Electrophysiological recording consisits of a series of techniques that allow us to measure and manipulate the electrical properties of neuronal cells. In this way, cells can be given a regular, known stimulus while the response to that stimulus is measured. The electrical currents being measured using these techniques are very small (in the range of pA) and so specialised amplifiers are needed. In this way, the function of single neurons, or whole populations, cell can be studied.
These techniques are extremely good at giving temporal information on neuronal function - i.e. when does a neuron respond to a stimulus, how big is that response. However, it can provide very little or no spatial information, i.e. where does the response occur? It is also very difficult to prise structural information from the data obtained. For this, other techniques are required.
Imaging techniques allow us to view the structure of cells and to see how they change in response to different stimuli. We can also study the localisation and movement of receptor proteins with the use of fluorescent proteins or immunolabels. The basis of all the imaging techniques we use is fluorescence.
Fluorescence is a property of some molecules in which light of one colour is absorbed that results in light of a different colour being emitted. The detection of this fluorescence is the basis of both fluorescence microscopy and LSCM.
Visible light is simply a form of electromagnitic radiation that we can see, unlike radio waves or X-rays. Light energy is 'packaged' into discreet bundles called photons. The energy contained within a single photon determines it's wavelength (i.e. colour). A wide range of molecules absorb light of different colours. The energy contained in these photons is transferred to electrons that become 'exited'. Electrons in this state are unstable and rapidly lose this excess energy. In fluorophres, this energy in lost in two stages - small amounts are lost first, then a large amount, resulting in the emission of a photon of light. The energy of the emitted photon is smaller than the absorbed photon. Because the photonic energy is related to the wavelength there is a difference in the colour light that is absorbed to that which is emitted. Thus, fluorophores that absorb blue light (short wavelength - high energy) usually emit green fluorescent light (longer wavlength - lower energy) and those that absorb green light usually emit red light (even longer wavelength - even lower energy). Fluorophores that absorb red light do not emit green
