RESUMO
The absence of imaging lenses after the specimen in the scanning transmission electron microscope (STEM) enables electron tomography to be performed in the STEM mode on micrometer-thick plastic-embedded specimens without the deleterious effect of chromatic aberration, which limits spatial resolution and signal-to-noise ratio in conventional TEM. Using Monte Carlo calculations to simulate electron scattering from gold nanoparticles situated at the top and bottom surfaces of a plastic section, we assess the optimal acquisition strategy for axial bright-field STEM electron tomography at a beam-energy of 300keV. Dual tilt-axis STEM tomography with optimized axial bight-field detector geometry is demonstrated by application to micrometer-thick sections of beta cells from mouse pancreatic islet. The quality of the resulting three-dimensional reconstructions is comparable to that obtained from much thinner (0.3-micrometer) sections using conventional TEM tomography. The increased range of specimen thickness accessible to axial STEM tomography without the need for serial sectioning enables the 3-D visualization of more complex and larger subcellular structures.
Assuntos
Tomografia com Microscopia Eletrônica/métodos , Microscopia Eletrônica de Transmissão e Varredura/métodos , Animais , Células Secretoras de Insulina/ultraestrutura , Camundongos , Método de Monte CarloRESUMO
AIMS: To image the distribution of drug molecules attached to single-wall carbon nanotubes (SWNTs). MATERIALS & METHODS: Herein we report the use of scanning transmission electron microscopy (STEM) for atomic scale visualization and quantitation of single platinum-based drug molecules attached to SWNTs designed for targeted drug delivery. Fourier transform infrared spectroscopy and energy-dispersive x-ray spectroscopy were used for characterization of the SWNT drug conjugates. RESULTS: Z-contrast STEM imaging enabled visualization of the first-line anticancer drug cisplatin on the nanotubes at single molecule level. The identity and presence of cisplatin on the nanotubes was confirmed using energy-dispersive x-ray spectroscopy and Fourier transform infrared spectroscopy. STEM tomography was also used to provide additional insights concerning the nanotube conjugates. Finally, our observations provide a rationale for exploring the use of SWNT bioconjugates to selectively target and kill squamous cancer cells. CONCLUSION: Z-contrast STEM imaging provides a means for direct visualization of heavy metal containing molecules (i.e., cisplatin) attached to surfaces of carbon SWNTs along with distribution and quantitation.
Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Nanomedicina/métodos , Nanotubos de Carbono/química , Platina/química , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Microscopia Eletrônica de Transmissão e Varredura , Modelos Biológicos , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Electron tomography provides three-dimensional structural information about supramolecular assemblies and organelles in a cellular context, but image degradation, caused by scattering of transmitted electrons, limits applicability in specimens thicker than 300 nm. We found that scanning transmission electron tomography of 1,000-nm-thick samples using axial detection provided resolution comparable to that of conventional electron tomography. We demonstrated the method by reconstructing a human erythrocyte infected with the malaria parasite Plasmodium falciparum.