Abstract
Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.
Highlights
Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM)
Protocols are being actively developed that will enable the study of large tissue s amples[15], such as whole fruit fly b rains[16,17,18], zebrafish b rains[19], or even mouse b rains[20,21] but these protocols cannot be used for the en block staining of whole insect head, since they do not provide uniform quality for a heterogeneous sample
We have developed a protocol based on simultaneous fixation with glutaraldehyde (GA) and osmium (OsO4)[31], sequential osmification and treatment of samples with potassium ferrocyanide (FeCN)[15], staining of samples with uranyl acetate (UA) with heating to 50 °C32, staining with lead aspartate (PbAsp)[33]
Summary
Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The success of specimen fixation and contrasting is largely determined by the rate of penetration of the reagents into the s ample[11,22], which is considerably slowed down in heterogeneous samples with the presence of a poorly permeable integument or of tissues with a high lipid content Such problems with sample preparation are noted for insects[23,24] and other arthropods 25, other invertebrates[25,26,27,28] and vertebrates[29], as well as plants 30. The 3D-EM methods in connectomics are most in demand; our attention was focused on the brains of the sampled insects
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