Abstract
A simple method for imaging biological tissue samples by electron microscopy and its correlation with super-resolution light microscopy is presented. This room temperature protocol, based on protecting thin biological specimens with methylcellulose and imaging with low voltage scanning electron microscopy, circumvents complex classical electron microscopy sample preparation steps requiring dehydration, resin embedding and use of contrast agents. This technique facilitates visualization of subcellular structures e.g. synaptic clefts and synaptic vesicles in mouse brain tissue and the organization of mitochondrial cristae in the zebrafish retina. Application of immunogold protocols to these samples can determine the precise localization of synaptic proteins and, in combination with super-resolution light microscopy methods clearly pinpoints the subcellular distribution of several proteins in the tissue. The simplicity of the method, including section collection on a silicon wafer, reduces artefacts and correlates protein location with sample morphology.
Highlights
Native-state preservation of the sample is the most critical factor to take into account when imaging at nanometer resolution[1,2]
We recently demonstrated that platinum shadowing can generate topographic contrast on thin Tokuyasu sections and combined it with immunogold or super-resolution microscopy[13,14]
We have developed a simple method which takes the advantages of the reported methods[13,14], collection of Tokuyasu ultrathin sections on wafers and correlation of light and electron microscopy and eliminated any additional source of external contrast agent
Summary
Native-state preservation of the sample is the most critical factor to take into account when imaging at nanometer resolution[1,2]. Tokuyasu cryo-section protocol, a well-established method, uses methylcellulose (MC) to protect the sample from the high vacuum in the electron microscope avoiding the steps of dehydration and resin embedding or critical point drying[8,9] Contrast enhancement is another important requirement for imaging biological samples. LVSEM increases the topographic contrast of the sample[15] and has been successfully used for strongly stained samples[16] as well as for completely untreated samples like bacteria[17] or cells[18] This method provides fine ultrastructural details, reduces the time required for preparation and minimizes possible artefacts by decreasing the number of preparation steps
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