live cell imaging

Position available: 2016 Honours Student Project in our Lab

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Great news we are currently looking for a new honours student for 2016.

The title of the project is “Developing novel biosensors to monitor DNA damage in cancer cells”.

Its a very exciting new project incorporating cutting edge microscopy and fluorescent biosensors.

If you think you have what it takes and are interested please feel free contact myself, or UNSW SoMS.
For more information on the UNSW honours program please visit: http://medicalsciences.med.unsw.edu.au/students/soms-honours/

Below is an example of the images that will be created during the project.

Using Thresholds to Measure and Quantify Cells in Image J

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I often get asked how to uses Thresholds to measure things in Image J.

There are some great guides on the web explaining how to use Thresholds in Image J, and here are a few that are well worth checking out [Link1][Link2].

Below are some of the Basic Steps for using Thresholds:

  1. Open your image and duplicate it (Image>Duplicate)
  2. On the duplicate go to Image>Adjust>Threshold
  3. Play with the sliders until all of your cells are red.
  4. Click ‘Apply’
  5. You should now have a ‘binary’ black and white image
  6. Now go to menu Process>Binary and select ‘fill holes’
  7. You may also want to select erode, dilate, open or close to optimise the binary image so that you have nice solid filling of your cells.
  8. Now go to menu Analyse>Set Measurements. Select all the things you want to measure.
  9. Critical steps: make sure that you select your original image (not the binary) in the ‘Redirect to:’ pull down Menu
  10. Also make sure the ‘Limit to threshold’ checkbox is ticked and also tick the ‘Add to overlay’ and ‘Display label’.
  11. Click ok to close the ‘Set Measurements’ box.
  12. Now go to Analyse>Analyse Particles
  13. Here you will need to play around with the size and circularity settings (bit of trial and error) in order to get accurate identification of your cells or ROIs. I suggest making duplicates before you start so that you can quickly try different things to see which one works best.
  14. Make sure you have the Display results tick box selected.
  15. Once you click ok you should have a the measurements box appear with all your measurements for each cell.
  16. You can copy and paste these into Excel or what ever program you like to use.
  17. Go get a coffee and cake you deserve it!

Good luck!

 

We will be at the Sydney Light Optical Users Meeting on July 24th 2014

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Great news, Cell Division Lab will be at the  Sydney Light Optical Users Meeting, hosted by Dr Pamela Young at Sydney University, this Thursday (24th of July).

I will be presenting a short seminar on “Imaging and Analysing Cell Division”.

If you would like to attend please contact Pamela asap. Her details are below!

Hope to see you there !

Sydney Light Optical Users Meeting July 2014

A brief Intro to Greatwall Kinase…The King of Mitosis

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Our favourite protein in the lab is Greatwall kinase. It was first discovered in 2004 to be critical for cell division in fruit flies (1,2) . The trail then went cold for a few years as to its exact function, but in 2009, while I was working as a post-doc in France, I was fortunate enough to be in the lab that uncovered its exciting mode of action. For cells to get into mitosis they must activate a key protein called cyclin dependent kinase 1 (Cdk1). I like to think of this as the accelerator in a car. So to get moving cells push on the gas!
And conversely to get out of mitosis you need to hit the brakes. These brakes are the phosphatases which reverse the action of kinases like Cdk1. That’s great but what is missing from this equation?
Well like any car it’s pretty useless without a driver to co-ordinate the accelerator and brakes. And this is where Greatwall (Gwl for short) comes in. It makes sure that when Cdk1 (accelerator) turns on that the breaks get turned off and vice versa (3,4). Without Gwl the cell gets into a lot of trouble very fast, which you can see in the image below. Here I depleted the human version of Gwl (a gene called MASTL) and watched what happened as cells tried to undergo mitosis (5). As you can see they don’t do a very good job… the result is cells fail to divide correctly, resulting in multiple defects and often cell death.

Gwl Figure

I hope you enjoyed part one of my feature on Gwl, and in part 2 I will into more details about this amazing and exciting new protein.

References:

1. Bettencourt-Dias, M. et al. Genome-wide survey of protein kinases required for cell cycle progression. Nature 432, 980–987 (2004).

2. Yu, J. et al. Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila. J Cell Biol 164, 487–492 (2004). [Link]

3. Vigneron, S. et al. Greatwall maintains mitosis through regulation of PP2A. EMBO J 28, 2786–2793 (2009). [Link]

4. Lorca, T. et al. Constant regulation of both the MPF amplification loop and the Greatwall-PP2A pathway is required for metaphase II arrest and correct entry into the first embryonic cell cycle. J Cell Sci 123, 2281–2291 (2010). [Link]

5. Burgess, A. et al. Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci USA 107, 12564–12569 (2010). [Link]