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Intracellular Chemical Imaging of Heme-Containing Enzymes Involved in Innate Immunity Using Resonance Raman Microscopy

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Biophysical Engineering Group, Faculty of Science & Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands, and Department of Experimental Immunohematology, Sanquin Research, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands
Cite this: J. Phys. Chem. B 2004, 108, 48, 18762–18771
Publication Date (Web):November 5, 2004
Copyright © 2004 American Chemical Society

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    Vibrational microspectroscopy has become a powerful tool in cellular biology because detailed information about the chemical composition of subcellular, femtoliter volumes can be obtained. Moreover, biological imaging techniques based on this type of spectroscopy avoid labeling methods and artifacts arising from them because the required contrast is generated from endogenous molecules. Here, we report the visualization, by confocal Raman microscopy, of the intracellular distribution of two enzymes important in the immune response of granulocytes (i.e., the NADPH oxidase subunit cytochrome b558 (cyt b558) in neutrophils and eosinophil peroxidase (EPO) in eosinophils). We excited these leukocytes with 413.1 nm laser light, allowing the Raman scattering signal from the heme-containing enzymes to be dramatically enhanced by resonance. In neutrophils, there is a nonnegligible contribution from the hemoprotein myeloperoxidase to the resonance Raman signal. The effect of photobleaching of the Raman signal at 413.1 nm excitation on the reconstructed Raman images is discussed. We also show how singular value decomposition can significantly reduce the noise that is present in the raw spectral data. Stimulation of the neutrophils, either by phagocytosis or by phorbol 12-myristate 13-acetate (PMA), resulted in intracellular redistributions of cyt b558. Spectra extracted from Raman images of PMA-activated neutrophils displayed a significantly higher level of cyt b558 reduction than spectra from resting neutrophils, indicating that cyt b558 reduction is linked with NADPH oxidase activation. The versatility of resonance Raman microscopy on leukocytes is further demonstrated by the visualization of EPO in single eosinophils. In conclusion, high-resolution cellular imaging based on resonance Raman spectroscopy enables the label-free visualization of the intracellular distribution of cyt b558 in neutrophils and EPO in eosinophils, two crucial enzymes in leukocyte innate immunity.


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     University of Twente.

     University of Amsterdam.

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