Video Script: Separation of a Cell

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Title: ....... Separation of a Cell
Length: ..... 1 min 30 secs
Credits: .... Andrew Noske, Horng Ou, Clodagh O'Shea, Mark Ellisman
URL: ........ http://www.youtube.com/watch?v=oFQZm7UEHFY

This video show a human cell undergoing mitosis (cell division) as imaged using 3D serial block face scanning electron tomography (SBFSEM). The cell's DNA is clearly visible thanks to a new probe for molecular labeling called miniSOG (Shu et al. 2011). This florescent protein is less than half the size of GFP and can be used to elucidate structures in both light microscopy and electron microscopy. Here it has been fused at the genetic level with the DNA associated protein Histone 2B.

Executive Summary

This page represents the script I wrote to showcase an "illustration" entry I put into the NSF International Science and Engineering Visualization Challenge in 2011 and made it into the finals and Science magazine. The video helped communicate the significance of the video. In particular, I showcase the newly engineered fluorescent protein miniSOG (mini Singlet Oxygen Generator) (Shu et al, 2011) which can be used in both electron microscopy (EM) and light microscopy (LM) to target and elucidate other proteins/structures in tissues/cells. In this video, miniSOG has been attached to a DNA associated protein, thereby allowing us to see areas of chromosomes (organized structures of DNA).

For the average person, what's most cool about this video isn't the protein, but instead the fact that we've captured a cell in the process of cell separation - also known as mitosis. In mitosis, a "mother" cell separates the chromosomes of its nucleus into two identical sets into two separate nuclei - which is what you can see here. At the end of the process the mother cell divides apart to form two identical "daughter" cells.


Script #1.2: Separation of a Cell

This animation "Separation of a Cell" shows a cell undergoing mitosis.

0:070:07

The cell you are looking at is a cancerous human cell line, 0:030:10 imaged using Serial Block Face Scanning Electron Microscopy, with a Gatan 3View system to cut successive slices. 0:090:19 But what's most interesting about this image, is the use of protein tagging using miniSOG.

0:060:25

MiniSOG, or mini Singlet Oxygen Generator, is a small and newly engineered fluorescent marker that works both in light microscopy and electron microscopy. 0:110:36 This protein is 106 amino acids, less than half the size of green fluorescent protein, and can be fused at the genetic level to a target protein in much the same way as GFP.

0:130:49

In this case, miniSOG is associated with a DNA binding protein Histone 2B, thus allowing us to mark the DNA of the chromosomes - something we usually can't see under electron microscope.

0:161:05

To produce this final 3d model, the images slices were manually traced using IMOD software and then final rendering done using Cinema 4D and ePMV. This image represents one of our first test images in a collaboration between the Mark Ellisman group at the University of California, San Diego, and the Clodagh O'Shea group at the Salk Institute.

0:211:26


Characteristics:

  • Small florescent protein - 13.9 kDa
  • Genetic engineered into a target protein in similar manner to GFP
  • Upon excitation produces reactive state oxygen, which polymerizes diaminobenzidine, which has an affinity to osmium thus appears electron dense in the EM


OLD VERSIONS

Script #1.1: Separation of a Cell (older long version)

This animation "Separation of a Cell" shows a cell undergoing mitosis and represents our illustration entry into the NSF International Science & Engineering Visualization Challenge.

The cell you are looking at is a cancerous human cell line, and to image this in 3D we used Serial Block Face Scanning Electron Microscopy, using a Gatan 3View system whereby successive slices are cut from a block using an automated diamond knife inside the microscope. But what's most interesting about this image is the use of a protein tagging using miniSOG.

MiniSOG, or mini Singlet Oxygen Generator, is a small and newly engineered fluorescent marker for correlative microscopy. This protein can be fused at the genetic level to a target protein - much the same way green fluorescent protein does. The two big differences however: firstly that its 14 kilo-Daltons, half the size of GFP, and secondly this marker works both in light microscopy and electron microscopy.

MiniSOG can be fused to many other different proteins, but in the case of this image, MiniSOG has been targeted to a molecule called Histone 2B, which is found in chromatin, thus allowing us to see DNA - something we can't usually see clearly in electron microscope images.

This image represents one of our very first test images in a collaboration between the Mark Ellisman group at the University of California, San Diego, and the Clodagh O'Shea group at the Salk Institute.... and were lucky enough to capture a cell in the middle of cell division.

To produce the final 3d model you see here, manual image segmentation was done using IMOD from the University of Boulder Colorado, and final rendering done using Cinema 4D plus a special plugin called ePMV from SCRIPPS to render the molecule. Special thanks also goes out to all these people listed - especially the NSF for providing the wonderful opportunity to showcase our science as part of their International Science & Engineering Visualization Challenge.


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