Jong-Man Jeong

Ultrastructures of Ptilota filicina (Rhodophyta) by High Pressure Freezing(HPF): Comparison of HPF Fixation and Chemical Fixation

In preparation of the biological samples for electron microscopy, the chemical fixation by glutaraldehyde, paraformaldehyde, and OsO4 has been generally used for a long time. However, the chemical fixation method has some problems: the infiltration time is a little bit long and the ultrastructure of cell or tissue transforms before complete fixation of sample. So, recently, cryo-fixation is considered more often in biomedical field. In this study, we compared High Pressure Freezing (HPF) method with chemical fixation method using a algal sample (Ptilota filicina J. Agardh), which was difficult to fix using chemical fixation method. In chloroplast, the ultrastructure of thylakoid lamella and phycobilisome can not show clearly by chemical fixation. In this study we could observe the ultrastructure of thylakoid lamella and phycobilisome of chloroplast very clearly using HPF fixation. An improved images of ultrastructures of nucleus, mitochondrion and floridean starch could obtain. These results suggest that HPF method is very useful method in algal specimen for electron microscopy.

Applicable technical method for lower temperature freeze-substitution of cryo-fixed cells

Cryo-fixation and freeze substitution followed by microscopy are commonly used sample preparation methods for visualizing the morphology of intracellular organelles. Freeze substitution is an especially important preparative step because it enables the preservation of intracellular structures in cryo-fixed cells close to the living states for visualization. In this study, we describe a novel freeze substitution method and device that uses both dry ice and liquid nitrogen as coolants rather than dry ice alone. We found that our approach achieved lower temperatures for longer periods of time than conventional devices and therefore allows major improvements in preservation quality, ultimately facilitating high contrast imaging of intracellular organelles. The technical approaches that are realized by this valuable approach are simple, rapid, and applicable for visualizing cellular events of interest.

The use of artificial structure as an initial reference model for projection-matching three dimensional reconstruction: visualization of macromolecular structure using cryo-electron microscopy

Projection-matching single particle three dimensional reconstruction following to electron microscopy is the one of common methods to visualize three dimensional structures of target macromolecules or protein-protein interaction at medium or near-atomic resolution. In this report, we demonstrated the potentiality of using artificial structure as an initial reference model for its computational processing by describing an example experiment in which we obtained ~ 2000 single particle images from cryo-electron microscopy. The reconstruction using the model provided correct matching parameters of single particle experimental images with projection images of the model, ultimately resulting in generating informative three dimensional volume. Comparative analysis confirmed that the resultant volume has a remarkably similar structure to the proposed atomic model. These results provided suggestive evidence for using this technical approach as a useful analytical tool for determining macromolecular structure in the model independence way. In addition, the finding goal can be used for evaluating high resolution structure processed from the reference model of atomic structure since it is an ideal method to eliminate any possibilities of model-bias.