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Note d’application

Application of silicone immersion objectives to long-term 3D live-cell imaging of mouse embryo during development


Long-term 3D live-cell imaging of a mouse embryo during development

Advances in microscopy have revealed numerous phenomena that occur during embryonic development, and this has become a major research focus in the field of developmental biology. In particular, as confocal microscopes have come into more general use, this has enabled researchers to obtain sharp three-dimensional fluorescence images of proteins, DNA, and other molecules in the zygote and of individual cells during embryonic development.
With research in this area making rapid progress, confocal microscopy is being used for prolonged 3D live-cell imaging to capture dynamic change over time as well as for three-dimensional fluorescence imaging. Olympus developed silicone immersion objectives that enable high-contrast[PG1] , long-term 3D live-cell imaging.
This application note introduces an example of high-contrast 3D live-cell imaging during the in vitro development of a mouse embryo from the zygote to blastocyst stage over approximately 4 days using a silicone immersion objective.
This application note is based on a study conducted by Dr. Kazuo Yamagata, Associate Professor in the Department of Genetic Engineering, Faculty of Biology-Oriented Science and Technology at Kinki University and his collaborators that was published in Stem Cell Reports in June 2014.

The use of silicone immersion objectives for long-term 3D live-cell imaging of a MethylRO mouse early embryo during preimplantation development

1)Creating MethylRO mice to visualize epigenetic changes in living cells

Researchers generated a fluorescent probe by fusing a red fluorescent protein to the methyl-CpG binding domain (MBD) of methyl-CpG binding domain protein 1 (MBD1), which recognizes methylated DNA. They then inserted the gene by targeting the ROSA26 locus, which is known for its ubiquitous gene expression, and generated the MethylRO mouse, a genetically modified mouse strain expressing this probe throughout the entire body.

メチローマウス
Figure 1. Neonate MethylRO mouse for visualization of methylated DNA (yellow arrows).
When irradiated with the excitation light, the entire body glows red through a filter (right panel).

2)Long-term 3D live-cell imaging of an early embryo during preimplantation development using a 60X silicone immersion objective

Dr. Yamagata and his collaborators used a confocal microscope for time-lapse 3D live-cell imaging of cells from a MethylRO mouse (Fig. 1) during early development of the preimplantation embryo over approximately 4 days.
The researchers used an Olympus silicone immersion objective designed for live cell observation. Silicone oil has a refractive index (ne≈1.40) close to that of living tissue (ne≈1.38). Spherical aberration, which occurs with oil or water immersion objectives due to a refractive-index mismatch with biological samples, is reduced in silicone immersion objectives, allowing researchers to achieve deep, high-contrast fluorescence imaging at a greater depth. In addition, silicone oil does not dry out or become solid, compared to water or oil immersion, when used in a warm (37 °C) environment over 4 days, thereby supporting long-term, stable high-resolution 3D live-cell imaging.
Dr. Yamagata’s team previously used an oil lens and water immersion objective, the former standard for deep observation in live-cell imaging. By switching to a silicone immersion objective, the researchers were able to view fluorescently labeled methylated DNA (mCherry-MBD-NLS) within the nuclei from the surface to the inner region over approximately 4 days from the one-cell zygote to blastocyst stage.

胚発生
Figure 2. Live-cell imaging of a MethylRO embryo during pre-implantation development.
Changes in methylated DNA (mCherry-MBD-NLS) within the nuclei were observed over approximately 4 days.

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Observation 82 h

 

Movie1. Time-lapse imaging over 82 hours of changes in methylated DNA in the nuclei (red: mCherry-MBD-NLS) and cells (green: CAG-EGFP).
Activation of embryonic genes (red) started from the two-cell stage in approximately 21 hours from the beginning of observation.

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Image dimensionnelle

 

Movie2. Three-dimensional image of methylated DNA (mCherry-MBD-NLS) within nuclei of a blastocyst

Figure 2 and Movie 1 above show that fluorescence photobleaching during long-term imaging was negligible, enabling clear imaging of methylated DNA within the nuclei. In Movie 2, a three-dimensional image of the entire 100 mm blastocyst was successfully obtained using an objective with both a high numerical aperture (1.3) and long working distance (0.3 mm)

Conclusion: The use of silicone immersion objectives is essential for the realization of deep, high-contrast 3D live-cell imaging over long periods of time.

Olympus’ line-up of 30/40/60/100X silicone immersion objectives offers both high numerical aperture (NA) and long working distance. Since the refractive index of silicone oil (ne≈1.40) is close to that of living tissue (ne≈1.38), spherical aberration induced by a refractive-index mismatch is reduced when observing thick tissues, thereby enabling high-resolution imaging. In addition, silicone oil does not dry out so there is no need to add more immersion liquid during an experiment. The silicone immersion objectives are compatible with the IX motorized inverted microscope series’ Z-Drift Compensation System IX-ZDC. With this system, researchers can obtain images that are constantly in focus and unaffected by temperature changes during long-term observation. In addition, the refractive index of silicone oil (ne≈1.40) and that of SCALEVIEW-A2 (ne≈1.38), an optical clearing agent supplied by Olympus, are comparable, minimizing refractive index mismatch when using this reagent. Silicone immersion objectives with high NA provide optimum performance when optically cleared specimens are used.

Imaging conditions
Imaging system: Research inverted microscope IX series
Objective: silicone immersion objective UPLSAPO60XS
Confocal scanner unit: CSU-X1 (Yokogawa Electric Corporation)
EMCCD camera: iXON3 DU897E-CS0 (Andor Technology)

This application note was prepared with the help of
Dr. Kazuo Yamagata, Associate Professor, Department of Genetic Engineering, Faculty of Biology-Oriented Science and Technology, Kinki University

For more details on the studies in this application note, please refer to the article below:

Ueda, Jun, Kazumitsu Maehara, Daisuke Mashiko, Takako Ichinose, Tatsuma Yao, Mayuko Hori, Yuko Sato, Hiroshi Kimura, Yasuyuki Ohkawa, and Kazuo Yamagata. "Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, MethylRO." Stem cell reports 2, no. 6 (2014): 910-924.

Products used for this application

Microscope confocal à disque rotatif et à très haute résolution

SpinSR10

  • Live cell super resolution imaging
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Le système de microscope inversé entièrement motorisé et automatisé

IX83

  • Système de plate-forme unique
  • Système entièrement motorisé
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Système de microscope pour imagerie de cellules vivantes

IXplore Live

  • Utilisation du contrôleur en temps réel d’Olympus pour l’obtention de données physiologiques pertinentes avec une perturbation minimale des cellules
  • Préservation de la viabilité des cellules lors de la prise d’images grâce à diverses options de contrôle environnemental
  • Maintien d’une mise au point précise et fiable lors d’expériences à prises d’images intermittentes à l’aide du système matériel de mise au point automatique d’Olympus (compensation de la dérive en Z)
  • Découverte de la forme réelle des cellules grâce aux systèmes optiques à immersion dans l’huile de silicone d’Olympus
Système de microscope automatisé

IXplore Pro

  • Observation multidimensionnelle automatisée et configuration facile des expériences
  • Amélioration de vos statistiques lors de vos analyses de plaques multi-puits
  • Acquisition d’images de fluorescence panoramiques pour des échantillons de grande taille, telles que des coupes de cerveau
  • Augmentation de la résolution et création de sections optiques avec déconvolution
Système de microscope pour imagerie confocale

IXplore Spin

  • Imagerie confocale rapide et à haute résolution avec système de disque rotatif
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  • Imagerie 3D précise offrant une meilleure récupération de la lumière grâce à des objectifs à immersion dans l’huile de silicone
  • Mise à niveau vers le système à très grande résolution IXplore SpinSR selon l’avancement de vos travaux de recherche ou de votre budget
Système de microscope à très grande résolution

IXplore SpinSR

  • Très grande résolution, jusqu’à une résolution XY de 120 nm
  • Viabilité des cellules prolongée lors d’imagerie confocale à prises d’images intermittentes en raison d’une phototoxicité et d’un blanchiment réduits
  • Basculement en une étape entre les observations champ large, confocales et à très grande résolution grâce au système IXplore SpinSR
  • Reconstruction 3D exacte grâce aux objectifs à immersion dans l’huile de silicone d’Olympus
Système de microscope pour imagerie TIRF

IXplore TIRF

  • Excellente imagerie TIRF multicolore pour les études de dynamique membranaire et la détection de molécules uniques
  • Colocalisation exacte de quatre marqueurs (ou moins) grâce au contrôle individuel de la profondeur de pénétration
  • Bénéficiez de l’excellent objectif TIRF d’Olympus doté d’une ouverture numérique de 1,7*, soit la plus grande au monde (* en date du 25 juillet 2017, à la connaissance d’Olympus)
  • Configuration intuitive d’expériences complexes à l’aide du gestionnaire d’expériences graphique (GEM)
Objectifs super apochromatiques

UPLSAPO-S/UPLSAPO-W

  • Compensation des aberrations sphériques et chromatiques et transmission élevée de la région du visible jusqu’à la région du proche infrarouge.
  • Indice de réfraction de l’eau ou de l’huile de silicone similaire à celui de la cellule vivante et très efficace pour l’imagerie de cellules vivantes.

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