Professor Jeff Lichtman examines the technique of fluorescence microscopy in terms of its benefits (e.g. exquisite resolution) and its drawbacks (e.g. confined by the wavelength of light).
One of the biggest benefits of fluorescent microscopy comes about by the marriage of something that nature did and something that humans do well. What humans do well we have built microscopes that can see fluorescence very well; What nature has done is it has through the course of evolution, generated animals that have created fluorescent pigments, proteins often, that are very useful in biology and there’s no pigment better then the green fluorescent protein, sometimes called GFP for green fluorescent protein which comes from jellyfish, and this is a protein that allows jellyfish to bioluminesce a green color when they are using a special mechanism inside their own bodies that generates a blue light that activates this green fluorescent protein. This protein can then be, its gene can be taken from the jellyfish and modified slightly and then put into other animals, and into the nervous systems of other animals to allow us to see the structure of the nervous system by virtue of using this fluorescent protein, and this is one of the amazing advances of fluorescence microscopy because it allows one to look into a living nervous system and see cells and parts of cells glowing, as they are doing their work, and that allows us to see things that would not be possible to see other ways. Now there are some drawbacks to fluorescence microscopy as well, the first of which is that microscopy techniques that use light are limited in what they can resolve; although light allows us to see things that are very small there are many aspects of the nervous system that are even smaller then the wavelength of light and are very difficult to resolve with standard fluorescence microscopy techniques and for those kind of things one needs to use electron microscopes which by and large cannot be used in living animals.
The other thing fluorescence microscopy has a hard time showing is the function of the nervous system. It is very good for the structure of the nervous system, but the function of the nervous system requires looking at things over time and the best tool in fluorescence microscopy to do this is to image changes in the level of an ion called calcium inside cells and calcium imaging is used to see when cells are excited and active and when cells are not active. Calcium imaging is the only tool we have in fluorescence microscopy right now that works well for studying the activity of the nervous system. It’s problem is that it is a bit slower than the actual signaling of the nervous system and so one would hope someday that fluorescence microscopy or another technique would give us the ability to image activity in real time essentially as fast as the activity is occurring rather then the slightly slow motion activity we see with calcium imaging which is better then nothing, but it is far from perfect.