Single-molecule research

Common analytical methods, such as fluorimetry, detect billions upon billions of molecules simultaneously, and what is observed is the average of all of the states that they are in. However, the average may not give a good picture as to what is happening in the system, i.e. there may be several different populations present, and these cannot be distinguished by looking at the average. An everyday example would be the traffic on a motorway; one could be given information about the average speed that the vehicles are moving at; however, this does not tell the whole story, since we know nothing about slow lorries in the left hand lane (or those in the middle lane attempting to pass vehicles travelling at the same speed), nor do we know anything about the rampaging German-made cars in the outside lane, flashing their lights at any smaller car daring to encroach in their territory. In other words, the average speed gives us some useful information (from which we can make some inferences, such as whether there is a traffic-jam), but it doesn’t allow us to really see what is happening at the single vehicle level.

Through single molecule techniques, we are able to observe individual molecules within a system and then classify them and see how they behave. Or, back to our example, we can look at each vehicle separately, and then depending on what we want to find out, we can group them into different categories and determine what they do; for example, we may wish to see how the proportion of vehicles that are lorries changes over time, and how this affects the overall average speed, or how many Ford Fiestas make it into the outside lane without being bullied out of the way etc. In the past few decades, single molecule research has taken off, and is now ubiquitous in most biological fields.

How to look at single molecules

There are several methods for looking at single molecules; however, my research involves using fluorescence to observe the molecules one at a time. Briefly, biological molecules can be tagged with a dye molecule, which when excited with a particular wavelength of light, can emit light that has a longer wavelength (for example, a dye excited with blue light may emit green light, allowing the molecule to be easily distinguished). The dye molecules are usually viewed on a fluorescence microscope, which essentially focuses laser light down to a tiny volume and then collects the fluorescence. It is possible to label different types of molecules with varying colours, allowing one to differentiate between them.


Super-resolution microscopy

Fluorescence microscopy allows users to dynamically observe phenomena in living cells; however, the wave nature of light and its associated diffraction restrict the resolution of light microscopy: When light of wavelength λ is focused by a lens with a numerical aperture NA, objects that are closer together than d = λ/(2NA) cannot be easily discerned. This is the Abbe diffraction limit, and it has until recently restricted the resolution of fluorescence microscopy to ~250 nm. New methods, collectively grouped under the term super-resolution microscopy, have been developed to overcome this physical limit, gaining over two orders of magnitude in precision, allowing direct observation of processes at spatial scales much more compatible with the regime that biomolecular interactions take place on. Radically, different approaches have so far been proposed, including limiting the illumination of the sample to regions smaller than the diffraction limit (targeted switching and readout) or stochastically separating single fluorophores in time to gain resolution in space (stochastic switching and readout). The latter also described as follows: Single-molecule active control microscopy, or single-molecule localisation microscopy (SMLM) allows imaging of single molecules which cannot only be precisely localised, but also followed through time and quantified.

 

The essential step leading to SMLM was the ability to control the activation of point emitters, and the techniques now distinguish themselves based on how emitters are spatially isolated. The first SMLM techniques were all demonstrated in 2006, and these were photo-activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and fluorescence photo-activation localization microscopy (fPALM). The techniques all work by finding the centre position of the point emitters by fitting to a mathematical function. As the point emitters “blink” on and off, they are each “localised” and a super-resolution image is generated by plotting the co-ordinates of the centre positions (see figure below). 


Alzheimer’s and Parkinson’s Disease

Alzheimer’s and Parkinson’s disease are chronic, long-lasting, and so far, incurable neurodegenerative diseases. There are over 800,000 people living with dementia in the UK today, and this number is expected to rise rapidly as the population ages. Alzheimer’s disease is the most common cause of dementia, and according to figures published by the Alzheimer’s research trust, dementia costs the UK £23 billion per year, the vast majority of which is spent on care, rather than research.

The pathological hallmark of these diseases is the presence of insoluble protein deposits in the brain, which are formed when specific protein molecules mis-fold and aggregate (clump together)  into highly ordered fibrils. In Alzheimer’s disease, the deposits are primarily made up of amyloid-beta, whereas in Parkinson’s, the major protein is alpha-synuclein. Rather than the fibrils themselves being toxic, much evidence now points towards the smaller, soluble oligomers formed in the initial stages of the process, as being the culprit. It is vitally important to characterise these oligomers and determine how they are formed, and more importantly, how they damage neurons.

During the aggregation process, the total concentration of oligomer may be much less than 1% of the total protein concentration. Therefore, in order to look at the oligomers, single molecule techniques must be used. Our methodology involves labelling half a population of the protein of interest (either alpha-synuclein, or amyloid-beta) with one colour, and the other half with another colour, and then observing the solution as the protein aggregates (within a test-tube). At the start, there will mainly be monomer and so each species will have only one colour dye. However, over time, the monomers will clump together to form oligomers, and so the species will be likely to have more than one colour. By looking at the progression of only the two-coloured species over time, it is possible to follow the oligomer formation process. The size/structure of the oligomers can also be determined by looking at the intensities of them. The conditions of the experiment can then be changed (i.e. by adding potential drugs, changing the biological environment etc.) and the effect on the aggregation observed.