Tuesday, 16 July 2013

Micro 3D Scanning - 1 Focal Depth

3D scanning is a very powerful tool, and it's value isn't limited to the objects and scenes you interact with in everyday life. The ability to precisely determine the 3D shape of tiny (even microscopic) objects can also be really useful.

 The 3D reconstructed shape of a tiny (0.8 by 0.3 mm) surface mount resistor on a printed circuit board. This was made using only a microscope; no fancy laser scanning required!

3D scanning through a microscope is a bit different to normal 3D scanning; mostly because when you look down a microscope at an object it looks very different to what you might expect from day-to-day life. The most immediately obvious effect is that out of focus areas are very out of focus, often to the point where you can barely see what is there. This effect comes down to the angle over which light is collected by the lens capturing the image; your eye or a camera lens in everyday life, or an objective lens when using a microscope.

Three images of the surface mount resistor. The three pictures are taken at different focus distances so different parts of the image are clear and others blurred. The blurred parts are very blurred!

In every-day-life when using a camera or your eyes distance from the lens to the object is normally long, it may be several metres or more. As a result the camera/your eye only collects light over a small of angle, often less than one degree. In comparison microscopes collect light from an extremely large range of angles, often up to 45 degrees. The angle must be this large because the objective lens sits so close to the sample. A wider angle of light collection makes out of focus objects appear more blurred. In photography terms the angle of light collection is related to the f-number, and large f-numbers (which have a large angle of light collection) famously have very blurred out of focus portions of the image.

The upshot of this is that in a microscope image the in focus parts of an image are those which lie very near (often within a few micrometers) to the focal plane. It is quite easy to automatically detect in focus parts of an image by using local image contrast (this is actually how autofocus works in many cameras) to map which parts of a microscope image are perfectly in focus.

In this series of images the most in-focus one is image 6 because it has the highest local contrast...

 ... using edge detection to emphasise local contrast in the image really highlights which one is perfectly in focus.

In this series of images the most in-focus one is image 55 instead.

The trick for focus 3D scanning down a microscope is taking the ability to detect which parts of an image are in focus, and using this to reconstruct the 3D shape of the sample. Going to the 3D scan is actually really easy:
  1. Capture a series of images with the focus set to different distances.
  2. Map which parts of each of these images are perfectly in focus.
  3. Translate this back to the focus distance used to capture the image.
This concept is very simple; if you know one part of an object is perfectly in focus when the focus distance is set to 1mm, that means it is positioned exactly 1mm from the lens. If a different part is perfectly in focus when the focus distance is 2mm, then it must be positioned 2mm from the lens. Simple!

It may be a simple idea, but this method gives a high quality 3D reconstruction of the object.

The reconstructed 3D shape of the resistor, using 60 images focused 0.01mm apart, mapped to a depth map image. The lighter bits stick out more from the surface, and the darker bits stick out less.

Using the depth map to reconstruct the resistor reconstructed in full colour in 3D! Pretty cool for something less than 1 mm long...

Does that seem impressive? Then check out the videos:

A video of the original focus series of images captured of the resistor.

The reconstructed 3D shape.

A 3D view of the resistor, fully textured.

This approach is, roughly speaking, how most 3D light microscopy is done in biological and medical research. It is very common practice to capture a focal series like this (often called a "z stack") to get this 3D information from the sample. 3D imaging is most useful in very thick samples where you want to be able to analyse the structure in all three dimensions, an example might be analysing the structure of a tumour. My research on Leishmania parasites inside white blood cells uses this approach a lot too. The scanning confocal fluorescence microscope was actually designed to maximise the value of this 3D effect by not only blurring out of focus parts of the image, but also eliminating the light all together by blocking it from reaching the camera.

Software used:
ImageJ: Image analysis.
Blender: 3D viewing and rendering.

Marista - my second professional font!

It has been over a year since I released my last font. This new one, Marista, has actually been sat on my hard disk for over 9 months so it is about time it saw the light of day! You can take a look at the interactive previews here: http://www.myfonts.com/fonts/zephyris/marista/

Marista is a bit of an unusual design. It is a monospaced font meaning every symbol is equal in width (like found on a typewriter; Courier is the classic example) but unlike most monospaced fonts it is designed to be a stylistic cursive script, within the constraints of the even letter width. It is actually based on a real and popular typewriter font used in the 1960s-70s, and I designed it to try and capture some of the light irregularities which characterise real typewritten fonts rather than their computer equivalents. The name itself is a derived from Maritsa, the name of the major Bulgarian typewriter company; it was a Maritsa typewriter sample which first introduced me to this text style.

It is a very distinctive, yet readable, font and I designed it with the intention to be used in a block of text. Both normal and italic variants are available, though the original typewriter fonts are more similar to the italic. At very large scales the subtle irregularities which make it look authentically typewritten can look awkward, but in a block of text this slight irregularity really captures the typewritten feeling. Try writing your next letter or invitation in it!

Software used:
Inkscape: Outline design
FontForge: Glyph coding and font generation

Wednesday, 3 July 2013

Cell Biology of Infectious Pathogens - Ghana 2013

For the last four years there has been a cell biology workshop in West Africa, organised by Dick McIntosh, an intense two week course aiming to help young African scientists around the master's degree stage of their careers. This course ran again this year, and was the first organised by Kirk Deitsch (malaria expert and a regular from the previous courses) and I was fortunate enough to be invited to teach the trypanosome half of the course. For its fifth incarnation the course returned to a location where it has previously been held, the Department of Biochemistry, Cell and Molecular Biology in the University of Ghana, and was organised with Gordon Awandare.

The focus this year was teaching basic cell biology and the associated lab techniques, emphasising how this helps understand and fight some of the major parasitic diseases in Africa: African trypanosomiasis (sleeping sickness), leishmaniasis and malaria. All three of these diseases impact Ghana and the surrounding countries and these diseases are of enormous interest to students embarking on a scientific career in Africa.

Of the three diseases we were teaching about malaria is by far the most well known, both locally and internationally. It is caused by Plasmodium parasites (which are single cell organisms) which force themselves inside the red cells in the blood to hide from the host immune system. Malaria is often viewed as the iconic neglected tropical disease, however in the last 10 years or so the understanding of the disease and efforts to find a vaccine and new drugs has improved vastly. Unfortunately it is still very common (we had one case in the participants on the course in the two weeks), drug resistance is rising, and it places a huge cost and health burden on the affected countries. It also impacts a huge area; almost all of sub-Saharan Africa is at risk.

Looking at Leishmania. One of the lab practicals was making light microscopy samples from non-human infective Leishmania using Giemsa stain.

Leishmaniasis and trypanosomiasis are caused by two related groups of parasites, Leishmania and trypanosomes (also single cell organisms), and if malaria is a neglected tropical disease the these are severely neglected tropical diseases. The two parasites live in different areas in the host, trypanosomes swim in the blood while Leishmania live inside macrophages, a type of white blood cell that should normally eat and kill parasites. In comparison to malaria fewer drugs are available, the drugs are less effective and several have severe side effects. Even diagnosis is thought to often be inaccurate. The impact of these diseases is less than malaria; human trypanosomiasis is thought to be relatively rate and leishmaniasis is confined to a semi-desert band just to the south of the Sahara. Trypanosomiasis does have a huge economic impact though, as it infects cattle and prevents milk and meat production, and cases of leishmaniasis are probably under-reported.

Staining trypanosomes. One practical was making immunofluorescence samples. In this sample the flagellum of trypanosomes was stained fluorescent green using the antibody L8C4.

So what did we teach?

The teaching was a mixture of lectures, small group discussions, lab practicals and lab demonstrations and we taught for 14 hours a day for 11 days; we could cover a lot of material! All the teaching was focused on linking basic cell biology to parasites and to practical lab techniques. Topics taught included how parasites avoid the host immune system, molecular tools to determine parasite species, light microscopy techniques, using yeast as tool to analyse cell biology of proteins from other species, host cell interaction of parasites, and many more.

Detecting human-infective trypanosomes. This gel of PCR products shows whether the template DNA was from a human-infective or non-human infective subspecies of T. brucei. If there was a DNA product of the correct size (glowing green) then the sample was human-infective.

A great example of how all the teaching tied together was polymerase chain reaction (PCR) to determine species. Human-infective trypanosomes have a single extra gene which lets them resist an innate immune factor in human blood which would otherwise kill them, and I taught about why this is important for understanding the disease and how it was discovered. This gene can be detected by PCR, and this technique is used to tell if a particular trypanosome sample could infect people. We ran a practical actually doing this in the lab.

PCR is a simple, adaptable and easy technique for checking any parasite for a particular species-defining or drug resistance gene, and we also taught how to use online genome sequence data to design PCR assays. We even worked through PCR assay design for many individual participant's personal research projects, really transferring the skills we were teaching to their current research. Finally we looked at papers using PCR techniques to critically analyse the experiment and assay design to help people avoid pitfalls in their own work.

This was a great demonstration of how basic cell biology and lab techniques can have real practical application with medical samples and help with surveillance of a disease. We designed all of the teaching to have this kind of practical application.

7x speed timelapse video of fish melanophores responding to adrenaline.

One practical with massive visual impact was the response of fish melanophores to adrenaline/epinephrine. Fish normally use these cells to change colour in response to stimuli and melanin particles (melanosomes) inside specialised cells (melanophores) run along the microtubules which make up a large portion of the cell cytoskeleton. We used it to demonstrate signalling; adrenaline can be used to stimulate movement of the melanosomes towards the centrosome.

This is really flexible experimental system for demonstrating the functions of the cytoskeleton, motor proteins and signalling pathways because the output (movement of the pigment particles) is so easy to observe with a cheap microscope or even a magnifying glass. This experiment was particularly chosen as it is a useful and accessible teaching tool for cell and molecular biology, and many of the course participants had teaching obligation in addition to their research.

Western blots in Western Africa. 100x timelapse of loading and running a SDS-PAGE gel.

We aimed to cover all the major molecular and cell biology techniques and had practicals doing microscopy, immunofluorescence, growing a microorganism (in this case yeast), PCR, agarose gel electrophoresis SDS-PAGE and Western blotting. The yeast practicals were particularly cool; using genetically modified cell lines the students analysed the function of p53, a transcription factor with a major role in recognising genetic damage and avoiding cancer, and how well it promotes transcription from different promoter sequences. These practicals taught growing yeast, temperature sensitive mutants, several types of reporter proteins in yeast and Western blotting, all concerning a transcription factor with huge clinical relevance in cancer!

Exploring DNA and protein structures through PyMol in a bioinformatics session.

Practicals weren't just limited to lab practicals though. We also ran interactive bioinformatics sessions looking at the kinds of data which are freely available in genome and protein structure databases online. These were also very popular, especially as so much data is available for free online.

All in all the course was a great success. The participants were all extremely enthusiastic, hard working and scarily smart! Feedback so far has also been very positive. I feel that courses like this can have a huge impact on the careers of young African scientists, and I sincerely hope that funding can be secured to continue running this type of course in the future.

You can also read more about this course at the ASCB website.

Software used:
ImageJ: Image processing and timelapse video creation.
Tasker for Android: Timelapse video capture.
Pymol: Protein structure analysis