Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT-MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3-D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.