Researchers have been using cryo-electron tomography to study cells up close, but the modality has its limitations because it only provides a small viewing window, so only small cells or partial peripheral sections of larger cells can be examined at high resolution.
By introducing focused ion beam (FIB) technology to the technique, researchers at Max Planck Institute of Biochemistry were able to remove specific material from cells, opening up a viewing window for cryo-electron tomography to peek inside.
From the announcement:
The entire cell or individual cell components are “shock frozen” and enclosed in glass-like ice, thus preserving their spatial structure. The transmission electron microscope then enables the acquisition of two-dimensional projections from different perspectives. Finally, the scientists reconstruct a high-resolution three-dimensional volume from these images. However, the electron beam can penetrate only very thin specimens (for example bacteria cells) up to a thickness of 500 nanometers. Cells of higher organisms are clearly thicker. State-of-the-art electron microscopic preparation techniques are therefore necessary to make also larger objects accessible for cryo-electron tomography. “The artefact-free and, in particular, targeted preparation of larger cells is a critical step,” explained Alexander Rigort, MPIB scientist. “With the traditional methods, we could never rule out that structures we wanted to investigate were changed.” The meaningfulness of the results was therefore limited, according to the biologist.
Using a focused ion beam microscope (FIB), researchers can now mill single layers of the frozen-hydrated cell and remove them in a controlled manner – thus rendering thin, tailor-made electron-transparent “windows”. An additional advantage of ion thinning is that mechanical sectioning artefacts are completely avoided.
Abstract in Proceeings of The National Academy of Sciences: Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography…