Martin Krzywinski / Genome Sciences Center / mkweb.bcgsc.ca Martin Krzywinski / Genome Sciences Center / mkweb.bcgsc.ca - contact me Martin Krzywinski / Genome Sciences Center / mkweb.bcgsc.ca on Twitter Martin Krzywinski / Genome Sciences Center / mkweb.bcgsc.ca - Lumondo Photography
Poetry is just the evidence of life. If your life is burning well, poetry is just the ashLeonard Cohenwatch

mouse veins: beautiful



Circos at British Library Beautiful Science exhibit—Feb 20–May 26


visualization + design

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Cover image accompanying our article on mouse vasculature development. Biology turns astrophysical. PNAS 1 May 2012; 109 (18) (zoom, PNAS)

Creating the PNAS Cover

One of my goals in life, which I can now say has been accomplished, is to make biology look like astrophysics. Call it my love for the Torino Impact Hazard Scale.

Recently, I was given an opportunity to attend to this (admittedly vague) goal when Linda Chang from Aly Karsan's group approached me with some microscopy photos of mouse veins. I was asked to do "something" with these images for a cover submission to accompany the manuscript.

When people see my covers, sometimes they ask "How did you do that?" Ok, actually they never ask this. But being a scientist, I'm trained me to produce answers in anticipation of such questions. So, below, I show you how the image was constructed.

The image was published on the cover of PNAS (PNAS 1 May 2012; 109 (18))

Tools

Photoshop CS5, Nik Color Efex Pro 4, Alien Skin Bokeh 2 and a cup of coffee from a Rancilio Silvia.

source images

Below are a few of the images I had the option to work with. These are mouse embryonic blood vessels, with a carotid artery shown in the foreground with endothelial cells in green, vascular smooth muscle cells in red and the nuclei in blue.

Of course, as soon as I saw the images, I realized that there was very little that I needed to do to trigger the viewer's imagination. These photos were great!

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Mouse carotid arteries. (zoom)

memories of star trek

Immediately I thought of two episodes of Star Trek (original series): Doomsday Machine and the Immunity Syndrome, as well as of images from the Hubble Telescope.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Enterprise is about to be consumed by a horror tube: a planet killer! (The Doomsday Machine)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Enterprise heads into a giant amoeba. Who eats whom? I'll let you guess. (The Immunity Syndrome)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Orion nebula (M42) as seen by the Hubble telescope. (zoom)

I though it would be pretty easy to make the artery images look all-outer-spacey. They already looked it.

centerpiece image

And then I saw the image below.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
A particularly spectacular image of a mouse carotid artery. I'm thinking 10 on the Torino scale. (zoom)

constructing the cover

background

The background was created from the two images shown here. The second image was sampled three times, at different rotations.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Images used for background. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Images used for background. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Layer composition for background elements. (zoom)

The channel mixer was used to remove the green channel and leave only red and blue.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Background elements for PNAS cover image. (zoom)

middle ground

The next layer was composed of what looked like ribbons of blue gas. This was created by sampling the oval shapes from the source images. Here the red channel was a great source for cloud shapes, and this was the only channel that was kept. The hue was shifted to blue and a curve adjustment was applied to increase the contrast.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
First set of middle ground elements, before adjustments. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
First set of middle ground elements, after channel adjustments. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Second set of middle ground elements. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Layer composition for middle ground elements. (zoom)

When the foreground and middle ground elements were combined, the result was already 40 parsecs away.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Background and foreground elements for PNAS cover image. (zoom)

foreground

The foreground was created from the spectacular comet-like image of a mouse artery. Very little had to be done to make this element look good. It already looked good.

I applied a little blur using Alien Skin's Bokeh 2 to narrow the apparent depth of field, masked out elements at the bottom of the image and removed some of the green channel. The entire blue channel was removed altogether (this gave the tail of the comet a mottled, flame-like appearance).

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Foreground element, before adjustments. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Foreground element, after channel adjustments. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Layer composition for foreground element. (zoom)

post processing

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Initial composition of background, middle ground and foreground elements. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
40% localized application of Nik's Tonal Contrast (Color Efex 4 plugin) to increase structure in red channel. (zoom)
Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
50% blend with Nik's Pro Contrast (Color Efex 4 plugin). (zoom)

And here we have the final image.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Final PNAS cover. Spacey! (zoom)

news + thoughts

Analysis of Variance (ANOVA) and Blocking—Accounting for Variability in Multi-factor Experiments

Mon 07-07-2014

Our 10th Points of Significance column! Continuing with our previous discussion about comparative experiments, we introduce ANOVA and blocking. Although this column appears to introduce two new concepts (ANOVA and blocking), you've seen both before, though under a different guise.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Nature Methods Points of Significance column: Analysis of variance (ANOVA) and blocking. (read)

If you know the t-test you've already applied analysis of variance (ANOVA), though you probably didn't realize it. In ANOVA we ask whether the variation within our samples is compatible with the variation between our samples (sample means). If the samples don't all have the same mean then we expect the latter to be larger. The ANOVA test statistic (F) assigns significance to the ratio of these two quantities. When we only have two-samples and apply the t-test, t2 = F.

ANOVA naturally incorporates and partitions sources of variation—the effects of variables on the system are determined based on the amount of variation they contribute to the total variation in the data. If this contribution is large, we say that the variation can be "explained" by the variable and infer an effect.

We discuss how data collection can be organized using a randomized complete block design to account for sources of uncertainty in the experiment. This process is called blocking because we are blocking the variation from a known source of uncertainty from interfering with our measurements. You've already seen blocking in the paired t-test example, in which the subject (or experimental unit) was the block.

We've worked hard to bring you 20 pages of statistics primers (though it feels more like 200!). The column is taking a month off in August, as we shrink our error bars.

Krzywinski, M. & Altman, N. (2014) Points of Significance: Analysis of Variance (ANOVA) and Blocking Nature Methods 11:699-700.

Background reading

Krzywinski, M. & Altman, N. (2014) Points of Significance: Designing Comparative Experiments Nature Methods 11:597-598.

Krzywinski, M. & Altman, N. (2014) Points of Significance: Comparing Samples — Part I — t-tests Nature Methods 11:215-216.

Krzywinski, M. & Altman, N. (2013) Points of Significance: Significance, P values and t-tests Nature Methods 10:1041-1042.

...more about the Points of Significance column

Designing Experiments—Coping with Biological and Experimental Variation

Thu 29-05-2014

This month, Points of Significance begins a series of articles about experimental design. We start by returning to the two-sample and paired t-tests for a discussion of biological and experimental variability.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Nature Methods Points of Significance column: Designing Comparative Experiments. (read)

We introduce the concept of blocking using the paired t-test as an example and show how biological and experimental variability can be related using the correlation coefficient, ρ, and how its value imapacts the relative performance of the paired and two-sample t-tests.

We also emphasize that when reporting data analyzed with the paired t-test, differences in sample means (and their associated 95% CI error bars) should be shown—not the original samples—because the correlation in the samples (and its benefits) cannot be gleaned directly from the sample data.

Krzywinski, M. & Altman, N. (2014) Points of Significance: Designing Comparative Experiments Nature Methods 11:597-598.

Background reading

Krzywinski, M. & Altman, N. (2014) Points of Significance: Comparing Samples — Part I — t-tests Nature Methods 11:215-216.

Krzywinski, M. & Altman, N. (2013) Points of Significance: Significance, P values and t-tests Nature Methods 10:1041-1042.

Have skew, will test

Wed 28-05-2014

Our May Points of Significance Nature Methods column jumps straight into dealing with skewed data with Non Parametric Tests.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Nature Methods Points of Significance column: Non Parametric Testing. (read)

We introduce non-parametric tests and simulate data scenarios to compare their performance to the t-test. You might be surprised—the t-test is extraordinarily robust to distribution shape, as we've discussed before. When data is highly skewed, non-parametric tests perform better and with higher power. However, if sample sizes are small they are limited to a small number of possible P values, of which none may be less than 0.05!

Krzywinski, M. & Altman, N. (2014) Points of Significance: Non Parametric Testing Nature Methods 11:467-468.

Background reading

Krzywinski, M. & Altman, N. (2014) Points of Significance: Comparing Samples — Part I — t-tests Nature Methods 11:215-216.

Krzywinski, M. & Altman, N. (2013) Points of Significance: Significance, P values and t-tests Nature Methods 10:1041-1042.

Mind your p's and q's

Sat 29-03-2014

In the April Points of Significance Nature Methods column, we continue our and consider what happens when we run a large number of tests.

Martin Krzywinski @MKrzywinski mkweb.bcgsc.ca
Nature Methods Points of Significance column: Comparing Samples — Part II — Multiple Testing. (read)

Observing statistically rare test outcomes is expected if we run enough tests. These are statistically, not biologically, significant. For example, if we run N tests, the smallest P value that we have a 50% chance of observing is 1–exp(–ln2/N). For N = 10k this P value is Pk=10kln2 (e.g. for 104=10,000 tests, P4=6.9×10–5).

We discuss common correction schemes such as Bonferroni, Holm, Benjamini & Hochberg and Storey's q and show how they impact the false positive rate (FPR), false discovery rate (FDR) and power of a batch of tests.

Krzywinski, M. & Altman, N. (2014) Points of Significance: Comparing Samples — Part II — Multiple Testing Nature Methods 11:215-216.

Krzywinski, M. & Altman, N. (2014) Points of Significance: Comparing Samples — Part I — t-tests Nature Methods 11:215-216.

Krzywinski, M. & Altman, N. (2013) Points of Significance: Significance, P values and t-tests Nature Methods 10:1041-1042.