Multi-photon microscopy

Under normal excitation, the energy of emitted fluorescent light is always less than that of the incident light and that, for instance, fluorophores that absorb red light do not emit green fluorescence. This holds true for almost all fluorescent applications.
However, rules are there to be broken, so there is a technique that does just this - Multi-Photon excitation. The generation of high energy fluorescence using low energy incident light is achieved by delivering multiple photons of excitation light to the same point in space in a sufficiently short time that the energy effectively is summed and so acts as a higher energy single photon.

The arrival of the first photon causes the electron to become excited, but not sufficiently to reach a more stable state. This excess energy is lost VERY quickly, but if a second photon is delivered rapidly enough, the electron acts as if a high energy single photon has been delivered. Fluorescence emission then acts as normal.

The timescale of electronic excitation is incredibly short. The second photon must arrive within <0.1 fs, so photons must be delivered in rapid succession. However, the power required to deliver such a rapid continuous stream of photons into a particular position in space is enormous, up to about 1 TW/cm2. To put it another way, the total output of 2000 500MW power stations would have to be converted into light and delivered to one square cm. So, in order to image a biological sample without utterly destroying it (and the building it's in!!), pulsed lasers are used. A typical multiphoton excitation laser is the Ti:Sapphire laser, which delivers pulses of photons of about 100 fsec duration separated by about 12 ns, at wavelengths ranging from ~700 - 1000 nm. Thus, although the pulses of light are of extremely high intensity, the average power delivered to the sample is relatively low.

The lasers required for this technique are very specialised and very expensive!. So why use this technique? There are a number of potential advantages of using multiphoton LSCM over conventional techniques. Firstly, high intensity red light scatters less than low intensity blue light, so objects of interest can be imaged in thicker sections of tissue than in conventional LSCM. Thicker slices are likely to be healthier, and the cells being observed are less likely to have been damaged in the preparatrion of the sample. Secondly, the lower overall energy of the excitation light means that less phototoxic damage is caused during viewing and less photobleaching is seen, extending the time that cells can be observed. Thirdly, multiphoton LSCM is inately confocal, i.e. no pinhole is required. Excitation of the fluorophore can only occur where the two photons can interact. Given the quadratic nature of the probability of two photons interacting with the fluorophore in the necessary timescale, excitation only occurs in the focal plane of the objective lens. This provides cleaner images.