Project Update Gary Johnston

1. Project Update Gary Johnston Project Update 30th March 2012

2. Overview • Aims – Very brief. • Implementation- Primary focus. • Results – touch on this only. Project Update 30th March 2012

3. Project Aims • Investigate the effects of ionising Radiation on cancer cells. • PC3(Highly metastatic), DU145(moderately metastatic) and PNT2(normal). • AFM response to investigate the mechanical effects. • Confocal images to look at overall actin cytoskeleton morphology. • Find if differences exist, and if so, model them. Project Update 30th March 2012

4. Implementation • Completed Automated Tasks. • Unwanted files - Removing bad AFM data. • Read Ibw files - Converting from Igor binary wave to force-indentation curves. • Elastic modulus – Hertz Eb and Extended moduli Eb,k1,k2 for the force indentation curves. • Contact point - Calculate reliably for curves fitting normal characteristics. • Database Creation - Based on the directory structure and results. • Database Script function – Performs a series of statistical tests in a methodical manner. • The Frontend – A user tool for the database. (Contains lots of statistical outputs and • visualisation methods for the data contained within the database). • Contact point Editor – Murphy’s law - For when automation goes wrong. Non-Automated. Project Update 30th March 2012

5. Unwanted files - Removing AFM data Approximately 40 subdirectories Each containing around 100 files to be selected. So around 20000 files are removed. Only the last files from the number are wanted, the unwanted readings occur when performing the AFM reading and the tip is too far from the cell. These are removed automatically from all the subdirectories. Project Update 30th March 2012

6. Force curves. • Important points. • They vary a lot. Same cell line. • No visible adhesion for all tested cells on • Fibronectin. PNT2, PC3 and DU145. This isn’t • always the case with other cell lines. F o r c e Human hair 40-50 microns wide. The graph to the right is a tenth of this. (FN) Indentation. • Scales for force are sub 20nN. Although this is measured to 20nN here typical scaled used for the data is normally less than half that at a maximum of 1.75mm. This is less than 1/20th of the width of a human hair, and in AFM circles, normal use is just 0.50mm. • Insanity is doing the same thing over and over again but expecting different results, Yet probe the same cell twice and you will get two different results, and if you wait for it to promote a strong stress response it could be quite different or just to be awkward not! • Interesting possibly true fact : Your fingernails grow one nanometer every second! Project Update 30th March 2012

7. Elastic Moduli Hertz Model Extended Hertzian model 𝐹=A0δ4+A1δ3+A2δ2 𝐹=A2δ2 • Linked data • Fitted in matlabby A/b • Rationale : Standard Polyfitrequires • constant. In more recent matlab versions • can be fitted using the curvefit toolbox but • my code also avoids scaling issues with the • matrices due to the scale (10-9)4. 0.5 Eb = Constant for particular cantilever tip.

8. Contact point. • Initially this was done based on the angle of a short length. (Redundant) • Changed to be based on smoothing and symbolic differentiation. • Partial Lin et al. Method. (Partial because the curves don’t adhere) • An ad-hoc method based on deviation from a localised straight line. • A Bayesian change pointmethod. (Rudoyet al. 2010) No method works perfectly all the time, each has weaknesses and strengths. A review of various methods is contained in Lin et al (2007) and for the Bayesian change point. (Rudoy et al. 2010) Care must still be taken and a quick look is still a good idea. Next up an example of why visually checking is advisable. Project Update 30th March 2012

9. The switching regression model : A metropolised Gibbs Rao-Blackwellizedsampler. (Rudoy et al. 2010). • Normally works well in identifying the contact point. • Care though needs to be taken with short start lines. • Takes a very long time. 5 Days pre-optimisation. • Still takes a complete day to do. • Needs some aftercare hence a contact point editor to visually check the points.

10. The importance of the contact point. • At 100nm the difference is over 300%. A 1nm difference is at least1.5%. Project Update 30th March 2012

11. Database Creation • Select the top level directory to create the database from. • (From the user point of view that is it) • The function itself uses most of the previous things I’ve just mentioned. • Remove the extra AFM files stored. (automated) • Read the IBW files (automated) • Work out the contact point by using one of the 4 methods. (automated, but selectable) • Work out the Elastic moduli based on the contact point. (automated) • Convert them to force indentation curves. (automated) • Store the result into an SQL database. (automated) Project Update 30th March 2012

12. Database Frontend Project Update 30th March 2012

13. Standard Graphs available from the frontend.

14. Standard Graphs available from the frontend. Standard Graphs available from the frontend. Andrews plot. Parallel plot. Project Update 30th March 2012

15. Non- Standard Frontend Graphs 3d rotating plots. Force contribution plot. Project Update 30th March 2012

16. Non- Standard Frontend Graphs Method for looking at time. The flock plot looks at the data in a different way by using the different times as a basis and moving between them using the shortest distance.

17. Non- Standard Frontend Graphs • Eb,k1,k2 as relative distances to look for patterns. • We can’t do actual distance (As scales are very different) • We can do ratio distances, to look for patterns. Left at rough stages as no benefit was seen for the current data. Project Update 30th March 2012

18. Script functions – some of the output. Shown below is the total energy. Blue is Eb, which is x2 Green is k2, which is x3 Red is k1, which is x4

19. Contact Point Editor – For when things go wrong! File selected Point Of Contact File selection Fit red Green point in fit for 50nm. File adjuster and saver

20. Problem Results : Weekly variation Issues. • Future Work. • Force Eb to be positive. (Doesn’t make physical sense to really be negative) • Fix Eb to be fitted over 50nm and then adjust k1,k2 accordingly. • Finite Element modelling of the median AFM force response and confocal images.

21. Recap. • Record the AFM data an Place into a database. • Select the top level directory. • Remove the extra AFM files stored. (automated) • Read the IBW files and convert them to force indentation. (automated) • Work out the contact point by using one of the 4 methods. (automated, but selectable) • Work out the Elastic moduli. (automated) • Store the result into a database. (automated) Frontend – To enable a user to select whatever plot and parameters they want to test. Contact point editor – To enable a user to determine if the point is out and to change it. Statistical script function – To automatically generate a host of stats. Project Update 30th March 2012

22. MatlabBits’n’Bobs. • I figured I’d take a little tangent at the end to talk briefly about matlab. • 1) Deployment of a matlab project makes it slower! • 2) http://www.mathworks.co.uk/matlabcentral/. Chances are it’s here already. • 3) Matlab does not contain the Anderson-Darling test for normality. • 4) X or Y axis naming the numbers. Often you don’t want 1,2,3 so use…set(gca,'XTickLabel',xaxis_labels); instead to replace the numbers with cell strings. xaxis_labels = {‘foo1’, ’foo2’, …. ‘foon’}; • 5) Speed-ups. • … There’s lots more but I’ll leave those for another time.