Resonance Raman imaging of the NADPH oxidase subunit cytochrome b 558 in single neutrophilic granulocytes

Henk Jan Van Manen, Natallia Uzunbajakava, Robin Van Bruggen, Dirk Roos, Cees Otto

    Research output: Contribution to journalArticleAcademicpeer-review

    34 Citations (Scopus)

    Abstract

    We have employed confocal resonance Raman (RR) imaging to visualize the subcellular distribution of the NADPH oxidase subunit cytochrome b558 in both resting and phagocytosing neutrophils. Our Raman microscopic technique is a label-free, chemical (vibrational) imaging method that can be applied to individual, intact cells, thus probing cytochrome b558 in its native environment. The Raman signal from cytochrome b558 is resonantly and selectively enhanced in neutrophils by using 413 nm excitation. Experiments on resting neutrophils show a cytoplasmic distribution of cytochrome b558, with several areas of high content. Upon phagocytosis of polystyrene particles, we found that part of the cytochrome b558 is translocated toward the ingested beads. This is in accordance with immunocytochemistry studies combined with electron and fluorescence microscopy. As compared to these methods, RR microscopy requires minimal sample preparation and disturbance. Moreover, it allows the determination of the redox state of cytochrome b558 inside the cell, which reflects its NADPH oxidase activity.

    Original languageEnglish
    Pages (from-to)12112-12113
    Number of pages2
    JournalJournal of the American Chemical Society
    Volume125
    Issue number40
    DOIs
    Publication statusPublished - 8 Oct 2003

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