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Figure 1. Overview of the sampling process. Cylindrical resin blocks were exhaustively sectioned in an ultramicrotome, taking a known fraction of the sections. The 200 nm sections sampled were placed fully on TEM grids and analyzed in a TEM. A known fraction of each section was analyzed for nanoparticle number and location. TEM: Transmission electron microscope.
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Figure 2. Cell pellets embedded in bottleneck BEEM® capsules.
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Figure 3. The method to center thin sections on transmission electron microscope finder grids.
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Figure 4. Fractionator principle and subsampling for transmission electron microscopy. TEM: Transmission electron microscopy.
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Figure 5. Nanoparticle counting process. (A) Illustration of the meander-like sampling pathway on transmission electron microscopy sections. (B) A typical transmission electron microscopy image overlaid by a sampling probe showing parts of a 3T3 fibroblast cell with internalized nanoparticles (dark areas). (C) Subcellular structures and internalized nanoparticles.
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Figure 6. Graytone image of a resin section showing cell transects and overlaid counting frames.
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Figure 7. Nanoparticle number as a function of sectioning distance determined by counting nanoparticles and cells in a systematic random manner via transmission electron microscopy.
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Figure 8. Intracellular distribution of nanoparticles. (A) Percentage of nanoparticles in different subcellular compartments. (B) Subsections of a cell imaged via transmission electron microscopy (not representative), and examples of nanoparticles contained in subcellular compartments. (i-iv) represents the link between the intracellular compartments and the percentage of nanoparticles found in each subcellular compartment.
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As nanoparticles become increasingly important in medical [1] and biological applications [2], accurate and statistically robust quantitative methods to measure particle number in biological systems are essential. Besides the technological potential of new nanomaterials, health and environmental safety concerns are growing [3]. Understanding of fundamental bio-nano interactions demands quantification as a basis for objectivity and reproducibility. There have been several attempts to quantify nanoparticle uptake by cells directly [4] using fluorescence microscopy of particles [5,6], quantum dots [7], magnetic nanoparticles [8,9] or through indirect methods measuring total accumulated particle mass (e.g., ion-coupled plasma mass spectrometry [10,11]. While fluorescence microscopy may provide...