Retarding Field Integrated Fluorescence and Electron Microscope

Yoram Vos, Ryan Lane, Chris J. Peddie, Anouk H. G. Wolters, Jacob P. Hoogenboom*

*Corresponding author for this work

    Research output: Contribution to journalArticleAcademicpeer-review

    4 Citations (Scopus)
    48 Downloads (Pure)

    Abstract

    The authors present the application of a retarding field between the electron objective lens and sample in an integrated fluorescence and electron microscope. The retarding field enhances signal collection and signal strength in the electron microscope. This is beneficial for samples prepared for integrated fluorescence and electron microscopy as the amount of staining material added to enhance electron microscopy signal is typically lower compared to conventional samples in order to preserve fluorescence. We demonstrate signal enhancement through the applied retarding field for both 80-nm post-embedding immunolabeled sections and 100-nm in-resin preserved fluorescence sections. Moreover, we show that tuning the electron landing energy particularly improves imaging conditions for ultra-thin (50 nm) sections, where optimization of both retarding field and interaction volume contribute to the signal improvement. Finally, we show that our integrated retarding field setup allows landing energies down to a few electron volts with 0.3 eV dispersion, which opens new prospects for assessing electron beam induced damage by in situ quantification of the observed bleaching of the fluorescence following irradiation.

    Original languageEnglish
    Pages (from-to)109-120
    Number of pages12
    JournalMicroscopy and Microanalysis
    Volume27
    Issue number1
    DOIs
    Publication statusPublished - Feb-2021

    Keywords

    • backscattered electron detection
    • correlative light and electron microscopy
    • electron beam induced damage
    • retarding field
    • SEM

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