Immuno-Fluorescence Scanning Electron Microscopy of Biological Cells
Phyllis J. Fisher,* William S. Wessels, Allan B. Dietz, and Franklyn G. Prendergast Mayo Foundation, Rochester, MN
*
fisher.phyllis@
mayo.edu
Introduction Scanning electron microscopy (SEM) can produce striking
three-dimensional images of biological cells and tissues with submicron resolution of surface morphology. Such cell surfaces are oſten complex blends of folds, extrusions, and pockets that may be necessary in the positioning of specific molecules within interaction range of each other. Tus, surface changes can have a spatial control over some molecular functions, and identifi- cation of select molecules at distinct morphological locations becomes critical to our understanding of total cell function. Immuno-electron microscopy has recently evolved into
a well-defined area of study with the use of colloidal metal nanoparticles [1–3]. Te colloids, which can be conjugated to antibodies, are available in a variety of metals and sizes that have been shown to distinguish between two specific antibody binding sites [4]. A natural extension of this line of study is the use of antibody-bound fluorophores, which are widely available in an extensive range of both antibody specificity and fluorescence emission wavelengths, and which can be excited to an emissive energy level by the electron beam of an SEM [5]. Such light production, resulting from bombardment of luminescent material by an electron beam, is called cathodo- luminescence (CL). In the field-emission gun scanning electron microscope
(FEG-SEM) an electron emission current is produced by an electric field concentrated at the sharp metal tip of the gun cathode (Figure 1). Te electron beam is accelerated down the column through a series of electromagnetic lenses and apertures, most of which are adjustable to some degree. Tis electron beam can provide over a thousand times the energy necessary to excite a fluorescent molecule. Te absorbance spectra of biological materials is similar, whether excitation is by electrons or photons— as in laser excitation in confocal microscopy [6]. Te photon collector
consists of a focusing mirror (Figure 1, M) set either annular to, or at an angle near, the opening of the objective lens. Light emitted by the sample is collected by this mirror and piped (L)
8
Figure 1: Diagram of a field emission gun, scanning electron microscope with cathodoluminescence attach- ments. (M) Light-gathering mirror, (L) light pipe connecting mirror, and (PMT) photomultiplier tube.
to a photomultiplier tube (PMT). Filters or a monochromator may be placed in the light path before the PMT. When the photon detector is independent of the secondary or backscatter detectors, concurrent images may be collected. Detection of photons during SEM imaging has been
a standard technique in geology and some materials work for several decades [7], yet it only began to emerge in the biological field since 1974 [8]. Within the last several years, development of biologically active fluorophores has exploded with a variety of over 600 currently available [9]. Today there are both primary and secondary antibodies, fluorescently labeled, with fluorescence efficiency and photostability greatly improved since their initial appearance [10, 11]. We find the relatively new semiconductor fluorophore, the quantum dot, the most useful in cathodoluminescence for its excellent quantum yield and especially for its stability under the electron beam [12]. Nevertheless, cathodoluminescence still poses several
problems for biological samples, including the reduction of background autofluorescence and dispersal of surface charges without quenching the fluorescence of the antibody label. We have developed techniques and protocols to overcome these difficulties and to maximize SEM detection of photon emission from specifically labeled proteins on biological cells. To illustrate this protocol we present here images from our work in the field of human glioblastomas (brain tumors).
Cell Handling and Labeling Glioblastomas release microvesicles (exosomes) consisting
of 50- to 500-nm diameter packets of tumor cell material that contain the protein EGFr (epidermal growth factor receptor) [13]. We collected the supernatant from a glioblastoma cell culture, centrifuged it to remove whole cells, and then incubated it with CD14+ monocytes, an immune system precursor to dendritic cells, which have been shown to accumulate near tumors [14] and which do not express EGFr [15]. Aſter several hours the monocytes, which were fixed and washed, were labeled with an antibody to EGFr and a secondary antibody conjugated with fluorescent quantum dots. Simultaneous fluorescence and electron imaging of these monocytes delineated specifically labeled glioblastoma exosomes, which attached to the veil-like membrane extensions of the cell (Figure 2). Te CD 14+ mononuclear cells (monocytes) were isolated
by negative selection immunoadsorption from human peripheral blood [16]. For those experiments involving tumor exosomes, the monocytes were incubated at 37°F, 5% CO2, with the supernatant of a U373 glioblastoma cell culture for two or more hours. Since biological samples for SEM imaging must be fixed and dried thoroughly, particular care must be taken toward maintaining the fluorescence of attached labels
doi:10.1017/S1551929510000805
www.microscopy-today.com • 2010 September
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