I was once told by a poster judge at a small conference that, “for a project titled SIMPLI, it’s not very simple is it?” as he perused my poster intently with what I noticed was a raised left eyebrow. There were a number of things I wanted to say in response to this comment that may not have done my score any favours. However since there was a £50 Amazon voucher on the line for the winner (and I had been eyeing a food processor for some time on my amazon wish list), I decided to choose a more appropriate response, which was “let me explain…
My PhD involves designing and building a new type of microscope that can be applied to cancer research which has been coined SIMPLI (Single Molecule Programmable Lifetime Spectroscopy). It is designed to illuminate a sample by totally internally reflecting a laser beam off of the boundary of the microscope objective and the surface the sample sits on. This is known as Total Internal Reflection Fluorescence (TIRF) microscopy. This generates an evanescent wave that only extends approximately 100 nanometres (100nm) into the sample. This length is 10000 times smaller than a millimetre.
When studying biological samples under a microscope you usually tag the thing you want to study with a fluorescent molecule. These molecules glow when illuminated with the right wavelength of light. This allows you to see the part of the cell you are interested in and (hopefully) nothing else. When illuminating the sample in a TIRF microscope, your fluorescent molecules have to be on the membrane of the cell otherwise the evanescent illumination will not reach them and you won’t be able to see anything. If you are studying something that is not biological but is still able to fluoresce, such as quantum dots, you just have to make sure the particles are on, or very close to, the sample surface. TIRF microscopes are sensitive enough to even image individual proteins or quantum dots, which typically have dimensions of between 1-10nm. It’s these single molecules that SIMPLI aims to study.
So, SIMPLI can illuminate a sample by totally internally reflecting laser light, whether it’s biological or not, and obtain an image. Unlike the cellular scale images that can be obtained with, for example confocal microscopy, images of single molecules can look very much like a clear night’s sky filled with stars, which in my opinion is just as beautiful. Conceptually speaking, the difference between the applications of TIRF and confocal microscopy here, is like the difference between looking at a book from a distance and picking it up to read it. Confocal microscopy can give you great information about what a cell looks like under certain conditions, but with TIRF microscopy you can begin to see some of the hidden processes that would otherwise be hidden, simply because they are too small.
What SIMPLI does with these images is much more interesting. One of the components of SIMPLI is called a spatial light modulator (SLM). These are devices that essentially allow you to shape the light that either passes through or reflects off its display. In SIMPLI’s case, the SLM used reflects light off of its display. These displays contain specialized pixels which I can control programmatically. This gives me the ability to change the direction in which parts of the image is reflected off of it. It’s vital that I can record the images I obtain from the illuminated sample, so in one direction off of the SLM I have a camera. This camera also acts like a feedback mechanism for the SLM. After some image processing I can find out which pixels on the SLM the light from the single molecules occupy. I can then change the direction of only these pixels on the SLM to reflect the light only from these molecules towards a different detector. This detector allows me to measure a property of the fluorescence called the fluorescence lifetime by counting the photons emitted by that molecule. This property alone can give an amazingly detailed insight into many aspects of the sample, such as the molecules microenvironment, whether the molecules are interacting with other molecules or whether any other chemical reactions are occurring.
So to put it more simply (…no pun intended), under total internal reflection illumination SIMPLI is able to measure the lifetime of single fluorescent molecules by sequentially cutting each molecule out of the image using an SLM. Much of the work I am currently doing involves developing the software that controls the microscope and designing applications specific to the required experiment. In time, hardware improvements will also be made that will improve the speed in which it can operate. With these improvements we then want study molecules that are known to be involved in preventing cells from dying after they have become cancerous, and provide more detailed information about their interactions on the single-molecule level, which could ultimately contribute in the development of more targeted therapeutics.”
Unfortunately, I missed out on winning that amazon voucher this time around, but I still had a smaller win. The height of my judges left eyebrow had reduced to what looked like a much more comfortable position.
Written by Justin Aluko