Immuno-Fluorescence SEM
Discussion With cathodolumines-
cence imaging of fluoro- phores
attached to
Figure 6: Confocal image of the same cell type as in Figure 5 prepared by the same protocol except for the addition of CMFDA (green) to the cell cytoplasm. Red Qdots 605, bound to anti-EGFr, light up the exosomes in this image.
specific antibodies on tumor cells, we have caught the release of exosomes and their subsequent attachment to the membrane veil-like structures of monocytes. Further studies with cathodoluminescence may reveal a time-dependent change of location of these exogenous bodies on or within the monocyte and, possibly, subsequent changes in the morphology of the monocyte itself. In
addition, we were able to see that, coincidentally with release of exosomes from the tumor cells, EGFr concentrations on the cell surface changed. In a correlative study this technique has revealed that the primary location of the monocyte surface protein CD14 is on the membrane veils, which suggests an extensive role for the veils in the monocyte physiology. We were unable to capture any of these morphologically specific states by confocal light microscopy. Te resolution of field-emission SEM is currently reported
at better than 1 nm [24, 25]. Tis, and the high quantum yield and submicron (15–20 nm) size of quantum dots and other new protein tags, along with the broad availability of fluorescent labels, will ultimately allow extensive morphological mapping of cell structures.
Figure 8: The surface of a highly veiled monocyte labeled with mouse anti-Human CD14/anti-mouse Qdot 605 (red), which is concentrated on the veil edges. (A) CL image. (B) Electron image from a mix of the upper and lower detectors. (C) Overlay of A and B. Scale bar = 1 mm.
With currently available SEM/CL instrumentation,
Figure 7: Developing exosomes on the cultured U373 glioblastoma tumor cell surface. Qdot 565 bound to anti-EGFr (green). (A) Cell with little EGFr on the surface. (B) Cell with large coverage of EGFr remaining. (C) Exosomes with EGFr on the surface of the cells.
2010 September •
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multiple labels can be imaged on a single sample [26]. Tus, this technique could also be applied to determining the relative orientations of functionally interactive molecules such as receptors, ion barriers, molecularly selective membrane pores, or cell synapses. It might also be used in determining
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