Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM

• Published in: Journal of microscopy (Oxford) Vol. 263; no. 2; pp. 142 - 147
• Main Authors: RIZZO, N.W., DUNCAN, K.E., BOURETT, T.M., HOWARD, R.J.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.08.2016
• Subjects: Biological ultrastructure; BSE; correlative light and electron microscopy; Electrons; FESEM; immunolocalization; Intracellular Space; Light; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Microtomy; Plant Cells - ultrastructure; Resins, Synthetic; scanning electron microscopy; scanning electron microscopy; Biological ultrastructure; correlative light and electron microscopy; immunolocalization; BSE; FESEM
• ISSN: 0022-2720; 1365-2818; 1365-2818
• DOI: 10.1111/jmi.12373

Summary We have refined methods for biological specimen preparation and low‐voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130–70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin‐embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals—the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon‐coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations. Lay Description Different types of microscopes offer varying levels of resolution, or size of the smallest detail of a sample that one can see. Using an electron microscope, one can resolve much finer detail than when using a light microscope; however, the maximum sample size for specimens in the two microscopes is very different. One can study a much larger area of a specimen with a light microscope than with an electron microscope because a light microscope allows a much bigger sample under the lens. This paper reports methods that allow examination of larger pieces of biological specimens by electron microscopy. The methods make it easier to find very small and perhaps rare details, not visible by light microscopy, in a piece of tissue that previously would have been too large to study by electron microscopy. Instead of needing to survey hundreds of samples, hunting for that one cell, stage of development or brief encounter between two different organisms, we can use these methods to locate our subject with the electron microscope at a low magnification, just as with light microscopy. Then, after finding our target, we can zoom in with a much higher level of magnification on the very same sample. The methods also allow us to merge light‐ and electron microscope‐specific information into a single image; we can identify, for example, a fluorescent component of a cell first with the light microscope and then, using the same sample, in the electron microscope. We can also use the method to analyze the distribution of specific molecules within the sample by using antibodies via a methodology called immunocytochemistry.