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Imaging and Cytometry Unit

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http://hdl.handle.net/10400.7/689

The Unit of Imaging and Cytometry (UIC) is composed of the following sub-units:

Advanced Imaging, headed by Gabriel G. Martins

Flow Cytometry, headed by Rui Gardner

Electron Microscopy, headed by Erin Tranfield

The UIC has been at the forefront of major technological developments at IGC, since its formal creation in 2003. In the last decade, spearheaded by José Feijó, the UIC has grown in personnel and technological spectrum to anticipate the demands of a growing base of users. Because cellular imaging and cytometry have been in high demand, and new systems and techniques are continuously developed, the facility expanded significantly to facilitate accessibility while introducing the latest innovations - in optical microscopy, electron microscopy and flow cytometry - to the whole research community.

The three sub-units now provide more dedicated and focused services on their technical areas, and continue development through a close collaboration with the "Equipment Unit", headed by Nuno Moreno, who was previously responsible for managing all microscopy equipment.

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Browsing Imaging and Cytometry Unit by Subject "Microspore"

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  • FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei
    Publication . Borges, Filipe; Gardner, Rui; Lopes, Telma; Calarco, Joseph P; Boavida, Leonor C; Slotkin, R; Martienssen, Robert A; Becker, Jörg D
    Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.
    2012-10-17Journal article Open access Show more