New SLAC Methodology Guides Higher Cell Slice Preparation for Cryo-ET Imaging

SLAC researchers develop an strategy to higher information the preparation of cell slices for cryogenic electron tomography imaging.

It is tough enterprise taking photographs of tiny buildings inside cells. One method, cryo-electron tomography (cryo-ET), shoots electrons via a frozen pattern. The pictures fashioned by the electrons that emerge permit researchers to reconstruct the interior structure of a cell in 3D with near-atomic decision. 

The factor is, this methodology would not work if a pattern is simply too thick. Electrons cannot penetrate via most cells, together with human cells. As a substitute, researchers use a beam of ions to mill these beefier cells right down to 200 nanometer-thick slices, however then one other problem arises: making certain the construction of curiosity – a ribosome or chloroplast, say – is definitely contained on this skinny slice. It typically takes a number of tries to seize the goal object in an eroded pattern. 

Now, by combining mild microscopy with ion beam milling, scientists on the Division of Vitality’s SLAC Nationwide Accelerator Laboratory recognized an extra sign in fluorescent mild that may information the milling course of. This system improves the accuracy of optically-guided milling by roughly an order of magnitude, which can permit cryo-ET to extra simply goal small, uncommon buildings inside cells, together with invading viruses. 

“Our strategy tells you the place your object is inside that closing skinny cell part very precisely and with a excessive success charge,” stated Peter Dahlberg, an assistant professor at SLAC and Stanford College. “This enchancment additionally enormously improves effectivity, as you spend much less time milling and lacking your targets.” Dahlberg developed this time-saving method, which was revealed in Nature Communications, along with his analysis associates Anthony Sica and Magda Zaoralová.

The group used what’s referred to as a tri-coincident system, which aligns the focal planes of three completely different devices: a scanning electron microscope, an ion beam for cell milling, and an optical microscope for observing tiny objects tagged with fluorescent chemical substances. In contrast to industrial techniques, which would not have co-aligned devices, this method permits researchers to observe fluorescence whereas milling. 

Though, tri-coincident techniques cannot usually resolve objects smaller than 200 nanometers resulting from elementary optical limitations of those devices, Dahlberg and his group turned to interference, a phenomenon of sunshine wave interactions, to beat this limitation. 

As an ion beam erodes the highest of a cell, fluorescent mild from the item of curiosity shines up from beneath. The pattern’s prime floor displays these mild waves, which then flip round to intrude with the incoming mild – very like ripples on a pond that cross and create extra advanced patterns. On this case, because the floor of the pattern is eroded, interference causes the fluorescent mild from tagged objects to dim and brighten. Sica wrote software program that may exactly find the fluorescent object primarily based on these patterns of dimming and brightening, permitting extra correct milling. 

To check out the strategy, the group imaged a 26 nanometer-wide virus infecting a human cell, demonstrating that this strategy can goal organic buildings beforehand off limits with cryo-ET. The method might be utilized to different viral particles and tiny, transient buildings concerned in cell division, in addition to different objects. 

“I wish to present the sphere that utilizing this impact and this tri-coincident geometry is the suitable option to goal one thing very small in cells of their native state,” Dahlberg stated.

Subsequent, the group desires to include superior mild microscopy strategies into the tri-coincident system to additional enhance the optical picture high quality and take essentially the most informative cryo-ET photographs attainable. 

“There’s loads of analysis and growth on the milling, however I wish to incorporate fancier, subtle fluorescence methods,” Sica stated. 

The analysis was funded partially by the DOE Workplace of Science, the Nationwide Institutes of Well being, and the Chan Zuckerberg Initiative DAF, an suggested fund of the Silicon Valley Group Basis.