Development of correlative light and electron microscopy to observe green fluorescent protein-labeled organelles embedded in resin using field-emission electron scanning microscope

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  • 光−電子相関顕微鏡法：蛍光タンパク質標識した細胞小器官を走査電子顕微鏡で捉える
  • 光-電子相関顕微鏡法 : 蛍光タンパク質標識した細胞小器官を走査電子顕微鏡で捉える
  • ヒカリ-デンシ ソウカン ケンビキョウホウ : ケイコウ タンパクシツ ヒョウシキ シタ サイボウ ショウキカン オ ソウサ デンシ ケンビキョウ デ トラエル

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Abstract

The use of fluorescent protein for live imaging is an effective method of determining the localization of various proteins and organelles in cells or in tissues. Fluorescence labeling of organelles is useful for the study and analysis of the ultrastructure of plant organelles. To obtain electron micrographs of fluorescently labeled organelles and cells, we developed a correlative light and electron microscopy and successfully visualized the green fluorescent protein (GFP)-labeled organelles in resin by using a combination of confocal laser scanning microscopy (LSM) and field-emission scanning electron microscopy (FE-SEM). Organs of an Arabidopsis thaliana plant expressing GFP-labeled organelles were fixed and then embedded in acrylic resin. The block was cut by the ultramicrotome, and 1 µm-thick sections were placed on a glass slide. Then the GFP fluorescence images were acquired by performing LSM. Subsequently, these sections were stained with heavy metals, and electron microscope images of stained sections were acquired by FE-SEM utilizing a high-sensitivity reflection electron detector. Finally, the ultrastructure of the GFP-labeled organelles was analyzed by merging the fluorescence image and the electron micrograph.

Journal

  • PLANT MORPHOLOGY

    PLANT MORPHOLOGY 28 (1), 15-21, 2016

    The Japanese Society of Plant Morphology

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