LAUSR

In this chapter, we describe the study of bone marrow megakaryocytes (MKs) using a high-resolution 3D imaging approach known as focused ion beam-scanning electron microscopy (FIB-SEM). The apparatus consists of a scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and an electron beam, used to image the milled surfaces. This produces a series of ultrastructural images which can be computationally reconstructed into three-dimensional (3D) volume images. Using this approach it is possible to characterize the 3D ultrastructure of MKs in their native bone marrow environment, to study subcellular organelle interactions in the context of a complete cell and to quantify specific features. This chapter provides protocols for sample preparation, image acquisition and 3D reconstruction, the whole procedure requiring about 7-8 days. It also describes a method combining light microscopy (LM) with FIB-SEM, a procedure called correlative light electron microscopy (CLEM), which allows the site-specific 3D imaging of MKs in tissues.