E3.75 PdgfraH2B-GFP/+ positive embryos obtained from intercrossing of PdgfraH2B-GFP/+ and CD-1 or F1 hybrid were used. Isolated single ICM cells in Blast medium containing 1:10000 CellMask Orange (Life Technologies) were loaded into the BSA coated polydimethylsiloxane (PDMS) confinement devices with a fixed roof height of 8 μm, 9 μm or 10 μm. Confinement devices were designed to restrict cells between two glass plates, trapping the cells in the z-direction but allowing free movement in x- and y-direction, as first demonstrated in (Le Berre et al., 2014 (link)). To adapt the device for the use with very small cell numbers, we modified confinement channels as described in Heuzé et al. (2011) (link) by replacing the channels with pillars to create a constant, well-defined roof height. Live imaging of the cells was performed with a 6x silicon objective (UPLSAPO60XS, Olympus) on an inverted microscope (Olympus FV1200) equipped with a humidified chamber at 37°C and 5% CO2. The bright field, GFP, and CellMask images of the cells were taken every one, two or three minutes for up to ten hours.
Uplsapo60x
Manufactured by Olympus
Sourced in Japan
The UPLSAPO60X is a high-numerical-aperture objective lens designed for use in microscopy applications. It features a long working distance and is optimized for use with Olympus' UPLSAPO series of lenses.
Automatically generated - may contain errors
Lab products found in correlation
Fv1200, by Olympus (5 mentions)
Fv1000 confocal laser scanning system, by Olympus (4 mentions)
Fvd10 spd spectral detector, by Olympus (4 mentions)
35 mm glass bottom dishes, by MatTek (3 mentions)
Metamorph software, by Molecular Devices (2 mentions)
Axioobserver z1, by Yokogawa (2 mentions)
Polystyrene beads, by Polysciences (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Ix81 microscope, by Olympus (2 mentions)
Cyanine3 (cy3), by Olympus (2 mentions)
Cellmask orange, by Thermo Fisher Scientific (1 mentions)
Csu x1, by Yokogawa (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 633 phalloidin, by Thermo Fisher Scientific (1 mentions)
Mabt336, by Merck Group (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Axioplan 2, by Zeiss (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Bx61wi, by Olympus (1 mentions)
Csu w1, by Yokogawa (1 mentions)
17 protocols using uplsapo60x
1
Confinement-based Live Imaging of ICM Cells
2
Dual-Color Imaging of C. elegans Embryos
3
Quantification of Matrix Degradation by Cancer Cells
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Gelatin was fluorescently labeled with Alexa-405-NHS ester and 35 mm glass bottom dishes (MatTek Corporation) were coated with Alexa-405-gelatin as previously described31 (link). 400,000 (4T1/67NR) or 300,000 (MDA-MB-231) cells were plated per dish and cells were fixed 18 h later with 4% paraformaldehyde (Alfa Aesar) for 10 min, permeabilized with 0.1% Triton X-100 (Calbiochem) for 5 min, blocked with 1% FBS/1% BSA (Sigma-Aldrich) in PBS (Gibco) for 3 h, incubated with anti Tks5 antibody (Millipore, MABT336) for 2 h, then with secondary antibody and Alexa Fluor 633 Phalloidin (Invitrogen) for 1 h.
Samples were imaged on a laser scanning confocal microscope (FV1200, Olympus) using a 60X objective (UPLSAPO60XS, 1.35 NA, Olympus). Stacks were collected at 1 µm z-step. To quantify matrix degradation, images were processed using a custom macro in Fiji. Briefly, slices from the stack were z-projected using the Max Intensity method, followed by thresholding of the signal in the gelatin channel, using the Automatic Threshold algorithm, and measuring the area of degradation spots using the Particle Analysis tool. To account for the differences in the cell density across fields of view, the total area of degradation in a field of view was divided by the total number of cell present in this field of view. Cells were counted using the F-actin staining.
Samples were imaged on a laser scanning confocal microscope (FV1200, Olympus) using a 60X objective (UPLSAPO60XS, 1.35 NA, Olympus). Stacks were collected at 1 µm z-step. To quantify matrix degradation, images were processed using a custom macro in Fiji. Briefly, slices from the stack were z-projected using the Max Intensity method, followed by thresholding of the signal in the gelatin channel, using the Automatic Threshold algorithm, and measuring the area of degradation spots using the Particle Analysis tool. To account for the differences in the cell density across fields of view, the total area of degradation in a field of view was divided by the total number of cell present in this field of view. Cells were counted using the F-actin staining.
4
Live-cell Imaging of Semi-in vivo Fertilization
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Unless otherwise indicated, all images were generated using an AxioCam HRc mounted on a Zeiss Axioplan 2 imaging microscope with the software AxioVision version 4.8; with an Axio Imager.Z1 equipped with an AxioCamMR3; or with a confocal microscope OLYMPUS BX61Wi with the software OLYMPUS FLUOVIEW version 3.1. Microscopic images were processed using Adobe Photoshop and Illustrator software.
For live-cell imaging of semi-in vivo fertilization, the following microscopy settings were used as described previously (Hamamura et al., 2014 (link); Gooh et al., 2015 (link)). Confocal images were acquired using an inverted microscope IX-83 (Olympus) equipped with a disk-scan confocal system (CSU-W1; Yokogawa Electric). A silicone oil immersion objective lens, UPLSAPO60XS (Olympus), mounted on a Piezo z-drive (P-721; Physik Instrumente) was used. Time-lapse and z-stack images were acquired every 5-10 min in seven planes (3 μm intervals). The exposure time of 488 nm laser was 250-300 ms for eGFP, and of 561 nm laser was 50-200 ms for mRFP and tdTomato. Images were processed by Metamorph version 7.8.4.0 (Universal Imaging) to display maximum-intensity projection images and to add pseudo-colors. The images and movies were edited by MacBiophotonics ImageJ software (http://www.macbiophotonics.ca/ ).
For live-cell imaging of semi-in vivo fertilization, the following microscopy settings were used as described previously (Hamamura et al., 2014 (link); Gooh et al., 2015 (link)). Confocal images were acquired using an inverted microscope IX-83 (Olympus) equipped with a disk-scan confocal system (CSU-W1; Yokogawa Electric). A silicone oil immersion objective lens, UPLSAPO60XS (Olympus), mounted on a Piezo z-drive (P-721; Physik Instrumente) was used. Time-lapse and z-stack images were acquired every 5-10 min in seven planes (3 μm intervals). The exposure time of 488 nm laser was 250-300 ms for eGFP, and of 561 nm laser was 50-200 ms for mRFP and tdTomato. Images were processed by Metamorph version 7.8.4.0 (Universal Imaging) to display maximum-intensity projection images and to add pseudo-colors. The images and movies were edited by MacBiophotonics ImageJ software (
5
High-Resolution 3D Imaging of Neuronal Structures
6
Measuring Cell-ECM Contact Angle
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Measurement of the contact angle was performed as previously described36 (link). Briefly, 35 mm glass bottom dishes (MatTek Corporation) were coated with fluorescently labeled gelatin as previously described31 (link) or with 50 µg/ml poly-L-lysine (Sigma-Aldrich) for 20 min and let to air dry. Cells were left to adhere for 5 h before imaging. Solitary cells, which had no physical interaction with nearby cells, were imaged on a laser scanning confocal microscope (FV1200, Olympus) using a 60X objective (UPLSAPO60XS, 1.35 NA, Olympus) equipped with an environmental chamber (In Vivo Scientific), with a 1 µm z-step. Orthogonal Views was used to measure the angle between the ECM and the main body of the cell: the contact angle.
7
4D Imaging of C. elegans Embryogenesis
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Embryos were collected from gravid hermaphrodites and mounted with polystyrene beads (Polysciences Inc) as described (Du et al., 2015 (link)). Embryos were imaged on a Zeiss AxioObserver Z1 inverted microscope frame with Yokogawa CSU-X1 spinning disk and an Olympus UPLSAPO 60xs silicone oil immersion objective. GFP and mCherry channels were acquired simultaneously on a pair of aligned EMCCD cameras (C9100-13). Image acquisition was performed using MetaMorph software (Molecular Devices). Embryos were imaged every 75 s, with 30 z slices at 1 μm apart. Lineage tracing and quantification of marker expression were done with the StarryNite and AceTree software as described (Du et al., 2015 (link)).
8
Contact Angle Measurement of Cell-ECM Interaction
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Measurement of the contact angle was performed as previously described (36) . Brie y, 35 mm glass bottom dishes (MatTek Corporation) were coated with uorescently labeled gelatin as previously described (31) or with 50 µg/ml poly-L-lysine (Sigma-Aldrich) for 20 min and let to air dry. Cells were left to adhere for 5 h before imaging. Solitary cells, which had no physical interaction with nearby cells, were imaged on a laser scanning confocal microscope (FV1200, Olympus) using a 60X objective (UPLSAPO60XS, 1.35 NA, Olympus) equipped with an environmental chamber (In Vivo Scienti c), with a 1 µm z-step. Orthogonal Views was used to measure the angle between the ECM and the main body of the cell: the contact angle.
9
Fingerprint hsSRS Imaging Setup
10
Fingerprint hsSRS Imaging Setup
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The experimental setup we used for fingerprint hsSRS imaging was published previously24 (link) (supplementary Fig. S1 ). In brief, two synchronized femtosecond lasers were chirped to about 2 ps using SF57 glass rods. One laser was fixed at 1040 nm wavelength and the other was tunable from 750 nm to 970 nm. We chose the center wavelength of the tunable laser to be at 914 nm and 890 nm for hsSRS imaging in the 1300 cm−1 region and 1600 cm−1 region, respectively. The wavenumbers were calibrated with oleic acid spectral peaks using linear fitting. Spectral resolution is about 20–30 cm−1. The temporal delay between the two pulsed lasers was controlled by a motorized stage (Newport MFA-PP). A 60X water immersion objective (Olympus UPLSAPO60X, NA = 1.2) was used to focus the lasers onto the sample, with typical optical power at the sample of 40 mW for the pump beam and 40 mW for the Stokes beam. Each SRS image has 512 × 512 pixels and takes 1.65 sec to acquire.
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