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Application of silicone immersion objectives to long-term live-cell imaging of plant zygote embryogenesis


Plant zygote embryogenesis

Studies on embryogenesis in the field of life science research have made progress together with advances in microscopy techniques. In particular, studies on animal embryogenesis have developed into research on stem cells such as embryogenic stem (ES) cells. In contrast, plant embryogenesis is far less understood than animal embryogenesis even though it has been studied for a long time. The major reason for this slow progress is the difficulty of live observation of the division process of the fertilized egg cell (zygote) because the angiosperm zygote is deeply embedded in the pistil, a maternal tissue.
The Optical Technology Group of ERATO Higashiyama Live-Holonics Project, Nagoya University, a research group led by Dr. Daisuke Kurihara, has been working on elucidating the plant reproductive system by primarily using live-cell imaging and microscopic cell manipulation techniques. Recently, they succeeded in live-cell imaging of the plant zygote embryogenesis process, a world first. This used to be considered to be very difficult. The results from a series of studies were published in Developmental Cell*, an American scientific journal, in July 2015. In this study, long-term live-cell imaging of plant zygote division and growth was made possible by development of a special medium and a new microdevice.
This application note introduces an example of long-term live-cell imaging of plant zygote embryogenesis using a silicone immersion objective, which is specially designed for live-cell imaging and was used in this study.

Application of silicone immersion objectives
Long-term live-cell imaging of Arabidopsis zygote embryogenesis

1)Establishment of a culture method for long-term real-time observation of embryogenesis in live ovules

Angiosperm embryogenesis occurs within the ovule, which is deeply embedded in the pistil (Fig. 1); therefore, it has been impossible to observe embryogenesis from zygote division in living material.
The Optical Technology Group of ERATO Higashiyama Live-Holonics Project, Nagoya University, a research group led by Dr. Daisuke Kurihara, used the model plant Arabidopsis thaliana, removed ovules from the pistils, and examined the composition of a medium that would enable embryogenesis in an ovule under in vitro culture conditions. By examining the medium composition that supports normal embryo development and ovule growth, a significant increase in the frequency of ovule survival and normal embryo development was observed when trehalose, an organic substance, was added to the culture medium for the ovary, a pistil tissue surrounding the ovule. Development of this new ovule culture medium allowed development of the embryo into a mature seed within the ovule outside the pistil, germination, and growth of the seedling into an adult plant (Fig. 2).

Schematic representation of an Arabidopsis flower and embryogenesis
Fig1. Schematic representation of an Arabidopsis flower and embryogenesis
After significant elongation of the zygote in the ovule, it divides asymmetrically into a smaller apical cell and a larger basal cell. The apical cell generates the embryo, which ultimately gives rise to the adult plant.

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Fig2. Growth of early embryos to seedlings in ovule culture media, and a seedling into an adult plant

Next, the group of Dr. Kurihara worked on development of a microdevice that enabled stable long-term imaging. Since the ovule is oval shaped, it moves during observation and may wander outside the microscopic field. Because of this, stable live observation of the process of embryogenesis occurring over several days was not possible. By improving their proprietary microcage array that can stably hold ovules, they developed a micropillar array. This is a new microdevice that can stably hold ovules for a long period without preventing their growth. A new system for stable long-term culture of the ovule in a fixed position was established by development of this new microdevice and the ovule culture medium.

Fixation of ovules with the microdevice 01Fixation of ovules with the microdevice 02Fixation of ovules with the microdevice 03

 Tilted-view  Top-view

Fig3. Fixation of ovules with the microdevice
A micropillar array of a uniform pillar distribution is used for stable long-term retention of ovules. In-depth observation of embryogenesis is possible because pillars hold ovules without preventing their growth. Scale bars represent 300 mm.

2)Use of silicone immersion objectives for long-term live-cell imaging of Arabidopsis zygote embryogenesis

The world’s first long-term and real-time observation of embryogenesis a zygote to late embryo was made possible by combining the ovule culture system described above and a microscope system capable of high-sensitivity imaging of the embryo, which is inside the ovule covered by multiple layers of cells.
The high-sensitivity microscope system used in this study was the fully-motorized research inverted microscope IX series with a 30´ silicone immersion objective, UPLSAPO30XS.
The silicone immersion objectives are specially designed for live-cell imaging with an optical design based on the refractive index of silicone oil (ne≈1.40), which is close to that of living tissue (ne≈1.38). The 30´ silicone immersion objective, UPLSAPO30XS, used in this imaging experiment has a high numerical aperture of 1.05 and long working distance of 0.8 mm, which is required for deep, high-definition imaging while retaining a wide field. Unlike water immersion objectives which require a water supply because of evaporation, it is not necessary to supply silicone oil because it does dry out at 37°C during operation over several days. In addition, the silicone immersion objectives are compatible with the Z-Drift Compensation System IX-ZDC of the fully-motorized research inverted microscope IX series. Live images that are constantly in focus can be obtained by using a silicone immersion objective with the Z-Drift Compensation System IX-ZDC.
As an example of live-cell imaging using a 30´ silicone immersion objective, UPLSAPO30XS, the process of Arabidopsis embryogenesis was stably observed in real time for 67 hours from the early embryo (4-cell stage) to the late embryo (Movie).

Schematic illustration of microdevice imaging with a silicone immersion objective
Fig4. Schematic illustration of microdevice imaging with a silicone immersion objective
Multi-point time-lapse imaging was acquired using a motorized XY stage

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Live-cell imaging of zygote division and embryogenesis

 


Movie. Live-cell imaging of zygote division and embryogenesis
The process of embryogenesis from the early embryo (4-cell stage) to the late embryo was recorded over 67 hours by taking images at 10-minute intervals. The movie captured formation of a round-shaped tissue from proembryo cells while changing the orientation of divisions, and formation of rod-like tissue from suspensor cells, which divide only transversely. The numbers indicate the time from the beginning of recording. Nucleus and plasma membrane are labeled green (H2B-GFP) and magenta (tdTomato-LTI6b), respectively.

Imaging conditions
Imaging system: Fully motorized research inverted microscope IX series
Objective: silicone immersion objective UPLSAPO30XS
Confocal scanner unit: CSU-X1 (Yokogawa Electric Corporation)
EMCCD camera: Evolve 512 (Photometrics)
Motorized XY stage: MD-XY30100T-Meta (Molecular Devices)
Piezo Z-focus drive: P-721 (Physik Instrumente)


In parallel with this study using a live-cell imaging technology, Dr. Kurihara’ research group also developed the ClearSee method, which is a new technique for optical clearing of a whole plant, and published their results in Development, an American scientific journal, in October 2015.
Global application of the newly developed live-cell imaging technology and the new optical clearing technique for plants is expected to bring about major progress in plant research.
 

This application note was prepared with the help of
Dr. Daisuke Kurihara, Group Leader, Optical Technology Group, ERATO Higashiyama Live-Holonics Project, Nagoya University

For more details on the studies in this application note, please refer to the articles below.

*Source: Dev Cell. 2015 Jul 27;34(2):242-51. doi: 10.1016/j.devcel.2015.06.008. Epub 2015 Jul 9.
Journal: Developmental Cell
Publication date: July 9, 2015
Title: Live-Cell Imaging and Optical Manipulation of Arabidopsis Early Embryogenesis
Authors: Keita Gooh, Minako Ueda, Kana Aruga, Jongho Park, Hideyuki Arata, Tetsuya Higashiyama, and Daisuke Kurihara

**Source: Development. 2015 Dec 1;142(23):4168-79. doi: 10.1242/dev.127613. Epub 2015 Oct 22.
Journal: Development
Publication date: October 22, 2015
Title: ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging
Authors: Daisuke Kurihara, Yoko Mizuta, Yoshikatsu Sato, and Tetsuya Higashiyama

Silicone immersion objectives for realization of deep, high-definition 3D live-cell imaging over a prolonged period

Olympus has a line-up of 30/40/60/100´ silicone immersion objectives (UPLSAPO30XS/UPLSAPO40XS/UPLSAPO60XS2/UPLSAPO100XS), which offer both a high numerical aperture 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 in the observation of thick living tissues, thereby enabling high-resolution imaging. In addition, silicone oil does not require the extra task of supplying immersion liquid because it does not dry out or becomes solid. The silicone immersion objectives are compatible with the Z-Drift Compensation System IX-ZDC of the fully-motorized research inverted microscope IX series. Long-term, stable high-resolution 3D imaging that is constantly in focus is achieved and is unaffected by temperature changes or the addition of reagent solution.

Products used for this application

El microscopio invertido modular completamente motorizado y automatizado

IX83

  • Exclusivo sistema de plataformas
  • Sistema completamente motorizado
  • Soluciones modulares
Sistema de microscopio para imágenes de células vivas

IXplore Live

  • Utilice el controlador en tiempo real de Olympus para los datos relevantes a nivel fisiológico con una mínima alteración celular
  • Mantenga la viabilidad celular durante el procesamiento de imágenes con varias opciones de control ambiental
  • Mantenga el enfoque de forma precisa y fiable en experimentos a intervalos gracias al sistema de enfoque automático (compensación de deriva en Z) del hardware Olympus
  • Descubra la forma real de sus células gracias a los objetivos de inmersión de silicona de Olympus
Sistema de microscopio automatizado

IXplore Pro

  • Observación multidimensional automática con una configuración sencilla de los experimentos
  • Mejore sus estadísticas con análisis de placas multipocillo
  • Adquiera imágenes panorámicas fluorescentes de grandes muestras, como las rodajas de tejido cerebral
  • Aumente la resolución y cree secciones ópticas con deconvolución
Sistema de microscopio para imágenes confocales

IXplore Spin

  • Procesamiento rápido de imágenes confocales de alta resolución gracias con un sistema de disco giratorio
  • Imágenes confocales en 3D a intervalos con menos fototoxicidad y blanqueo
  • Imágenes precisas en 3D con una admisión de luz mejorada usando objetivos de inmersión de aceite de silicona
  • Migre al sistema de súper resolución IXplore SpinSR en función de su proceso de investigación o presupuesto
Sistema de microscopio con súper resolución

IXplore SpinSR

  • Súper resolución XY de hasta 120 nm
  • Viabilidad celular prolongada en imágenes confocales a intervalos gracias a la reducción de la fototoxicidad y el blanqueo
  • Alterne entre las observaciones de campo amplio, resolución confocal y súper resolución en un solo paso con el sistema IXplore SpinSR
  • Reproducción precisa en 3D con los objetivos de inmersión de aceite de silicona de Olympus
Sistema de microscopio para procesamiento TIRF

IXplore TIRF

  • Excelente procesamiento TIRF simultáneo y multicolor para investigar la dinámica de las membranas y la detección de moléculas individuales
  • Colocalización exacta de hasta cuatro indicadores gracias al control de profundidad de penetración individual
  • Benefíciese de los extraordinarios objetivos TIRF, dotados de la apertura numérica más alta de 1,7* (*según los estudios de Olympus hasta el 25 de julio de 2017). 
  • Configure de forma intuitiva experimentos complejos con el Graphical Experiment Manager (GEM)
Objetivos superapocromáticos

UPLSAPO-S/UPLSAPO-W

  • Compensación de las aberraciones esféricas y cromáticas, más alta transmisión desde la región visible hasta la infrarroja cercana.
  • Índice de refracción de agua o aceite de silicona similar al de las células vivas como medio de inmersión y alta efectividad en el procesamiento de sus imágenes.

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