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!

 

Using ImageJ to Measure Cell Fluorescence

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Image J can be downloaded for free from here .
This guide can also be downloaded as a complete PDF here: Measuring Cell Fluorescence using ImageJ

Here is a very simple guide for determining the level of  fluorescence in a given region (e.g nucleus)

  1. Select the cell of interest using any of the drawing/selection tools (i.e. rectangle, circle, polygon or freeform)
  2. From the Analyze menu select “set measurements”. Make sure you have AREA, INTEGRATED DENSITY and MEAN GRAY VALUE selected (the rest can be ignored).
  3. Now select “Measure” from the analyze menu or hit cmd+m (apple). You should now see a popup box with a stack of values for that first cell.
  4. Now go and select a region next to your cell that has no fluroence, this will be your background.
    NB: the size is not important. If you want to be super accurate here take 3+ selections from around the cell.
  5. Repeat this step for the other cells in the field of view that you want to measure.
  6. Once you have finished, select all the data in the Results window, and copy (cmd+c) and paste (cmd+v) into a new excel worksheet (or similar program)
  7. Use this formula to calculate the corrected total cell fluorescence (CTCF).
    NB: You can use excel to perform this calculation for you.
    CTCF = Integrated Density – (Area of selected cell  X Mean fluorescence of background readings)

     
  8. Make a graph and your done. Notice that in this example that the rounded up mitotic cell appears to have a much higher level of staining, but this is actually due to its smaller size, which concentrates the staining in a smaller space. So if you just used the raw integrated density you would have data suggesting that the flattened cell has less staining then the rounded up one, when in reality they have a similar level of fluorescence.

How to Cite this if you wold like to:

We have used this method in these papers:

McCloy, R. A., Rogers, S., Caldon, C. E., Lorca, T., Castro, A., and Burgess, A. (2014) Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle 13, 1400–1412 [Link]

Burgess A, Vigneron S, Brioudes E, Labbé J-C, Lorca T & Castro A (2010) 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

But you can also find a similar method published here:

Gavet O & Pines J (2010) Progressive activation of CyclinB1-Cdk1 coordinates entry to mitosis. Dev Cell 18: 533-543

And here:

Potapova TA, Sivakumar S, Flynn JN, Li R & Gorbsky GJ (2011) Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited. Mol Biol Cell 22: 1191–1206

And my apologies to any others that I have not mentioned.

Our latest Publication has received a F1000Prime Recommendation !

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Great news our recent paper “Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel substrate dephosphorylation motifs., Molecular & Cellular Proteomics, 2015 (DOI: 10.3410/f.725545508.793507630), has been recommended in F1000Prime as being of special significance in its field by F1000 Faculty Member Angus Nairn.

Really glad to see that all our hard work and research is being found useful for the research community.

You can checkout the recommendation here and our original publication here

Our Latest Publication Accepted and Now Online!

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Great news our latest publication “Global phosphoproteomic mapping of early mitotic exit in human cells identifies novel substrate dephosphorylation motifs” has been accepted by the top Proteomics Journal Molecular & Cellular Proteomics.

You can currently download the unformatted version for free here [link]

And here is an still image from the paper showing live HeLa cells undergoing forced phosphatase dependent mitotic exit. The red colour is Histone H2B tagged with the fluorescent mCherry protein, and the Green is tubulin tagged with GFP (green fluorescent protein).

 

HeLa cells undergoing phosphatase dependent mitotic exit
HeLa cells undergoing phosphatase dependent mitotic exit

 

Please help ASMR convince the government to urgently inject funding into Medical Research

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Hi everyone,
I am honoured to have recently joined the National board of directors of the Australian Society for Medical Research (#ASMR). This is a fantastic society that has for decades fought for a better working environment and more sustainable investment in Medical research. We recently launched a campaigned to help restore much needed funding to the sector, which as many of you know this year is facing its worst level of grant success rates in years. Please read the information below, and using the template letter lobby your local, state and national MPs to help convince them of the urgent need and benefits of Medical Research.

In 2014, NHMRC project grant success rates were below 15% and are the lowest in our history. These funding rates mark a significant drop of over 8% since 2011. This year our sector faces its toughest challenge yet, with NHMRC project grant success rates predicted to drop below 10%. In addition, the NHMRC budget allocation in 2014 dropped below the 2008 investment. If we don’t act immediately, this reduced investment into health and medical research (HMR) will result in attrition of a world-class, highly skilled and productive workforce and also negatively impact our capacity to improve health outcomes.

Early this year ASMR made its pre-budget submission (attached to this update), requesting
·         an immediate injection into NHMRC,
·         that the Medical Research Future Fund (MRFF) is established,
·         that Government commit to incrementally increasing investment for health and medical research until it reaches 3% of the total health expenditure by 2023.

This evidence based model will ensure that health and medical research is positioned to be responsive to the future health challenges facing Australia.

We applaud the Government recognising the value of health and medical research with the announcement of the Medical Research Future Fund (MRFF), however, this is a long-term initiative and we as a sector will not see any significant positive effects for several years. Our biggest challenge at the present time is to secure an immediate injection into NHMRC to claw back the loss of intellectual capital which an 8% drop in NHMRC grant success rates since 2011 represents.

Today I officially request your support for our campaign to secure

An Immediate additional investment of $300M into the NHMRC in the 2015 Budget’

ASMR has and will continue to inform the Government of the gravity of this situation which threatens our ability to continue to provide the exceptional returns on investment our HMR brings to Australia in terms of both health and economic wellbeing.  As always, our argument for additional public investment into NHMRC is underpinned by evidence. .

We will continue strategic meetings in the coming weeks. But, we need you!

As a sector we cannot accept the current situation and we must unite with a clear message. I ask that each of you, write a letter in the next couple of weeks to your local MP, the health minister and the prime minister. We recommend sending the letters by email and hard copy by mail. We know that this remains a very powerful advocacy tool. I have attached a template letter and also an ASMR fact sheet to help you. Also, spread the word to your friends, family and professional networks. In our 2006 campaign Government received >10,000 letters and this had a huge impact on the Government at the time – investment into NHMRC was doubled!

It is not too late to act on influencing Government decisions, you can make a difference! Thanks for getting on board and I am optimistic that we can ensure a positive change.

Best wishes,

Phoebe

ASMR President

ASMR Logo
The Australian Society for Medical Research
Dr Phoebe Phillips | President
Level 7, Suite 702, 37 Bligh Street|  Sydney |  NSW  | 2000
T: 02 9230 0333 | f: 02 9230 0339 | m: 0415 928 211
Email: asmr@alwaysonline.net.au
Website: www.asmr.org.au
Snr. Exec Officer :  Catherine West

 

Please use the links below to download all of the factsheets and performed letters to help you lobby your local MPs.

PreBudgetSubmission2015

ASMR Fact Sheet_April2015

Researcher example letter

ASMR April Newsletter out Now!

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Screen Shot 2015-04-13 at 10.16.28 amI am pleased and honoured to announce that I recently joined the ASMR (Australian Society for Medical Research) National Board of Directors, and have taken on the Newsletter portfolio.

With that said, I am very proud to announce that the latest ASMR newsletter is now available on-line for FREE !

You can download and view it here

There are some amazing articles in the April edition, including articles covering topics on current state of funding, past-present and future of NHMRC, scientific advocacy, the challenges faced by clinician-scientists, indigenous health and ASMR activities.

Please feel free to share and repost the article as much as you like.

Andrew

 

Congratulation to Sam our PhD student on his 1st Author Publication!

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Great news, we recently published a collaborative paper in the journal Cell Division with the Lab of Dr Liz Caldon here at the Garvan.
The title of the paper is “Cyclin E2 is the predominant E-cyclin associated with NPAT in breast cancer cells”, and you can find it online here

It also marked the inaugural 1st Author publication for our PhD student Sam!

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#ACCM15 a huge Success!

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Originally posted on The Australian Cell Cycle Community:

The 15th Australian Cell Cycle meeting was held last week (March 30th to April 1st) at the Powerhouse Museum, Sydney Australia. We had over 80 registered attendees from across Australia and Asia, making this the largest meeting to date. Megan, Tony and myself would like to take this opportunity to thank all the attendees who gave fantastic oral and poster presentations. We would also like to thank all of the Sponsors, whom without their generous support this meeting would not have been able to go ahead.

We are now planing for the 16th ACCM, which is tentatively scheduled for 2017. Make sure you click the subscribe button (top right of page) to keep up to date with the latest Australian Cell Cycle news.

Please feel free to become a member of the Australian Cell Cycle Community by registering here.

Looking forward to seeing you all again at #ACCM16!

Andrew…

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Last Chance #ACCM15 Poster & Registration Deadline Closing Soon!

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Originally posted on The Australian Cell Cycle Community:

Don’t miss out on what is set to be a fantastic meeting, with a stellar line up of International and Australian scientists. Be quick as there are limited places, and only a few spots left.
The Poster Abstracts Deadline is Friday 23rd of January
Registration closes on Friday 13th of February.
To Register please click here.
15th ACCW 2015 FlyerFinal

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