High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non-conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre-coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross-section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals. Lay Description: In the recent years, gold nanoparticles are widely investigated as a diagnostic and therapeutic tool in medical and research applications. Nanoparticles are very small particles between 1 and 100 nanometers in size (1 nanometer equals 1 milionth of a millimeter). At such a small size, individual nanoparticles cannot be distinguishable with conventional optical microscopy hence electron microscopy (SEM) is applied.However, visualization of biological materials in SEM requires a complicated preparation procedure. Since biological material is non-conductive, negative charge is built on the sample under the bombardment of the electron beam in the electron microscope. In order to avoid charging artefacts, the sample is typically coated with a thin metallic layer. However, the finest conductive metal coating available is usually composed of a few nanometer size clusters, which may hinder the almost identical metal nanoparticles used in the sample itself. Therefore SEM monitoring of metal nanoparticles within cell or tissues is incompatible with the conventional preparation methods.In the present work we show that charging artefacts related to non-conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive carrier. By growing the cells on glass pre-coated with a conductivelayer as chromium, we were able to observe the uptake of 10 nm gold nanoparticles inside the living cells. By mounting a skin cross section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.