Okey, first blog of the year and I must admit it has been a while since I made another blog, however I deem it’s time to get back into some blogging action!
Last blog I got a question about why I would be working with fluorescent proteints and an even better question what are they used for? As I explained in my comment in my first blog I will try to give you some more background information without getting to technical about the “whys” and “hows” of fluorescent proteins used in this thesis.
Let’s start of with a question. What is purpose of a microscope?
Plain and simple: to make the invisible, visible right? To magnify, measure and to describe samples in detail.
What if I told you that we can only make the invisible, visible untill a certain point. What I mean by this is that the most fundamental limitation of microscopy resolution is light. Yes, light, the light we wake up to, the lightbulb we put on in the evening and countless other examples. The one thing that all these sources of light have in common is they are bound by the laws of optics. The wave-like character means it can only be focused to a point with dimensions of the same order of magnitude as its wavelength. In common language: on the detector of any microscope, a very small dot will appear as a spot of a few hundreds of nanometers, limiting further detail (see figure below). This concept is known as point-spreading. (If you’re interested in microscopy I suggest you can find some basic information regarding microscopy at the following site: http://zeiss-campus.magnet.fsu.edu/articles/basics/index.html).
Point spreading is unavoidable and while many attempts have been made to negate its effect, the ‘secret’ to breaking this limit is not and try to prevent or avoid point spreading but to resort to the properties of ,yes you guessed it, fluorescent proteins.
Fluorescent proteins or fluorophores have generally “2 states”. The excited state by absorbing light or electromagnetic radiation (on state) and the “off-state” when they are not excited. When the electrons are excited they will fall back to their ground state emitting a photon and this process is called fluorescence.
Based on this process, the general idea of this superresolution microscopy is to quickly activate and deactivate them (on and off blinking) capturing up to 100,000 images in the meantime, with the intention of capturing only a single “blink” per position in each image. The random blinking on-off in this massive number of images captured allows for incredibly precise localisation of single molecules and increasing the overal resolution. An example is the figure below.
This is superresolution microscopy (with fluorophores) in a nutshell.
I hope this clarified a little bit what the purpose is of making these fluoroscent proteins in my thesis. If you want any more information or got any questions regarding this topic, feel free to leave a comment! Allright I think that’s enough blogging action for today, see you readers next week!