A major complicating factor in whole cell electron microscopy is the need to embed and separate samples before imaging, which makes experimental procedures complicated and may introduce artifacts.
The Poseidon in situ system allows imaging of fully hydrated samples with electron microscopy. In this experimental study, images of live yeast cells were recorded with scanning transmission electron microscopy (STEM) using the Poseidon system.
In this experiment, live wild type and a septin mutant (spn3 Δ) strain of schizosaccaromyces pombe (s. pombe) yeast cells were incubated for 60 minutes in 10 µM of live/dead indicator dye (FUN-1). A droplet of s. pombe cell suspension was dispensed on the lower E-chip™ to load the Poseidon system holder. After placing a second E-chip on top, the chamber was sealed.
STEM images were recorded using a Philips/FEI CM200 TEM/STEM operating at 200 kV, and fluorescent images were recorded before and after electron microscopy. The thickness of liquid in the imaged areas was measured to be 3 ± 1 µm. An electron dose of 22 e-/nm2 was used to record images of the wild type and mutant strains.
Figure A shows the STEM image of wild-type yeast cells. A gold nanoparticle and internal structures such as the cell wall (1), the primary septum (2), the secondary septum (3), a cell membrane invagination (4), lipid droplet (5), a peroxisome (6), and an unclassified vesicle (7) are all visible.
Correlative light and electron microscopy (CLEM) was also shown. Figure B depicts a phase contrast light microscopy image of the mutated yeast cells that included the live/dead indicator dye, and Figure C shows the corresponding fluorescence image. Dead cells display a bright green fluorescence, and living cells display a punctuated red fluorescence.
Therefore, the cells that exhibit red fluorescence were alive at the start of STEM imaging. Figure D shows the corresponding STEM image of the cells in the dashed box. A resolution of 32 ± 8 nm was achieved, which provides a bridge between the resolution achieved with light microscopy and traditional biological TEM techniques.
Using the Poseidon liquid in situ system, live cells in a fully hydrated state can be imaged with electron microscopy. In this experiment, the resolution achieved was enough to determine the ultrastructural details of pristine yeast cells without plastic embedment and freezing.
This information has been sourced, reviewed and adapted from materials provided by Protochips.
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