Cells and Worms - 2. The Shirt

Last post I talked about how seeing how many worms overlap if you drop them on a patch of ground, how (somehow) this was vaguely related to my scientific research, and that the simulation of this process even generates quite nice pictures. If you thought that was geeky, then this takes geekyness to a whole new level!

Part of my research has been into the shapes of trypanosome parasites. Trypanosomes that cause disease in people are fairly widely known (you might have heard of sleeping sickness, Chagas disease, or leishmaniasis) but trypanosomes don't just infect people. Trypanosome species have also been found infecting animals from sharks to penguins, crocodiles to elephants. There is even one species named after Steve Irwin (the crocodile hunter) that infects koalas!

A scanning electron microscope image of Trypanosoma brucei, the trypanosome which causes sleeping sickness.

In short, I did some research to test whether there were particular characteristic shapes of trypanosomes (length, width, etc.) that look like they might help the parasite survive in the bloodstream of different host animals. I made a big database of properties of trypanosome shape and, using the scripts I made to draw nicely tesselated trypanosome shapes I talked about in the last post, I put together a compelling summary of just how varied trypanosome shapes from different host species are are:

The science behind this picture suggests some interesting adaptation to help the parasites swim within their host bloodstream, but that's enough about the science. To me this pattern was just begging to be on a shirt, an abstract design with a biological twist!

Spoonflower is a fantastic online service where you can order customised fabric, wallpaper and other prints. So that is exactly what I did, and after some sewing (that I didn't do myself) I am now the proud owner of the world's only 100% scientifically accurate trypanosome shirt, featuring 27 different trypanosome species.

Scientists always say that research can take you down unexpected paths. This path from wriggly worms, through an image generating script, through research into trypanosome shape, to the world's only trypanosome shirt was quite an unexpected one!

Software used:
ImageJ: Automated trypanosome drawing.
Inkscape: Conversion to vector graphics for printing.

Cells and Worms - 1. The Theory

If you scatter 100 worms on a patch of soil 1 meter by 1 meter how many worms will fall on top of another worm? This might seem like a really pointless question, but it is surprisingly relevant to biological research using microscopes. It's also a surprisingly hard question to answer because worms are very wriggly! However, even this dry, theoretical, research problem provides the tools for making fun illustrations...

My work involves a lot of automated image analysis; taking a picture from a microscope and automatically analysing it to extract scientific data. To make sure an automated analysis is reliable you have to think about all the likely problems that might turn up, and with cells and microscopes a common problem is when two cells are lying on top of each other. The problems this causes are easy to imagine; if there are two cells with one nucleus lying on top of each other then it might look like one cell with two nuclei.

For some types of cells it is quite easy to work out how likely two are to touch or lie partly on top of each other when they are scattered randomly over a microscope slide. An example of an easy case is where all cells are circular and the same size; the approximate calculation is quite simple. Unfortunately the cells I work on are more worm-like in shape, about 17 microns long and 2 wide... if you scatter these cells over a slide how many will end up touching?

To work out the answer simulation is vital; the maths is just too complicated to do it analytically. A simulation of worm-like shapes proved to be quite simple:

1. Pick a random starting point, direction and curvature.
2. Start drawing a curved line from that point.
3. Occasionally re-randomise the curvature.
4. Stop once you have reached the length of the cell.
5. Draw the profile of the cell shape along that curve.

Following these simple rules and tweaking the parameters (e.g. the minimum and maximum curvature, frequency of randomising curvature, etc.) gives a simple algorithm for drawing a worm-like shape. With a bit of tweaking it could draw cells that look like trypanosomes. Using this drawing tool it was possible to measure the chance of a cell touching or lying on top of another cell already on the microscope slide. Just repeat the drawing process thousands of times and detect whether the newly drawn cell intersects with any previously drawn ones. Problem solved.

This process gave me the answer I needed, but it also provided a tool for drawing trypanosome-like shapes. Better than that, it was easy to adapt it to make sure no two cells overlapped and they fitted neatly together over the image... And just like that a dry, theoretical, research problem turned into a beautiful image:

This was also easy to adapt to other worm-like shapes, like earthworms:

Software used:

ImageJ

Cheeky

Human cheek cells are a classic subject of school microscopy. It is easy to collect some by gently scraping the inside of your cheek. This is a high resolution phase contrast image of one of my cheek cells, put together using focus stacking of a 4 by 4 montage of 57 focus slices using one of my ImageJ macros. The detail of the nuclear structure, the granular contents of the cytoplasm and the structured surface of the cell really jump out.

This cell is quite large for mammalian cells, about 75 μm across, and around 10 times larger than the single cell Leishmania parasite I currently do much of my research on. If you have sharp eyesight you can even see human cheek cells by eye (although only just) when they are spread on a slide.

Like most mammalian cells, cheek cells are essentially transparent. If you use a microscope in the most basic way, essentially as a giant magnifying glass, shining light straight through the sample towards your eye, you see something like this:

Bright field micrograph of a human cheek cell.

This picture has even had the contrast artificially enhanced. Practically it is tough to even find the cells on the slide and get them in focus!

For many years the best alternative was oblique or dark field microscopy. Here you deliberately avoid shining light straight through the sample, and instead make sure that only light scattered by structures in the sample can collected by the objective lens and get up to your eye.

Dark field micrograph of a human cheek cell.

Images by dark field microscopy can be hard to interpret, and are typically limited to fairly low resolution.

More complex methods based on interference of light travelling through the sample were developed in the 20th Century. These methods, phase contrast and differential interference contrast, were a revolution. They allowed completely new approaches for looking at the biology of cells, particularly live cells and dynamic processes like cell division. They were such a revolution that the inventor of phase contrast microscopy, Frits Zernik, was awarded the Nobel prize in Physics in 1953 for this work.

Phase contrast micrograph of a human cheek cell.

DIC micrograph of a human cheek cell.

It was not until the development of the famous green fluorescence protein, for fluorescence microscopy in live cells in the 1990s, that there was another discovery which improved the capacity for live cell microscopy to the same extent as phase contrast and DIC.

Software used:
ImageJ

Diatomaceous Anaglyphs

I was trying to work out what the least friendly, most jargon rich blog title I could possibly write something interesting about... I think "Diatomaceous Anaglyphs" is a pretty good effort!

Diatomaceous means "of diatoms"; a type of single celled organism which grows beautiful shells made of silica. In this case these are the shells of dead diatoms from millions of years ago which settled out of the sea, made beds of diatom shells then got compressed into a soft rock. This rock is mined and processed for use in various areas of industry and is called diatomaceous earth.

Anaglyphs are the name of the red/blue 3D images you get which can be looked at using some glasses with coloured celophane instead of lenses. With the surge in 3D films at the cinema you can find more and more 3D stuff online using anaglyphs.

When I combined the I made diatomaceous anaglyphs, which are just plain beautiful (you can also view the whole album on my Flickr):

For those of you not geeky enough to have red/blue 3D glasses this is an animated version:

Software used:
ImageJ: Image alignment and processing

Diatomaceous Zoom

[watch in higher quality here]

I posted a video before about the magnification range of a scanning electron microscope... This is the update with a slightly more interesting sample! Last time I was using 10 micron beads to confirm the calibration of the scope, this time it is fossilised diatoms; tiny single celled plankton with beautiful shells.

The video starts at 25x magnification, the whole field of view is about 2mm across.



And it starts zooming in...



And in...



And in...



Until it gets to 200000x magnification, nearly 1000x greater than at the start of the video. Now the whole field of view is only about 2um across. That is 100 times narrower than a human hair.

Then it zooms back out again so you can see what you were looking at!

Software used:
ImageJ - Generating zoom series, converting individual images to a video.

Colouring SEMs

Scanning electron microscopes (SEMs) are the source of some of the most iconic science pictures... The problem is that they only work in black and white.

SEMs don't use light to create the image, instead a beam of electrons is fired at the surface and the ones which bounce back or are reemitted are detected. This gives a (very cool) looking picture that would be impossible to get with light but means that colours aren't detected...

The distinctive look of SEM images is because of the way edges of objects in the image appear; unlike most visible light photos the edges of objects are lighter than the middle. By detecting which way the slopes in the image are facing we can fake different coloured light falling onto the sample, I use a red light from the top, a green light from the bottom left and a blue light from the bottom right. This makes the image really come alive and gives it an even stronger sense of 3D.

Technically this colourisation method is mapping hues to the angle of orientation edges in the micrograph. The saturation of the illumination is based on the roughness of the texture at that point in the image and the value (brightness) is simply copied from the original micrograph.

The ImageJ macro I wrote to do this can be downloaded here.

Software used:
ImageJ

Image credit:
http://commons.wikimedia.org/wiki/File:Coleus_leaf_trichomes_SEM.jpg (public domain)

SEM Zoom!

Scanning electron microscopes have an amazing range of magnifications, from around 20x to 20000x! It is very hard to give a sense of this range of scales, so have a look at this video instead... It starts at 25x, about 6mm across the whole field of view, and zooms in to 12000x, about 12um across the whole field of view. The circular objects are glass beads 10um across, for comparison a red blood cell is around 8um across.

Software used:
ImageJ - video generation from a series of SEM images
FFMpeg - video transcoding

Extended Depth of Field

One of the tricky things with microscopy and macro photography is the depth of field, as you start magnifying a sample you need to collect as much light as possible to generate the image with a sensible exposure time. Unfortunately this requires a large aperture, and this creates a very shallow depth of field...



This micrograph of a diatom clearly shows the problem, it is impossible to get the whole sample in focus in one image. Fortunately there are ways around it; by analysing the image for sharp edges it is possible to find which image is the most in-focus and the whole image can then be reconstructed only using the in-focus patches. This process is called focus stacking and generates an extended depth of field. Good free implementations of focus stacking are hard to come across, so I wrote one; you can download the ImageJ macro here.
Using the same technique on macro photography (processing the red, green and blue channels separately) gives a similarly impressive result. The three starting images:


And the extended depth of field result:
Software used:
Image processing: ImageJ

Webcam Microscopy

Just a very short post sadly, real life is pressing down on me again... But check out my latest Instructable; with a normal webcam and 5 minutes you can make a >100x microscope...

Paper Autofluorescence

Microscopes are fun... Small things are cool! This is paper in all its fibrous glory; illuminated in ultraviolet paper autofluoresces at blue wavelengths and looks amazing! This was created from a 3x3 array of images, see the links below for higher resolutions:
6400x4800 at Wikipedia
Single screen wallpapers at Deviantart
Dual screen wallpapers at Deviantart

Software used:
The GIMP