For analysis of wound healing and cell migration by live imaging, we used a LCV100 (Olympus) equipped with a UAPO 40×/340× objective lens (Olympus), an LED light source, a DP30 camera (Olympus), and DIC optical components and interference filters, except Video 2 , for which we used an inverted fluorescence microscope (IX-81, Olympus) equipped with a spinning disk confocal imaging unit (CSU-X1, Yokogawa), a 40×/1.35 oil-immersion objective (UApo/340, Olympus), and electron-multiplying charge coupled device (EMCCD; iXon+, Andor Technology). To observe actin dynamics, we isolated stable lines of wild-type and αEcat KO Caco-2 cells expressing LifeAct-RFP. A wild-type line of these transfectants was additionally transfected with Nap1-GFP in a transient way. These cells were observed using an inverted fluorescence microscope (IX-81, Olympus) equipped with a spinning disk confocal imaging unit (CSU-X1, Yokogawa), a 40×/1.35 oil-immersion objective (UApo/340, Olympus), and a 561-nm laser (Sapphire LP, Coherent) for RFP excitation or a 488-nm laser (Sapphire LP, Coherent) for GFP excitation. We took fluorescence images with multiple z-stacks by EMCCD (iXon+, Andor Technology) at the specified time intervals and then made maximum-intensity Z projections.
Uapo 340
Manufactured by Olympus
Sourced in Japan
The UApo/340 is a high-performance objective lens designed for use in microscopy applications. It features a numerical aperture of 1.15 and a working distance of 0.25 mm, making it suitable for a variety of sample types and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Csu x1, by Yokogawa (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Alexa fluor 594 donkey anti mouse igg h l, by Abcam (1 mentions)
Alexa fluor 594 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Abcam (1 mentions)
Anti neun, by Merck Group (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Easyratiopro system, by Horiba (1 mentions)
Deltaram x monochromator, by Horiba (1 mentions)
Easyratiopro, by Horiba (1 mentions)
Csu x1, by Olympus (1 mentions)
Lcv100, by Olympus (1 mentions)
40 uapo 340 objective lens, by Olympus (1 mentions)
Dp30 camera, by Olympus (1 mentions)
Dv 885k, by Oxford Instruments (1 mentions)
Deltavision microscope system, by Cytiva (1 mentions)
P35g 1.5 10 c, by MatTek (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
10 protocols using uapo 340
1
Visualizing Wound Healing and Cell Migration
2
Immunofluorescence Staining of Neural Cells
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Cells were fixed in 4% paraformaldehyde/PBS (Nacalai tesque) for 15 min, permeabilized with 0.25% Triton X-100 (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) in PBS for 10 min and blocked with 1% bovine serum albumin (BSA, A9647, Sigma-Aldrich) in PBS for 1hr at room temperature (RT). Cells were incubated with primary antibodies (1:500 dilution in 1% BSA) overnight at 4°C and with secondary antibodies (1:1000 dilution in 1% BSA) for 1 h at RT. Primary antibodies used were: anti-NeuN (Cat# MAB377, Millipore, Tokyo, Japan, RRID:AB_2298772 ), anti-GFAP (Cat# ab53554, Abcam, Tokyo, Japan, RRID:AB_880202 ), and anti-Iba1 (Cat# 019-19741, FUJIFILM Wako Shibayagi Corp, Shibukawa, Japan; RRID:AB_839504 ). Secondary antibodies used were: Alexa Fluor 594 donkey anti-mouse IgG H&L (Cat# ab150108, Abcam, RRID:AB_2732073 ), Alexa Fluor 488 chicken anti-goat IgG H&L (Cat# A-21467, Molecular Probes, RRID:AB_141893 ), and Alexa Fluor 594 donkey anti-rabbit IgG (H+L) (Cat# A-21207, Thermo Fisher Scientific, RRID:AB_141637 ). Nuclei were stained with DAPI (sc-3598, Santa Cruz Biotechnology, Dallas, TX, USA). Images were obtained with an inverted microscope (IX71, Olympus, Tokyo, Japan) with a 20× objective (UApo/340, N.A. 0.75, Olympus) and a cooled-CCD camera (ORCA-ER, Hamamatsu Photonics, Hamamatsu, Japan).
3
Fluorescence Imaging of Mammalian Cells
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HeLa and MIN6 m9 cells cultured in 35-mm dishes were washed twice and imaged in modified Ringer’s buffer (RB: 140 mM NaCl, 3.5 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM HEPES, 2 mM NaHCO3, 5 mM glucose for HeLa and 3 mM glucose for MIN6 m9 cells, respectively). MIN6 m9 cells were cultured in the medium containing 1 g/L glucose for overnight, and incubated in RB for 30 min at 37 °C before imaging. Dishes were mounted on a stage that was heated and maintained at 37 °C and imaging was performed using an inverted microscope (IX-71, Olympus, Tokyo, Japan) equipped with an oil-immersion objective lens (UApo/340, 40×, NA = 1.35, Olympus), an intermediate magnification lens (1.6×), and an EM-CCD camera (Evolve, Photometrics, Tucson, AZ, USA). Images were acquired using a xenon lamp, 545–580 nm excitation filter, 585 nm dichroic mirror and 610 nm emission filter (Olympus). The exposure time of the EM-CCD camera was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). Images were acquired every 5 sec for 30 min. Data analysis of the acquired images was performed using MetaMorph software and the fluorescence intensity of the cells was quantified. After subtracting the background, basal fluorescence intensity, normalised to 100%, was calculated as the average fluorescence intensity between 90 and 120 sec after the beginning of image acquisition.
4
Calcium Imaging of Lysosomal Release
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Tet-On HEK-GCaMP3-ML1 stable cells were used for Ca2+ imaging. Doxycycline (0.01 µg/ml) was applied 20–24 h before the experiments to induce GCaMP3-ML1 expression. Cells were trypsinized and plated on coverslips 4–6 h before experiments. GCaMP3 fluorescence intensity at 470 nm (F470) was monitored and recorded at RT (21–23°C) using a EasyRatio Pro system (Photon Technology International), which includes an invert microscope (Olympus 1X71) with a 20×/0.75 objective lens (Olympus UApo/340), a high-resolution CoolSNAP HQ2 CCD camera (Photometrics), and a high-speed DeltaRam X monochromator (Photon Technology International) operated with EasyRatioPro software 1.12.121.86 (Photon Technology International). During recording, cells were bathed in Tyrode’s solution containing 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 20 mM Hepes, pH 7.4. Lysosomal Ca2+ release was measured by briefly switching to a 0 Ca2+ solution, which contained 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA, and 20 mM Hepes, pH 7.4; free Ca2+ concentration is estimated to be <10 nM based on Maxchelator software (Shen et al., 2012 (link)). Alterations of Ca2+ levels were normalized and shown as ΔF/F0, where F0 is baseline F, and ΔF is changes in F upon treatment.
5
Quantifying Amino Acid Fluorescence Intensities
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Microscope fluorescence images were acquired on an inverted microscope (Olympus IX-70) using a 40x, 1.15 NA water immersion objective (Olympus UApo/340) and a 1.5x tube lens. Images were recorded with a high-sensitivity camera (Andor Ixon, DV-885K) cooled to -60°C with preamplifier gain set to 3.6x, but without EM gain. Epifluorescence illumination was provided by a 385 nm LED (Thorlabs M385L2, 750 mA drive current) passing through a standard DAPI filter cube (Chroma 31000: AT350/50 ex; 400 dclp dichoroic; D460/50 em). Integration times for epifluorescence images varied from 0.5 s to 20 s, and are indicated in the figure caption for S2 Fig in S1 File . Fluorescence images were processed to provide intensity distributions and contrast suitable for visualization. For quantification of relative dbAF intensities from different crystals the average fluorescence intensities was measured over a large “region of interest”, thereby integrating over the noticeable variations in dbAF intensities. Background fluorescence measured over a comparable area off the crystals was subtracted and the resulting difference values were divided by the exposure time used to collect the images. The resulting relative dbAF intensities for amino acid crystals were plotted against the peak dbAF intensity of the same amino acid measured in solution.
6
Imaging of Alexa546-labeled shRNA Endocytosis
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CHO cells were seeded on a 35-mm glass-bottom dish (MatTek, P35G-1.5-10-C) at a concentration of 2 × 104 cells per dish (the volume of medium was 150 μL per dish). On the next day, TatU1A-Alexa546/FAM-shRNA complexes prepared as described above in 100 μL T buffer were loaded onto the cells and incubated for 2 h in a CO2 incubator. The cells were washed with 150 μL T buffer twice and then observed in T buffer. Live cell imaging was performed using a DeltaVision microscope system (Applied Precision) placed in a temperature controlled room (37 °C)34 (link). Light irradiation and subsequent time-lapse imaging were performed through the Olympus UApo/340 oil-immersion objective lens (40×, NA 0.65-1.35) at the NA value of =0.65. For the excitation of Alexa546, light at the wavelength of 529–556 nm (InsightSSI) was used at an irradiance of 1.47 W/cm2 for 6.8 s (approximately 10 J/cm2). Immediately after the irradiation, time-lapse images were obtained every 5 s. For measuring the fluorescence intensities at endosomal dots in the time-lapse images, ImageJ software (National Institutes of Health, Bethesda, MD) equipped with the SpotTracker 2D plug-in was used.
7
Confocal Microscope Imaging Protocol
8
Imaging Pollen Tube Growth
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Pollen tubes were grown through cut styles on pollen tube growth medium for 4.5 h at 22 °C in the dark. Confocal images were acquired using an inverted microscope (IX81; Olympus) equipped with a spinning disk confocal scanner (CSU-X1; Yokogawa Electric Corporation, Japan), 561-nm LD lasers (Sapphire; Coherent, Santa Clara, CA, USA), and an EM-CCD camera (Evolve 512; Photometrics, Tucson, AZ, USA). A 40× water immersion objective lens (UApo/340; Olympus, Japan) and 1.6× intermediate magnification changer were used.
9
Quantifying Cellular Uptake of Cy3-labeled Compounds
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KB, HeLa, and HT-1080 cells were
grown to confluence on LabTek 8-well microscope slides with glass
coverslip bottoms at a density of 50 000 cells per plate. The
cells were grown in folate-depleted media for 24 h prior to incubation
with 4 μM solution of Cy3-labeled25 for 4 h at
37 °C. For blocking studies, cells were grown in media containing
folic acid for 24 h prior to incubation with25 under
the same conditions mentioned above. The medium was removed and the
cells were washed three times with DPBS. Cells were visualized using
an Olympus FV1000 confocal laser scanning microscope with 543 nm (HeNe)
laser excitation and Cy3 emission filter under 40× magnification
(Olympus UApo/340, 1.15 NA water objective).
grown to confluence on LabTek 8-well microscope slides with glass
coverslip bottoms at a density of 50 000 cells per plate. The
cells were grown in folate-depleted media for 24 h prior to incubation
with 4 μM solution of Cy3-labeled
37 °C. For blocking studies, cells were grown in media containing
folic acid for 24 h prior to incubation with
the same conditions mentioned above. The medium was removed and the
cells were washed three times with DPBS. Cells were visualized using
an Olympus FV1000 confocal laser scanning microscope with 543 nm (HeNe)
laser excitation and Cy3 emission filter under 40× magnification
(Olympus UApo/340, 1.15 NA water objective).
10
Live-Cell Imaging of Transfected Cells
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Cells were transfected with plasmids using Effectene as described above. After transfection reagents were removed, the prewarmed observation medium (phenol red-free DMEM containing 10% FBS and 25 mM Hepes buffer, pH 7.3) was added to the cells. Then, the cells were subjected to time-lapse live-cell imaging. Images were obtained every 10 min (1 μm × 7 z-stacks for a single time point unless otherwise specified) using an oil-immersion objective lens UApo/340 (×40, NA = 1.35, Olympus, Tokyo, Japan) on a DeltaVision fluorescence microscope system (GE Healthcare Japan, Tokyo, Japan) in a temperature-controlled (37 °C) room or through a water-immersion objective lens C-Apo40 (×40, NA = 1.2, Carl Zeiss, Jena, Germany) on a confocal fluorescence microscope system LSM-880 (Carl Zeiss), maintained at 37 °C using a stage warmer.
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