 66 (link)]. All experiments were conducted at room temperature (~ 25 °C).
Du897
Manufactured by Oxford Instruments
Sourced in United Kingdom
The DU897 is a precision laboratory equipment designed for advanced analysis and measurement applications. It features high-performance capabilities for accurate data collection and processing. The core function of the DU897 is to provide reliable and consistent results in a variety of research and testing environments.
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Lab products found in correlation
N sim, by Nikon (8 mentions)
Nis elements software, by Nikon (8 mentions)
Csu x1, by Yokogawa (7 mentions)
Eclipse ti e, by Nikon (5 mentions)
Ti e microscope, by Nikon (5 mentions)
Cfi apo tirf 100 h 1, by Nikon (5 mentions)
Eclipse ti, by Nikon (5 mentions)
Nis element, by Nikon (5 mentions)
Apo tirf 100 na 1.49 plan apo oil objective, by Oxford Instruments (4 mentions)
N sim module, by Nikon (4 mentions)
Axio zoom v16, by Zeiss (4 mentions)
Ti e inverted microscope, by Nikon (3 mentions)
A1rsi, by Nikon (3 mentions)
Apo tirf 100x na 1.49 plan apo oil objective, by Oxford Instruments (3 mentions)
Tetraspeck bead, by Thermo Fisher Scientific (3 mentions)
N sim system, by Nikon (3 mentions)
Eclipse ti inverted microscope, by Nikon (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Dmk 23u274, by Imaging Source (3 mentions)
Improvision ultraview vox system, by PerkinElmer (2 mentions)
90 protocols using du897
1
Dual-Color TIRF Microscopy Protocol
2
Single-Molecule Imaging of PSEN1 Dynamics
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Single-molecule imaging was done live in GFP-PSEN1 or NCT-SNAP/GFP-PSEN1 in rescued PSEN1 sKO or PSEN1 and 2/NCT tKO MEFs, respectively, using Total Internal Reflection (TIRF) Microscopy on an Olympus IX71 microscope equipped with a 100 × 1.7 N.A. objective lens. Despite near-physiological levels of expression, levels were higher than the density limit of 2 spots/µm² for single-molecule imaging. Therefore, a circular region of 10–12 µm diameter in the center of the imaging field was photobleached using a focused laser steered by a set of scanning mirrors, similar to the PhotoGate approach (Madl et al., 2010 (link); Belyy et al., 2017 (link)). After the initial photobleaching, several rings were drawn with the focused laser at the edge of the bleached region at intervals of 5–10 s to control the re-population with unbleached molecules. For imaging, an iris in the TIRF illumination pathway was closed to eliminate glare from outside of the central region. Movies of 700 frames were recorded at 34 Hz with a back-illuminated EMCCD camera (Andor iXon DU-897).
3
Imaging Protocols for Live-Cell Microscopy
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For imaging experiments, cells were plated on 35mm glass bottom dishes at low density (MatTek Corp, Ashland, MA). Immunofluorescence and live cell imaging were carried out one day after transfection. Spinning disc confocal (SDC) microscopy was performed using the Improvision UltraVIEW VoX system (Perkin-Elmer) built around a Nikon Ti-E inverted microscope, equipped with PlanApo objectives (40x1.0-NA 1.0 and 60X1.49-NA) and controlled by Volocity (Improvision) software. Excitation light was provided by 488-nm/50-mW diode laser (Coherent) and 561-nm/50-mW diode laser (Cobolt), and fluorescence was detected by EM-CCD camera (C9100–50; Hamamatsu Photonics).
Total internal reflection fluorescence (TIRF) microscopy was performed on a setup built around a Nikon TiE microscope equipped with 60X1.49-NA. Excitation light was provided by 488-nm (for GFP), 561-nm (for mCherry/mRFP/mdsRed) and 640-nm (for iRFP) DPSS lasers coupled to the TIRF illuminator through an optic fiber. The output from the lasers was controlled by an acousto-optic tunable filter and fluorescence was detected with an EM-CCD camera (Andor iXon DU-897). Acquisition was controlled by Andor iQ software. Images were sampled at 0.20 Hz with exposure times in the 100–500 ms range. SDC microscopy was carried out at room temperature (20–25°C) and TIRF microscopy at 37°C.
Total internal reflection fluorescence (TIRF) microscopy was performed on a setup built around a Nikon TiE microscope equipped with 60X1.49-NA. Excitation light was provided by 488-nm (for GFP), 561-nm (for mCherry/mRFP/mdsRed) and 640-nm (for iRFP) DPSS lasers coupled to the TIRF illuminator through an optic fiber. The output from the lasers was controlled by an acousto-optic tunable filter and fluorescence was detected with an EM-CCD camera (Andor iXon DU-897). Acquisition was controlled by Andor iQ software. Images were sampled at 0.20 Hz with exposure times in the 100–500 ms range. SDC microscopy was carried out at room temperature (20–25°C) and TIRF microscopy at 37°C.
4
Total Internal Reflection Fluorescence Microscopy Protocol
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TIRF microscopy
experiments were performed on a home-built system
based on a Zeiss Axiovert 200 microscope equipped with a 100×,
NA = 1.46 Plan-Apochromat objective (Zeiss). TIR illumination was
achieved by shifting the excitation beam parallel to the optical axis
with a mirror mounted on a motorized table. The setup was equipped
with a 488 nm diode laser (iBeam smart 488, Toptica), a 532 nm diode-pumped
solid state (DPSS) laser (Spectra physics Millennia 6s), and a 647
nm diode laser (Obis LX 647, Coherent). Laser lines were overlaid
with an OBIS Galaxy beam combiner (Coherent). Direct analog laser
modulation (488 and 647 nm) or an Acousto-optic modulator (Isomet)
(532 nm) were used to adjust laser intensities (1–3 kW cm–2) and timings using an in-house developed package
implemented in LABVIEW (National Instruments). A dichroic mirror (Di01-R405/488/532/635-25x36,
Semrock) was used to separate excitation and emission light. Emitted
signals were split into two color channels using an Optosplit II image
splitter (Cairn) with a dichroic mirror (DD640-FDi01-25x36, Semrock)
and emission filters for each color channel (FF01-550/88-25, ET 570/60,
ET 675/50, Chroma) and imaged on the same back-illuminated EM-CCD
camera (iXon Ultra, DU897, Andor).
experiments were performed on a home-built system
based on a Zeiss Axiovert 200 microscope equipped with a 100×,
NA = 1.46 Plan-Apochromat objective (Zeiss). TIR illumination was
achieved by shifting the excitation beam parallel to the optical axis
with a mirror mounted on a motorized table. The setup was equipped
with a 488 nm diode laser (iBeam smart 488, Toptica), a 532 nm diode-pumped
solid state (DPSS) laser (Spectra physics Millennia 6s), and a 647
nm diode laser (Obis LX 647, Coherent). Laser lines were overlaid
with an OBIS Galaxy beam combiner (Coherent). Direct analog laser
modulation (488 and 647 nm) or an Acousto-optic modulator (Isomet)
(532 nm) were used to adjust laser intensities (1–3 kW cm–2) and timings using an in-house developed package
implemented in LABVIEW (National Instruments). A dichroic mirror (Di01-R405/488/532/635-25x36,
Semrock) was used to separate excitation and emission light. Emitted
signals were split into two color channels using an Optosplit II image
splitter (Cairn) with a dichroic mirror (DD640-FDi01-25x36, Semrock)
and emission filters for each color channel (FF01-550/88-25, ET 570/60,
ET 675/50, Chroma) and imaged on the same back-illuminated EM-CCD
camera (iXon Ultra, DU897, Andor).
5
Fluorescence Imaging of Microbial Colonies
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Fluorescence images of colonies were acquired following growth using a Nikon Eclipse Ti-E inverted fluorescence microscope equipped with a DU897 electron-multiplying charge-coupled device (EMCCD) camera (Andor) using μManager v. 1.4 (52 (link)) or a Nikon TE-2000 or Zeiss Axio Zoom.V16 microscope. Colonies sandwiched between two agar surfaces and the colonies in the corresponding control experiments were imaged after 7 days at 4°C. Colonies that did not show sufficient fluorescence at this point were imaged again after 26 more days at 4°C.
Edges of colonies were imaged with a 20X objective on a Nikon Eclipse Ti-E inverted fluorescence microscope equipped with a DU897 camera (Andor) using μManager v. 1.4.
Edges of colonies were imaged with a 20X objective on a Nikon Eclipse Ti-E inverted fluorescence microscope equipped with a DU897 camera (Andor) using μManager v. 1.4.
6
Spinning-Disc Confocal Microscopy Imaging
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Cells were imaged in complete medium (unless stated otherwise) at an acquisition rate from 5-s to 1-min intervals using a spinning-disc confocal microscope (Ultraview VoX; PerkinElmer) attached to an inverted microscope (IX81; Olympus), equipped with a 100× oil-immersion objective (1.40 NA, UPlanSApo), an EMCCD camera (C9100-13; Hamamatsu Photonics) for image acquisition, and Volocity software (PerkinElmer) to control the acquisition protocol. Fixed samples and live cells were also imaged with a Nikon confocal A1R system and Nikon SIM attached to a Ti-E inverted microscope (Nikon) with Perfect Focus System using a 100× oil immersion objective (1.40 NA, CFI Plan-ApochromatVC). The cameras (Neo sCMOS and DU-897; Andor Technology) were used to acquire images for confocal A1R and SIM systems, respectively, with NIS-Elements AR software (Nikon) to control the acquisition protocol. For z-stack images, cells were imaged at a step size of 0.2–0.5 µm with a total height of 15–20 µm.
7
Reconstitution of neurotransmitter release
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General procedures were based on previous literature35 (link),36 (link). In brief, glass coverslips were coated with PEG and PEG-biotin (with a ratio of 9:1) and assembled into a flow chamber. Neutravidin (0.2 mg/ml; Pierce) were coated in each channel. The PM-vesicles were firstly immobilized on PEG/PEG-biotin surface during a 20-min incubation at 25 °C. After an extensive buffer wash step to remove the unbounded supported liposomes, 40 µM (total lipids) SV-vesicles and 1 µM Munc13-1 C1-C2B-MUN fragment were flowed into the channels and incubated for 30 min at 30 °C. Before imaging, unbounded SV-vesicles and proteins were washed out by a two-step buffer wash. Imaging was carried out with a Nikon Ti series inverted microscope equipped with TIRF illuminator (Nikon), beam splitter (Cairn Research), and an EMCCD camera (Andor iXon DU-897). Total internal reflection was achieved by a 1.49 NA ×100 oil-immersed objective (Nikon). PM-vesicle density was initially checked upon excitation at 640 nm. SV-vesicles were counted by the number of fluorescent spots from DiI dyes upon excitation at 532 nm. Images were processed by home-written MATLAB (MathWorks) script.
8
Mitochondrial Membrane Potential Imaging
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Cells were cultured on glass-bottom dishes in the presence of JC-1 (2 µM; Molecular Probes), a cationic dye that accumulates in energized mitochondria, for 30 min. Images of cells in PBS were acquired using a confocal microscopy system (A1Rsi; Nikon) with an inverted microscope (Eclipse Ti-E; Nikon), a 40× NA 0.95 Plan Apochromat objective (Nikon), and an electron multiplying charge-coupled device 16-bit 512 × 512 camera (DU897; Andor Technology) at 37°C with 5% CO2. Both 488- and 561-nm lasers were used for imaging monomers and J-aggregate forms, respectively. Images were acquired using NIS-Elements imaging software (version 4.10.00). Acquired images were cropped and fluorescence intensities were quantified using ImageJ software (version 1.45f).
9
TIRF Microscopy for Flagellar Motor Dynamics
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A Nikon Ti-E microscope with a 100 mW, 514-nm laser (Cobalt Fandango) focused in the back focal plane of a 60× TIRF objective was used to generate evanescent fields. An Andor iXon DU897 camera was used for capturing TIRF (total internal reflection fluorescence) images while a CCD camera (DCC1545M-GL, Thorlabs Inc.) was used for capturing phase contrast images. Strains carrying fliCcys were labeled with maleimide dye as described elsewhere (Blair et al., 2008 (link)). The fliM-eYFP-fliM internal fusion was gifted to us by the Berg lab. The allele carried a [Gly Gly][YFPSer…YFPLys][Ser Gly Gly] insertion between codons 15 and 16 of fliM. Tethered motor assays indicated that the fusion motors were fluorescent and functional.
10
Dual-color FRAP Microscopy Setup
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The microscopy setup consisted of an epifluorescence microscope (Ti-E, Nikon), an objective lens (40× CFI Plan Apo Lambda, 0.95 NA, Nikon), a relay optics box for dual-color imaging (GA03; G-Angstrom, Japan), and an electron multiplier-type CCD camera (EM-CCD, iXon DV887 or DU897; Andor Technology PLC, UK). A slit was placed at the imaging surface of the microscope in the relay optics. A dichroic mirror (FF458-Di02, Semrock) located just outside the slit split the optical pathway after the imaging surface into two pathways for cyan (CFP) and yellow (YFP1G) fluorescence. The two pathways converged on the acceptance surface of the EM-CCD camera side by side. Band-pass filters were set for each pathway (467–499 nm for CFP and 510–560 nm for YFP). Multiphoton fluorescence recovery after photobleaching (MP-FRAP) experiments were conducted as described elsewhere [17 (link)]. Image analyses were carried out in the ImageJ software (NIH, USA).
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