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From fast-freeze to frame

BioPhotonics
Apr 2010
Caren B. Les, caren.les@photonics.com

MARTINSRIED, Germany – Researchers at Max Planck Institute of Biochemistry have obtained three-dimensional images of the vesicles and filaments involved in the communication among neurons in the brains of mammals. Using cryoelectron tomography, a microscopy technique based on the ultrafast freezing of cells, they acquired 3-D images of synapsis, the cellular structure in which the communication takes place.

“I found particularly interesting that so little was known on the mechanisms of synaptic transmission, which is the basic process by which neurons communicate with each other and with muscle cells, allowing nervous and motor function,” said team leader Rubén Fernández-Busnadiego. “One of the key aspects in the understanding of synaptic transmission is the structural organization of the molecular complexes and organelles that perform this task. Only electron microscopy has sufficient resolution to image these structures, and only cryoelectron tomography can provide a three-dimensional picture of them in their native environment.

“Cryoelectron microscopy is based on the cryofixation of the specimens – such fast freezing that water molecules do not have time to rearrange into crystals and remain in a solid but vitreous state. Thus, biological samples can be observed in the electron microscope in their fully hydrated, native state. Conversely, conventional sample preparation methods are based on sample dehydration. Water, the main component of cells, is completely removed by this process, introducing artifacts that affect image interpretation. In cryoelectron tomography, micrographs of vitrified frozen-hydrated samples are recorded at different angles and then merged computationally to reconstruct the original specimen in 3-D.”

He said samples thinner than 0.5 to 1.0 µm can be vitrified by plunge-freezing; i.e., immersed directly into a cryogen at 77 K with a simple apparatus. Thicker samples must first be frozen in special machines that apply jets of 2000-bar liquid nitrogen (high-pressure freezing); then the samples are sliced into thin sections (“cryosectioning”). He noted that this procedure is extremely demanding because samples are very labile and must remain at temperatures below ~100 K. The resulting vitrified samples can be imaged in microscopes equipped especially for that task and for allowing rotation of the sample inside the microscope column for tomographic studies.


Shown is a tomographic synapse from the mammalian hippocampus. Note the compression of the features resulting from the cryosectioning process. The scale bar equals 100 nm. Photos courtesy of Rubén Fernández-Busnadiego, Max Planck Institute of Biochemistry.


“The main advantages of cryoelectron tomography are that samples are imaged in their native, fully hydrated conditions and that three-dimensional information is provided. The main disadvantages are high cost and difficulty, compared to conventional electron microscopy, and the radiation sensitivity of the samples, which results in very noisy data, complicating interpretation. Also, the difficulties of sample preparation limit the spectrum of samples that can be analyzed,” Fernández-Busnadiego said.

For this study, the researchers treated synapses from the mammalian brain cortex with various pharmacological agents, then vitrified and examined them in the electron microscope. In the resulting cryoelectron tomograms, they observed the fine ultrastructure of the synapse, in which hundreds of ~40-nm synaptic vesicles accumulate to release neurotransmitters. They found that most of these vesicles were linked to each other and to the active zone – the area of the neuronal cell membrane where vesicle fusion actually occurs – by small filaments, and that these filaments suffered important rearrangements in different functional states of the synapse.


A tomographic slice of a mammalian synapse is depicted. Scale bar equals 100 nm.


In particular, the number of filaments that interconnected synaptic vesicles was significantly reduced upon synaptic stimulation, indicating that they play a role in vesicle mobilization and release. The study also showed that the exact configuration of the filaments that tether the vesicles to the active zone indicates the availability of the vesicles to be released. In summary, their results indicate that these small filaments have an important function in synaptic communication.

“The main implications of our study may be that the current ultrastructural picture of the synapse, obtained from electron microscopy studies on dehydrated samples, needs to be revised, and that the short filaments present in the presynaptic terminal play a very important role in synaptic transmission,” Fernández-Busnadiego said.

Cryoelectron tomography is now a mature technique, he added, but improvements in the hardware could push up its resolution limit. For this goal, cameras of higher sensitivity and microscopes allowing more elaborate sample-tilting geometries are needed, he said, noting that it also is important to advance in the automation of the process to reach higher throughput. As for the software, improved 3-D reconstruction algorithms also will increase resolution.


This image represents a 3-D rendering of a mammalian synapse. The synaptic vesicles (yellow) are extensively connected to each other and to the cell membrane (purple) by short filaments (red and blue, respectively). The material of the synaptic cleft is shown in light green and the postsynaptic density, in orange.


“In terms of sample preparation, new methods for thinning down thick vitrified samples – focused ion beam milling, cryoplaning – are needed to expand the catalog of problems that can be addressed by cryoelectron tomography. The technique is best suited for the study of thin – <0.5 µm – cellular structures in high resolution. Given the current advance of different thinning methods, this thickness limitation is likely to disappear in the future,” Fernández-Busnadiego said.

The study is reported in the Jan. 11, 2010, issue of the Journal of Cell Biology.


GLOSSARY
tomography
Technique that defocuses activity from surrounding planes by means of the relative motions at the point of interest.
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