This study demonstrates that a combination of unconventional electron microscopy techniques provides a quantitative means of assessing the degree of monodispersity of gadolinium (Gd) diethylenetriamine pentaacetic acid-conjugated polyamidoamine (PAMAM) dendrimers, which are designed for diagnostic imaging and delivering chemotherapeutics. Specifically, analysis of images acquired in the scanning transmission electron microscopy mode yields the distribution of molecular weights of individual dendrimers, whereas analysis of images acquired in the energy-filtering transmission electron microscopy mode yields the distribution of Gd atoms bound to the dendrimer nanoparticles. Measured compositions of Gd-conjugated G7 and G8 PAMAM dendrimers were consistent with the known synthetic chemistry. The G7 dendrimers had a mass of 330 ± 4 kDa and 266 ± 4 Gd atoms (± standard error of the mean). The G8 dendrimers had a mass of 600 ± 8 kDa and 350 ± 5 Gd atoms (± standard error of the mean). This approach will be particularly attractive for assessing the mass, composition and homogeneity of metal-containing organic nanoparticles used in nanomedicine. Keywordsdendrimer; EFTEM; energy-filtered transmission electron microscopy; gadolinium; mass measurement; scanning transmission electron microscopy; STEM The ability to create molecular nanostructures of controlled size and chemical functionality offers a powerful approach for the design of multifunctional biomedical nanocarriers containing image contrast-enhancing agents and therapeutic drugs. For any nanoparticle used in the diagnosis and treatment of disease, it is necessary to establish the degree of monodispersity in terms of size, molecular weight and composition (Figure 1) [1]. Dendrimers, a class of regularly branched polymeric nanostructures (Figure 2), present particularly attractive attributes as biomedical nanocarriers [2][3][4]. First, dendrimers can be synthesized to possess large numbers of chemically functional surface groups, which makes them suitable for the attachment of various chelating agents, fluorescent labels, cell-targeting ligands and drugs.

Extended abstract of a paper presented at Microscopy and Microanalysis 2008 in Albuquerque, New Mexico, USA, August 3 – August 7, 2008

No abstract

The first demonstration that electron energy loss spectroscopy (EELS) could be applied in biology to detect very small levels of calcium and other elements was made by Andrew Somlyo and his colleagues at the University of Pennsylvania twenty-five years ago [1]. In fact, many important developments originated from that research, particularly the work in collaboration with Henry Shuman. The laboratory's pioneering achievements included (i) the first electronic parallel recording system based on an array detector [2]; (ii) the first measurement of trace elemental concentrations [3,4]; (iii) the first demonstration that an electron spectrometer could be adapted to serve as a post-column imaging filter [5,6]; and (iv) the first digitally controlled system for acquiring spectrum-images in the scanning transmission electron microscope (STEM) [7]. The essential features of all four of these advances have been incorporated into today's EELS instrumentation and data acquisition software. The fact that these systems are now being applied mainly to characterize materials rather than biological structures demonstrates the wide impact of the technological developments in Andrew Somlyo's laboratory.As originally appreciated by Henry Shuman and Andrew Somlyo, all of the above developments are essential for successful application of EELS to biological systems, which present a formidable challenge in terms of their radiation sensitivity and the small numbers of atoms of specific elements that are found in cellular structures [8]. The current state of the art in EELS instrumentation includes: spectrum-imaging in the STEM [9]; energy filtering electron microscopy (EFTEM) using in-column or post-column filters equipped with highly efficient charge-coupled device (CCD) detectors [10]; sophisticated methods for spectral and image analysis [11][12][13]; and most recently attempts to collect 3-D elemental distributions from sectioned cells by energy-filtered electron tomography [14].The highest analytical sensitivity in EELS of biological structures is obtained in the STEM, where the electron probe can be focused to nanometer dimensions [15]. It has been shown that it is feasible to map single atoms of certain elements such as calcium and iron [16], although the very high required dose of more than 10 8 electrons nm -2 results in severe radiation damage and makes it difficult to achieve this sensitivity except for 'destructive' analysis of isolated macromolecules, which can first be imaged by dark-field STEM at low electron dose.In a cellular context, the relevant measure of sensitivity is often the minimum detectable atomic fraction of an element, which can be translated into the minimum detectable number of millimoles per kilogram. By using the techniques developed in the Somlyo laboratory [1][2][3][4][5][6][7], it is possible to detect the biologically important element, calcium, in subcellular compartments of freeze-dried cryosections with a precision of 10 atomic parts per million, corresponding to about 1 millimole per kilogram dry...