For live imaging, embryos are placed in 5 cm glass-bottom dishes (MatTek) under a CellDiscoverer 7 (Zeiss) equipped with a 20x/0.95 objective and an ORCA-Flash 4.0 camera (C11440, Hamamatsu) or a 506 axiovert (Zeiss) camera. Using the experiment designer tool of ZEN (Zeiss), we set up nested time-lapses in which all embryos are imaged every 5 h for 10 min with an image taken every 5 s at 2 focal planes positioned 10 μm apart. Embryos are kept in a humidified atmosphere supplied with 5% CO2 at 37°C. mTmG embryos are imaged at the 2-and 16cell stage using an inverted Zeiss Observer Z1 microscope with a CSU-X1 spinning disc unit (Yokogawa). Excitation is achieved using a 561 nm laser through a 63x/1.2 C Apo Korr water immersion objective. Emission is collected through 595/50 band pass filters onto an ORCA-Flash 4.0 camera (C11440, Hamamatsu). The microscope is equipped with an incubation chamber to keep the sample at 37°C and supply the atmosphere with 5% CO2. Surface tension measurements are performed on a Leica DMI6000 B inverted microscope equipped with a 40x/0.8 DRY HC PL APO Ph2 ( 11506383) )objective and Retina R3 camera and 0,7x lens in front of the camera. The microscope is equipped with an incubation chamber to keep the sample at 37°C and supply the atmosphere with 5% CO2.
C11440
Manufactured by Hamamatsu Photonics
Sourced in Japan
The C11440 is a high-performance charge-coupled device (CCD) image sensor produced by Hamamatsu Photonics. It features a large effective area, high quantum efficiency, and low noise characteristics, making it suitable for a variety of scientific and industrial applications that require high-quality imaging.
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Lab products found in correlation
Uplsapo60xw, by Olympus (8 mentions)
Ibeam smart 488, by Toptica (8 mentions)
Hcimage software, by Hamamatsu Photonics (3 mentions)
Eclipse ti, by Nikon (3 mentions)
Orca flash 4, by Hamamatsu Photonics (2 mentions)
Lambda 10 3, by Sutter Instruments (2 mentions)
Brightlineff01 600 37, by IDEX Corporation (2 mentions)
Brightlineff01 676 29, by IDEX Corporation (2 mentions)
Ba510 550, by Olympus (2 mentions)
Imager z2, by Zeiss (2 mentions)
Plan apo, by Hamamatsu Photonics (2 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Axio imager, by Zeiss (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Apotome, by Zeiss (2 mentions)
Bx63 microscope, by Olympus (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
Fish gelatin, by Merck Group (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
57 protocols using c11440
1
Live Imaging of Embryonic Development
2
Xenopus Chromosome Assembly Assays
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Chromosome assembly assays using Xenopus egg extracts were performed as described previously (Kinoshita et al., 2015 (link)) with minor modifications. For standard add-back assays, Δcond extracts were supplemented with purified recombinant complexes (35 nM) and preincubated at 22°C for 15 min. Xenopus sperm nuclei were then added at a final concentration of 0.8–2.0 × 103 nuclei/µl and incubated at 22°C for another 120 min to assemble chromosomes. Alternatively, mouse sperm nuclei prepared as described previously (Shintomi et al., 2017 (link)) were added at a final concentration of 0.4–1.0 × 103 nuclei/µl and incubated at 22°C for 150 min. After incubation, the reaction mixtures were fixed and processed for immunofluorescence analyses. For topo II–depleted add-back assays, Δcond Δtopo II extracts were used instead of Δcond extracts. Immunofluorescence analyses were performed as described previously (Kinoshita et al., 2015 (link)), except that an Olympus BX63 microscope equipped with a UPlanSApo 100×/1.40 NA oil immersion lens and an ORCA-Flash 4.0 digital complementary metal oxide semiconductor camera C11440 (Hamamatsu Photonics) were used for observations. CellSens Dimension software (Olympus) was used for image acquisition. Primary antibodies used for immunofluorescence in this study were as follows: anti-mSMC4, anti-histone H3, and anti–topo II.
3
Imaging of T-cell Synaptogenesis Assay
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The fixable CMAC dye CellTracker Blue (ThermoFisher) was applied in some experiments to label live target cells before engagement with T cells. AbTCR + co-stim T-cells and target cells were mixed in equal proportions at high density (4 × 106/mL) in complete growth media, centrifuged at 40 × g for 5 min and incubated at room temperature for 30 min to promote synaptogenesis25 (link). Cells were gently re-suspended in the same media and 50 µL (2 × 105 cells) were applied to poly-lysine coated glass coverslips and allowed to adhere for 15 min at room temperature. Adhered cells were then fixed and quenched as described previously26 (link). PBS with 3% BSA, 1% normal goat serum (Jackson ImmunoResearch) 1% cold water fish gelatin (Sigma Aldrich) and 0.03% TX100 (Sigma Aldrich) was used for all blocking and subsequent antibody incubation steps. Primary antibodies were applied overnight in a humidification chamber at 4 °C and secondary antibodies were applied for 90 min at room temperature. Stained cells were mounted on slides with ProLong Gold antifade reagent (ThermoFisher). Microscopy was carried out on an Olympus IX73 with a 60X UPlanSApo objective (NA 1.35) and images were collected with a Hamamatsu camera C11440 using HCImageLive v4.3 software and processed with Photoshop CC2019.
4
Microscopic Analysis of Parasite Egress and Invasion
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Egress and invasion were monitored by differential interference contrast (DIC) microscopy using a Nikon Eclipse Ni light microscope fitted with a Hamamatsu C11440 digital camera. Egress videos were performed using one population of parasites stained briefly with Hoechst, as described previously [33 (link)]. Invasion videos were performed using schizonts purified from DMSO- or RAP-treated ACβ-HA:loxP or PKA-HA:loxP cultures mixed with uninfected erythrocytes. DIC images were taken every 150 ms for at least 8 min, and the resulting time-lapse videos were processed using Nikon NIS Elements AR analysis software.
5
Live-Cell Imaging of PKA Activation
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Images were acquired on an inverted microscope (IX81, Olympus) with a heated incubator that maintained the chamber at 37 °C and 5% CO2 (WELS, Tokai-Hit). Images were acquired for either 15 or 25 minutes at 1-minute intervals. Drugs were added at the 5-minute and 15-minute point. PKA activation was achieved through treatment with 50 µM forskolin and 100 µM IBMX, unless otherwise noted. For the experiment demonstrating reversibility, the media was replaced with new media containing 40 µM H89 at the 15-minute point, as residual forskolin and IBMX led to an incomplete inhibition of PKA. Images were acquired with a CMOS camera (C11440, Hamamatsu) in a 60x oil objective (Plan Apo N, Olympus) with the appropriate excitation and emission filters driven by a filter wheel controller. Metamorph was used to control the hardware associated with the microscope which includes the motorized stage (MS-2000, Applied Scientific Instrumentation), filter wheels (Lambda 10-3, Sutter Instruments), and LED light source (pE-300, CoolLED). To measure response with the FRET reporter, cells were excited in the CFP channel, and images were acquired in the YFP channel.
6
In Vitro Scratch Wound Assay for NIH-3T3 Cells
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NIH-3T3 cells (50 000/well) were seeded in a 48-well plate 48 h before the assay. To prevent cell proliferation during the assay, cells were treated with 10 μg/ml Mitomycin C (Sigma-Aldrich) for 2 h, then a scratch was introduced in the middle of the well by a 20 μl pipette tip. The well was rinsed once with DMEM and replaced with DMEM supplemented with the corresponding treatment (lipids were acquired from Avanti Polar Lipids or produced internally (43 (link))). Then, the plate was transferred to the live-cell imaging microscope (Olympus IX81) fitted with an incubator (humidified atmosphere, 37°C, 5% CO2) and a motorized stage. Phase-contrast images were taken at 6.4× magnification every 30 min for 48 h, with the Hamamatsu (C11440) detector at 1 mega pixel (1,024 × 1,024 pixel) 16 bit.
7
SIM Imaging of Nano-Drug Delivery
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SIM imaging was performed
using a customer three-color system built around an Olympus IX71 microscope
stage, as previously described.40 (link),46 (link) Laser wavelengths of
488 nm (iBEAMSMART- 488, Toptica), 561 nm (OBIS 561, Coherent), and
640 nm (MLD 640, Cobolt) were used to excite the fluorescence in the
samples. A 60 Å∼/1.2 numerical aperture (NA) water immersion
lens (UPLSAPO 60XW, Olympus) focused the structured illumination pattern
onto the sample. This lens captured the samples’ fluorescent
emission light before being imaged onto an sCMOS camera (C11440, Hamamatsu).
Raw images were acquired with HCImage software (Hamamatsu). MCF7 cells
were treated with PP75 coacervate-like nano system and free Dox (both
with 6.4 μg/mL Dox concentration) for various time periods and
then stained with LysotrackerTM according to the protocol provided
by Thermofisher before they were imaged by SIM. Reconstruction of
the SIM images with LAG SIM Resolution-enhanced images were reconstructed
from the raw SIM data with LAG SIM, a custom plugin for Fiji/ImageJ
available in the Fiji Updater. LAG SIM provides an interface to the
Java functions provided by fair SIM.47 (link)
using a customer three-color system built around an Olympus IX71 microscope
stage, as previously described.40 (link),46 (link) Laser wavelengths of
488 nm (iBEAMSMART- 488, Toptica), 561 nm (OBIS 561, Coherent), and
640 nm (MLD 640, Cobolt) were used to excite the fluorescence in the
samples. A 60 Å∼/1.2 numerical aperture (NA) water immersion
lens (UPLSAPO 60XW, Olympus) focused the structured illumination pattern
onto the sample. This lens captured the samples’ fluorescent
emission light before being imaged onto an sCMOS camera (C11440, Hamamatsu).
Raw images were acquired with HCImage software (Hamamatsu). MCF7 cells
were treated with PP75 coacervate-like nano system and free Dox (both
with 6.4 μg/mL Dox concentration) for various time periods and
then stained with LysotrackerTM according to the protocol provided
by Thermofisher before they were imaged by SIM. Reconstruction of
the SIM images with LAG SIM Resolution-enhanced images were reconstructed
from the raw SIM data with LAG SIM, a custom plugin for Fiji/ImageJ
available in the Fiji Updater. LAG SIM provides an interface to the
Java functions provided by fair SIM.47 (link)
8
Visualizing Synchronized Parasite Egress
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Egress was visualized by differential inference contrast (DIC) light microscopy as described previously (26 (link)) using a Nikon Eclipse Ni microscope with a Hamamatsu C11440 digital camera. The PKG (cGMP-dependent protein kinase G) inhibitor compound 2 (4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-α]pyridine-3-yl]pyrimidin-2-amine) was used to tightly synchronize parasites prior to egress, as described previously (26 (link)). To visualize egress in both DMSO- and RAP-treated cultures concurrently, one culture was stained with 1 µg/ml Hoechst stain (Thermo) for 5 min before washing and pooling the cultures and imaging, as previously described (30 (link)). The percentage of parasites undergoing egress and the time to egress were quantified from nine videos, and the statistical significance of any differences between the DMSO- and RAP-treated parasites was calculated using paired t tests.
9
Mesenteric Microvascular Permeability Evaluation
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Fluorescein isothiocyanate (FITC)–bovine serum albumin (BSA) was used to evaluate albumin leakage across the mesenteric venular wall using inverted intravital microscopy (C11440, Hamamatsu, Shizuoka, Japan). Briefly, under anesthesia, the rat abdomen was opened via a midline incision. The ileocecal portion of the mesentery (10 to 15 cm from the caudal mesentery) was exteriorized and mounted on a transparent plastic stage under moist condition at 37 °C. Fluorescence intensity in the venules (IV) and perivenular interstitium (IP) was recorded at 0, 1, 3, and 6 min after an intravenous injection of FITC–BSA (50 mg/kg) using Image-Pro Plus 5.0 software. FITC–BSA leakage was estimated by dividing IP by IV, and the ratio of FITC–BSA leakage at a given time point to that of the baseline was designated as the ratio of FITC–BSA leakage at that point. For the mesenteric microvessel networks measurement, 50 mg/kg FITC–BSA was injected intravenously and allowed to circulate for 5 min; the fluorescence intensity of FITC–BSA was recorded and the FITC–BSA+ area per vessel was quantified.
10
Visualizing Autolysosomes in Tobacco Cells
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TBY-2 suspension tobacco cells (200 µL) were centrifuged at 4000× g for 5 min and the pellet was resuspended in 200 µL of 100 mM potassium phosphate buffer (PK) pH 6.5 containing 1 µM of Lysotracker Deep Red (Invitrogen, Thermo Fisher, Madrid, Spain) as previously described [36 (link)]. The mixture was incubated in the dark and at room temperature for 6 min. Subsequently, the preparation was centrifuged at 1000× g for 1 min, the supernatant was discarded, and the pellet was washed three times with PK buffer, pH 6.5 at 1000× g for one minute, and the final precipitate was resuspended in 150 µL of the same buffer. Fifty microliters were added to the slide and the structures were visualized in a Leica DM6 motorized fluorescence microscope equipped with Nomarski optic and a digital Hamamatsu C11440 camera. For the detection of autolysosomes, the preparations were observed using a band pass (BP) 545–580, DM 600, and a BA filter of 610. At least ten fields (approximately 20 cells per field) were evaluated in each of the three independent experiments carried out. Image analysis was performed using ImageJ software (Softonic, NIH, Bethesda, MD, USA).
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