For imaging we used a modified inverted Leica DM IRE2 microscope equipped with an oil immersion lens of a numerical aperture of 1.4 (UPLSAPO 100xO, Olympus). Two continuous wave lasers provided the excitation (LightCube Revolution, 770, R 639 nm, 1.5 W, HB-Laser) and activation (OBIS 405 LX, Coherent) light of 639 nm and 405 nm wavelength, respectively. Both laser beams were combined by a dichroic mirror (zt 442 RDC, AHF analysentechnik). A safeguard slit confined the excitation and activation light within the field of view of 33 µm × 33 µm. The intensities in the focal plane were 0.6 kW/cm for excitation and 0.07 kW/cm for activation. For detection, the fluorescence was separated from the excitation and activation light by a dichroic mirror (zt 642 rdc, AHF analysentechnik). A band pass filter (705/100 ET, AHF analysentechnik) in front of the recording EMCCD-camera (iXon 897, Andor) defined our detection bandwidth and a notch filter (zet635NF, AHF analysentechnik) blocked residual excitation light. With an electronically tunable bandpass filter (AOTFncC-VIS-TN, AA Optoelectronics) we reliably turned on the excitation laser within a time window of ~10 µs which was important for our measurement protocol. Images were acquired with the software Imspector (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany).
Ixon 897
Manufactured by Oxford Instruments
Sourced in United Kingdom, United States
The IXon 897 is a high-performance electron-multiplying charge-coupled device (EMCCD) camera designed for low-light imaging applications. It features a back-illuminated sensor with high quantum efficiency and low noise characteristics, enabling the detection of weak signals with high sensitivity. The IXon 897 is capable of capturing images at high frame rates and can be used in a variety of scientific research and industrial applications, such as fluorescence microscopy, bioluminescence studies, and astronomy.
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Lab products found in correlation
Csu x1, by Yokogawa (9 mentions)
Eclipse ti, by Nikon (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Metamorph 7, by Molecular Devices (3 mentions)
Inu zilcs f1, by Tokai Hit (3 mentions)
Em01 r488 568 15, by IDEX Corporation (3 mentions)
Uplansapo, by Olympus (3 mentions)
Axiovert 200, by Zeiss (2 mentions)
Te2000 u, by Nikon (2 mentions)
Uplsapo 60xw water immersion lens, by Olympus (2 mentions)
Xcellence software v 1, by Olympus (2 mentions)
Fb450 40, by Thorlabs (2 mentions)
Optiphot 100, by Nikon (2 mentions)
Lf 206 ls, by Uvitec (2 mentions)
Alpha plan fluar, by Zeiss (2 mentions)
Di03 635 t1, by IDEX Corporation (2 mentions)
Ff01 582 64, by IDEX Corporation (2 mentions)
Ff01 680 42, by IDEX Corporation (2 mentions)
Nis elements 4, by Nikon (2 mentions)
N sim, by Nikon (2 mentions)
87 protocols using ixon 897
1
Super-Resolution Imaging Microscope Setup
2
Quantitative Analysis of Neuronal Transcription Factors
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All images were captured with an epi-fluorescence microscope (Axiophoto with 20×/0.5 or 40×/0.75 objective lens, Carl Zeiss; Ti-E with 20×/0.75 or 40×/0.95 objective lens, Nikon) attached with a CCD camera (DP70, Olympus; CoolSNAP HQ2, Photometrics) or an EM-CCD camera (iXon897, Andor Technology). Fluorescence intensities of CREB and FOS expressions were quantified using ImageJ software. The background fluorescence intensities in the cytoplasm, which lack these transcription factors, were subtracted from the fluorescence intensities in the nucleus, and then the expression levels were determined by the ratio of the subtracted nuclear intensities to the cytoplasmic intensities. Measurements of dendrite morphology were performed using NeuronJ plugin [15 (link)].
3
Fluorescence Intensity-Based FRET Analysis
4
Three-Dimensional Spheroid Imaging Setup
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Three-dimensional live spheroid imaging was performed on a home-built setup based on an Axiovert 200 microscope body (Carl Zeiss Microscopy, Jena, Germany). Confocal imaging was achieved by means of a spinning disk unit (CSU-X1, Yokogawa, Musashino, Tokyo, Japan). The confocal image was acquired on an emCCD camera (iXon 897, Andor). IQ-software (Andor) was used for basic setup-control and data acquisition. Illumination of CFP- and GFP-labeled spheroids was performed with two different lasers of wavelengths 405 (CrystaLaser, Reno, NV, USA) and 488 nm (Coherent Inc., Santa Clara, CA, USA). Accurately controlled excitation intensity and excitation timing were achieved using an acousto-optic tunable filter (AA Optoelectronics, Orsay, France). Light was coupled into the confocal spinning-disk unit by means of a polarization maintaining single-mode fiber (OZ Optics, Ottawa, Canada). The fluorescent signal was collected by a 10×/0.3 air objective (Carl Zeiss Microscopy, Jena, Germany). Three-dimensional images of PDAC/PSC spheroids were obtained by imaging 100 µm z-stacks (every 1 µm) using a piezo system (Physik Instrumente, Karlsurhe, Germany).
5
Single-Molecule FRET Imaging Setup
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All FRET experiments were carried out on a custom-built prism-type TIRF setup built using a TE2000-U (Nikon) base. The sample was excited by an Ar/Kr ion laser (488 nm, Coherent), a Nd/YAG laser (532 nm, CrystaLaser) and a HeNe laser (633 nm, Laser 2000). Laser intensity as well as excitation duration (15–300 ms) was controlled by an acousto-optical tunable filter (AOTF.nC-TN, Pegasus Optik), which was synchronized to an EM-CCD camera (iXon+ 897, Andor) used for dual-color detection through a custom-built, real-time control unit. Fluorescence was collected by a 60× water immersion objective (CFI Plan Apo VC, Nikon, N.A. 1.2), spectrally separated by a dichroic mirror (630 DCXR, AHF) and filtered by the bandpass filters HQ550/88 and HQ715/150 (AHF). Fluorescence was detected on the EM-CCD camera split into two channels according to wavelength and an image series was recorded (Supplementary Figure S2A ).
6
High-Resolution Live-Cell Imaging Protocol
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Cell imaging (U2OS) was carried out on an Olympus IX83 microscope equipped with three EMCCD cameras (Andor iXon 897) mounted on a 4-camera splitter, four lasers (405 nm, 488 nm, 561 nm and 647 nm), mounted with a ×1.6 magnification adapter and ×60 apochromatic oil objective lens (NA 1.5), resulting in a total of ×96 magnification. The microscope stage incubation chamber was maintained at 37 °C with CO2 and humidity supplement. A laser quad-band filter set for TIRF (emission filters at 445/58, 525/50, 595/44, 706/95) was used to collect fluorescence signals simultaneously. Data acquisition was carried out with CellSens 4.1.1 software. Localization precision was ~5 nm in 4 s, ~6 nm in 16 s, and ~10 nm in 80 s82 . The video was recorded at 136 ms per frame with a total of 96 frames and 100 ms exposure time. Image size was adjusted to show individual nuclei, and intensity thresholds were set on the basis of the ratios between nuclear foci signals to background nucleoplasmic fluorescence.
7
Immunolabeling Actin Isoforms in Cells
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For immunolabeling the actin isoforms, cells were grown on glass coverslips and fixed in 1% paraformaldehyde solution (Sigma, St. Louis, MO, USA) in DMEM medium (PanEco, Moscow, Russia) containing HEPES buffer (Sigma, St. Louis, MO, USA) for 15 min, then rinsed with PBS and fixed for an additional 5 min with methanol at −20 °C. Before fixing, some coverslips were incubated with nocodazole at a concentration of 0.01 μM for 30 min at 37 °C and 5% CO2.
Actin filaments were stained with murine monoclonal antibodies against cytoplasmic β- or γ-actin [1 (link)]. Anti-mouse antibodies conjugated with Alexa 488 or Alexa 561 fluorescent dyes (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies.
The samples were embedded in Moviol and examined using an N-SIM microscopic system ((Nikon Instech Co., Tokyo, Japan) with an immersion objective 100×/1.49 NA, excitation laser wavelengths of 488 nm and 561 nm. Image stacks (with a z-axis step of 0.12 μm) were acquired with an EMCCD camera (iXon 897, Andor, effective pixel size 60 nm) in the 3D-SIM mode. Serial optical sections of the same cell, taken in the wide field mode, were processed using the AutoQuant blind deconvolution algorithm. Image acquisition and SIM reconstruction were performed using the NIS-Elements 4.2 software (Nikon Instech Co., Tokyo, Japan).
Actin filaments were stained with murine monoclonal antibodies against cytoplasmic β- or γ-actin [1 (link)]. Anti-mouse antibodies conjugated with Alexa 488 or Alexa 561 fluorescent dyes (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies.
The samples were embedded in Moviol and examined using an N-SIM microscopic system ((Nikon Instech Co., Tokyo, Japan) with an immersion objective 100×/1.49 NA, excitation laser wavelengths of 488 nm and 561 nm. Image stacks (with a z-axis step of 0.12 μm) were acquired with an EMCCD camera (iXon 897, Andor, effective pixel size 60 nm) in the 3D-SIM mode. Serial optical sections of the same cell, taken in the wide field mode, were processed using the AutoQuant blind deconvolution algorithm. Image acquisition and SIM reconstruction were performed using the NIS-Elements 4.2 software (Nikon Instech Co., Tokyo, Japan).
8
Imaging NK Cell Receptor Dynamics
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For imaging of NK cell receptors
at the membrane, we used total internal reflection microscopy setup
on a Zeiss Elyra PS1 microscope with ZEN 2012 Black software. An alpha
Plan-Apochromat 100× oil immersion objective of numerical aperture
1.46 was used for imaging, and fluorescence was excited with 488 and
633 nm laser light and detected in two channels (band-pass filter,
495 to 575 nm; long-pass filter, 655 nm) with an electron-multiplying
charge-coupled device camera (Andor iXon 897) at 36 ms frame-to-frame
intervals; sequences of 6000 frames were acquired per cell. Image
size was 12.8 μm with 128 × 128 pixels recorded at 100
nm pixel size.
at the membrane, we used total internal reflection microscopy setup
on a Zeiss Elyra PS1 microscope with ZEN 2012 Black software. An alpha
Plan-Apochromat 100× oil immersion objective of numerical aperture
1.46 was used for imaging, and fluorescence was excited with 488 and
633 nm laser light and detected in two channels (band-pass filter,
495 to 575 nm; long-pass filter, 655 nm) with an electron-multiplying
charge-coupled device camera (Andor iXon 897) at 36 ms frame-to-frame
intervals; sequences of 6000 frames were acquired per cell. Image
size was 12.8 μm with 128 × 128 pixels recorded at 100
nm pixel size.
9
Imaging Kinesin Dynamics in C. elegans
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Microscopy images were acquired using a custom-built epi-illuminated wide-field fluorescence microscope operated by a MicroManager software interface (μManager, MicroManager 1.4, www.micromanager.org ; Edelstein et al. 2014 ) and built around an inverted microscope body (Eclipse Ti; Nikon, Amsterdam, Netherlands) fitted with a 60× water-immersion objective (CFI Plan Apo IR 60× water immersion, numerical aperture 1.27; Nikon). Excitation light was provided by a diode-pumped solid-state laser (Calypso 50, 491 nm; Cobolt, Solna, Sweden). Images were captured with an electron-multiplying charge-coupled device camera (iXon 897; Andor, Belfast, UK). One camera pixel corresponded to 92 nm × 92 nm in the image plane.
The C. elegans strain expressing EGFP-tagged OSM-3 kinesin motor proteins (Snow et al. 2004 (link)) was a kind gift of Jonathan M. Scholey (University of California, Davis, Davis, CA). Fluorescence imaging in living C. elegans was performed by anesthetizing adult worms (maintained at 20°C) in M9 containing 5 mM levamisole (tetramisole hydrochloride, L9756; Sigma-Aldrich, St. Louis, MO) and immobilizing them between a 2% agarose pad and a coverslip. Samples were imaged at room temperature (21°C) at 152 ms/frame.
The C. elegans strain expressing EGFP-tagged OSM-3 kinesin motor proteins (Snow et al. 2004 (link)) was a kind gift of Jonathan M. Scholey (University of California, Davis, Davis, CA). Fluorescence imaging in living C. elegans was performed by anesthetizing adult worms (maintained at 20°C) in M9 containing 5 mM levamisole (tetramisole hydrochloride, L9756; Sigma-Aldrich, St. Louis, MO) and immobilizing them between a 2% agarose pad and a coverslip. Samples were imaged at room temperature (21°C) at 152 ms/frame.
10
Calcium Dynamics Monitoring in Cells
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