Abstract
Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.
Highlights
Correlative light and electron microscopy (CLEM) combines the advantages of Electron Microscopy (EM) and Light Microscopy (LM) by spatially aligning imaging data sets of the same region of interest
Polymer films such as Aclar are increasingly used for CLEM [14,15,16,17,18,19] and provide additional advantages as they are easier to handle during sample processing [20], and are compatible with cryopreservation by High Pressure Freezing (HPF) [21]
We attempted to perform the optical imaging without the glass coverslip support, but found that gluing Aclar film over a hole drilled into tissue culture plasticware does not support the weight of tissue culture medium and results in a curved film which restricts optical imaging (Figure 1A)
Summary
Correlative light and electron microscopy (CLEM) combines the advantages of Electron Microscopy (EM) and Light Microscopy (LM) by spatially aligning imaging data sets of the same region of interest. The primary challenge of CLEM sample preparation is the relocation of a target identified by optical microscopy This is aided by marking the sample with a reference grid either by attaching a TEM finder grid to the sample [9], depositing a layer of gold [10] or carbon [11] through a TEM finder grid, etching the sample surface with a needle or focussed ion beam [12], or using commercially available photo-etched glass coverslips [2]. Photoetched griddedglass coverslips have been used successfully for CLEM including in combination with the Tokuyasu technique [13] Polymer films such as Aclar are increasingly used for CLEM [14,15,16,17,18,19] and provide additional advantages as they are easier to handle during sample processing [20], and are compatible with cryopreservation by High Pressure Freezing (HPF) [21]
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