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Immuno-Fluorescence SEM


reduce the possibility of damage to the fluorophore and the biological sample. A more efficient light-gathering system than the one used here is available (Gatan, Inc., Warrendale, PA). Tat instrument includes an annular parabolloidal mirror, a high-efficiency PMT, and is also available with either a monochromator or filters for multi-color imaging. Confocal imaging was executed on a Zeiss LSM 510 using


a C-Apochromat 63× / 1.2 W corrected objective, 488-nm laser excitation, and 543-nm and 633-nm emission filters.


Results Comparison with light microscopy. Glioblastoma


exosomes, imaged by cathodoluminescence (Figure 5), appear as spherical bodies, falling within the size range measured by other techniques and were observed to bind to the membrane extensions (veils) of monocytes. Te same type of cell, in the same experimental procedure (except for the addition of the cytoplasmic dye CMFDA [Invitrogen]), was mounted on a coverslip dish, rather than the aluminum, and imaged by confocal microscopy (Figure 6). In this case the exosomes are not individually resolvable nor is their association with the monocyte veils apparent—a difficulty we have consistently encountered in our confocal studies. Glioblastoma exosomes were also fixed during the process


Figure 4: A monocyte (grayscale) containing tumor cell exosomes labeled with Qdot 565 bound to anti-EGFr (green) imaged at two different probe current levels, demonstrating intensity and signal concentration dependence on probe current. The exosomes (usually 50- to 500-nm diameter) on this cell have apparently fused into large masses, typical of membrane vesicular action [27]. (A) Intensity scale bar (Red = Max., Violet = Min.). Scaled intensity image of (C) with a vertical 45° tip backward. (B) Scaled intensity image of (D). (C) CL image with 20 µA probe current. (D) CL image with 37 µA probe current. (E) Secondary electron image from the upper detector. (F) Overlay of D & E. Scale bar = 1 mm.


voltage affects the size of the interaction volume (and, thus, the depth of penetration) as well as the CL intensity. A higher probe current results in a larger spot (beam) size, which yields a CL image with a higher intensity and concentration of signal [22] (Figure 4). Reduction of either of these parameters will


of release from the tumor cell (Figure 7). Although during early cell culture we’ve imaged the protein EGFr covering the entire tumor cell, aſter several days of growth, the protein concentrates into exosomal structures, which pinch off of the cell surface. As a demonstration of a different type of cell surface


labeling, we used the surface protein CD14 of human monocytes. Cathodoluminescence images of these monocytes, labeled with Qdot 605 bound to anti-CD14 (Figure 8) show a high concentration of the protein on the membrane veils (sheet- like extensions of the surface membrane), especially on the veil edges. Tis is consistent with previous studies [23] that show the veils as the active site of T-cell binding and the location of high concentrations of other T-cell-interacting proteins. Fluorophores. We have tested materials as diverse as


Figure 5: Tumor cell exosomes (green, B & C) adhere to the membrane extension of a monocyte. (A) Electron image (a mix of upper and lower detectors). (B) CL image of fluorescent Qdot 565 bound to anti-EGFr (see Cell Handling & Labeling). (C) Overlay of A & B. Scale bar = 1 mm.


10


phosphors, green fluorescent protein (GFP), quantum dots, and the common fluorescent tags attached to antibodies and proteins (Figure 9). All have responded well in this system though with variable stability under the electron beam and quantum yield (the ratio of photons emitted to photons absorbed). Te common organic fluores- cent dyes readily bleach under the conditions used here, whereas quantum dots have shown excellent stability. At 10–20 nm in diameter they are about the size of fluorescent proteins, are available in an extensive library of bioconjugates, and have been repeatedly proven effective [12].


www.microscopy-today.com • 2010 September


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