Abstract
SummaryIntegrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high‐resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via ‘smart tracking’. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence.
Highlights
Recent technological advances in electron microscopy have allowed the acquisition of extended volume data sets at high resolution (Peddie & Collinson, 2014)
One of these methods is known as array tomography, whereby an array of ultrathin sections cut through resin-embedded cells or tissues are imaged sequentially with a scanning electron microscope (SEM) to build up a 3D stack of images through the volume
The field of correlative light and electron microscopy has enabled the mapping of functional information onto high-resolution ultrastructural electron microscopy data, by detecting fluorescent biomarkers in the context of cell structure (Kopek et al, 2012; Bell et al, 2013; Loschberger et al, 2014; Johnson et al, 2015; Bykov et al, 2016; Mateos et al, 2016; Wolff et al, 2016)
Summary
Recent technological advances in electron microscopy have allowed the acquisition of extended volume data sets at high resolution (Peddie & Collinson, 2014). One of these methods is known as array tomography, whereby an array of ultrathin sections cut through resin-embedded cells or tissues are imaged sequentially with a scanning electron microscope (SEM) to build up a 3D stack of images through the volume (Micheva & Smith, 2007; Wacker & Schroeder, 2013; Hayworth et al., 2014). It is possible to perform integrated array tomography inside the ILEM using in-resin fluorescence (IRF) sections, in which both fluorescent and electron signals have been preserved (Peddie et al, 2014, 2017) This technique delivers data from both modalities with almost perfect alignment
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