C. elegans embryo imaging was carried out at 20 °C on a Nikon Eclipse Ti-E microscope equipped with a 100x Plan-Apo 1.40 NA objective (Nikon, Tokyo, Japan) and CSU-X1 spinning-disk confocal head (Yokogawa Corporation, Tokyo, Japan). Focus drift during image acquisition was corrected using Nikon’s Perfect Focus System (PFS). Embryo samples were excited using 491 and 568 nm DPSS-Laser (Roper Scientific, France) and images acquired using Prime95B CMOS camera (Photometrics, Tucson, AZ). Image acquisition was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA). All images are single confocal planes acquired with the camera set at BIN1. Equatorial planes from control or cav-1 RNAi embryos co-expressing Histone::mCherry and GFP::PH-PLC1δ were acquired every 10 seconds post anaphase onset. Image analysis was carried out using Fiji.
Dpss laser
Manufactured by Teledyne
Sourced in France
The DPSS-Laser is a compact, solid-state laser that generates coherent light through the process of diode-pumped solid-state lasing. The core function of this laser is to provide a reliable and stable source of optical energy for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Csu x1 spinning disk confocal head, by Yokogawa (4 mentions)
Metamorph software, by Molecular Devices (3 mentions)
Perfect focus system, by Nikon (2 mentions)
Eclipse ti e microscope, by Nikon (1 mentions)
Plan apochromat oil immersion objective, by Nikon (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Csu w1 spinning disk, by Nikon (1 mentions)
4 protocols using dpss laser
1
C. elegans Embryo Imaging Protocol
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C. elegans embryo imaging was carried out at 20 °C on a Nikon Eclipse Ti-E microscope equipped with a 100x Plan-Apo 1.40 NA objective (Nikon, Tokyo, Japan) and CSU-X1 spinning-disk confocal head (Yokogawa Corporation, Tokyo, Japan). Focus drift during image acquisition was corrected using Nikon’s Perfect Focus System (PFS). Embryo samples were excited using 491 and 568 nm DPSS-Laser (Roper Scientific, France) and images acquired using Prime95B CMOS camera (Photometrics, Tucson, AZ). Image acquisition was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA). All images are single confocal planes acquired with the camera set at BIN1. Equatorial planes from control or cav-1 RNAi embryos co-expressing Histone::mCherry and GFP::PH-PLC1δ were acquired every 10 seconds post anaphase onset. Image analysis was carried out using Fiji.
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C. elegans embryo imaging was carried out at 20 °C on a Nikon Eclipse Ti-E microscope equipped with a 100x Plan-Apo 1.40 NA objective (Nikon, Tokyo, Japan) and CSU-X1 spinning-disk confocal head (Yokogawa Corporation, Tokyo, Japan). Focus drift during image acquisition was corrected using Nikon’s Perfect Focus System (PFS). Embryo samples were excited using 491 and 568 nm DPSS-Laser (Roper Scientific, France) and images acquired using Prime95B CMOS camera (Photometrics, Tucson, AZ). Image acquisition was controlled by MetaMorph software (Molecular Devices, Sunnyvale, CA). All images are single confocal planes acquired with the camera set at BIN1. Equatorial planes from control or cav-1 RNAi embryos co-expressing Histone::mCherry and GFP::PH-PLC1δ were acquired every 10 seconds post anaphase onset. Image analysis was carried out using Fiji.
2
Imaging and Viability Analysis of C. elegans
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Larvae or embryos collected from gravid hermaphrodite, mounted onto 3% agarose padded-glass slide, closed with coverslip and sealed with wax. Normaski images shown in Fig. 1A, B and S2B were captured using a Nikon Ti Eclipse widefield microscope equipped with DIC 1.40NA oil condenser and a charged-coupled device camera Cool Snap HQ2 (Photometrics). All other imaging were done using spinning disk confocal system composed of a Nikon Ti Eclipse microscope with a CSU-X1 spinning disk confocal head (Yokogawa), DPSS-Laser (Roper Scientific) at 491 and 568 nm excitation wavelength and an Evolve Rapid-Cal electron multiplying charged-coupled device camera (Photometrics). For both microscopes, Metamorph software (Molecular Devices) was used to control acquisition. Projected images were created using Fiji.
After 24 hour DIC recording, wild type, pigv-1(qm34) and pigv-1(qm34) embryos expressing ERM-1::GFP were scored as viable or lethal and each category is further classified into four subcategories; i.e.: without visible defects, with cysts and rupture, with cyst only and with rupture only.
After 24 hour DIC recording, wild type, pigv-1(qm34) and pigv-1(qm34) embryos expressing ERM-1::GFP were scored as viable or lethal and each category is further classified into four subcategories; i.e.: without visible defects, with cysts and rupture, with cyst only and with rupture only.
3
Imaging Techniques for Nematode Embryogenesis
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Larvae or embryos collected from gravid hermaphrodite were mounted onto 3% agarose-padded glass slide, closed with a coverslip and sealed with wax. DIC images shown in Figs 1A , 3A, 3B, 3C , 8A, 8C, 8E and S1A were captured using a Nikon Ti Eclipse widefield microscope equipped with DIC 1.40NA oil condenser and a charged-coupled device camera Cool Snap HQ2 (Photometrics). All other images and movies were acquired using a spinning disk confocal system composed of a Nikon Ti Eclipse microscope with a CSU-X1 spinning disk confocal head (Yokogawa), DPSS-Laser (Roper Scientific) at 491 and 568 nm excitation wavelengths and an Evolve Rapid-Cal electron multiplying charged-coupled device camera (Photometrics). For both microscopes, Metamorph software (Molecular Devices) was used to control acquisition. Projected images were created using Fiji. All imaging was done at 20°C in an environmental chamber encompassing the microscope stage heated by a JCS temperature controller (Shinko Technos Co, Japan) within a microscope room kept at 18°C by a CITEC precision air conditioning unit.
4
Imaging Spermatheca Dynamics in C. elegans
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For DIC and fluorescence microscopy imaging of spermatheca dynamics, day 1 adult hermaphrodites with one row of embryos were mounted on glass slides with 10% agarose pads, which served to immobilize them, and 1.38 µl of M9 medium. Image acquisition was performed at 20°C on a Ti-Eclipse inverted microscope (Nikon) equipped with a CSU-X1 spinning-disk confocal head (Yokogawa Electric Corporation), DPSS-Laser (Roper Scientific) of 491- and 561-nm excitation wavelengths and an Evolve electron multiplying charged-coupled device camera (Photometrics). Focus drift during time-lapse acquisition was corrected using Perfect Focus System (Nikon). Acquisition control was performed using MetaMorph (Molecular Devices). Ovulation and embryo transit movies were acquired with a 60× 1.4 NA oil-immersion Plan-Apochromat objective (Nikon). Live super-resolution imaging of protein localization in the spermatheca (Fig. 6, A and B ; and Fig. S5 ) was performed on a Ti-2 Eclipse with a Live-SR module (Gataca systems, France) mounted on the light path of a CSU-W1 spinning disk, a 100× 1.45 NA oil-immersion Plan-Apochromat objective (Nikon, Japan), and a Prime95B SCMOS camera (Photometrics). Actin stress fibers in the spermatheca (Fig. 2 H and Fig. 7 A ) and embryos images (Fig. 1 B and Fig. 5 C ) were acquired with a 100× 1.45 NA oil-immersion Plan-Apochromat objective (Nikon).
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