Two-Photon Microscopy – Single Beam Methods
Two-photon imaging was developed partly as a response to these challenges (Denk et al., 1990). In this system, fluorophores are excited by absorption of a pair of photons each with wavelength double that of a photon capable of individually exciting a given fluorophore alone. Only at a very small (roughly 1 cubic micron) region surrounding the exact point of focus is light effectively absorbed to fully excite fluorescence. Thus, the non-linear absorption of light naturally provides axial resolution. This therefore limits photobleaching as well as any possible photodamage to points where the signal is acquired from. Two-photon imaging allows high spatial resolution imaging deep into tissues, including in vivo imaging in the mammalian brain (Denk et al., 1994), and also provides a means by which to carry out photochemical manipulations of cells and circuits (Denk, 1994).
Time lapse imaging using two-photon systems has traditionally relied on the same serial scanning as confocal imaging has and therefore suffers from the same time limitations on signal acquisition. While faster scanning regimes (as discussed below) represent a simple and common approach to this problem, such approaches reduce dwell time per pixel and can compromise signal-to-noise ratio. Furthermore, simply increasing excitation power is frequently not an option because above a certain intensity chromophore saturation and biological tissue photodamage occur. Consequently many experimenters use a laser power just below the photodamage and/or chromophore saturation threshold to maximize signal without inducing damage. In some cases this means much of the available laser power is not used, though in deep tissue imaging this is often not the case since more power must be used to overcome scattering.

Pulse splitting
A few methods aimed at improving two-photon imaging utilize on alteration of the excitation beam itself. Of these, the technique of splitting ultrafast pulses in time by increasing repetition frequency (Ji et al., 2008) stands out and allows application of greater total laser power to the sample without oversaturation or photodamage. Pulse splitting utilizes a beam splitter with delaying mirrors to split each laser pulse into two smaller pulses. Each new pulse excites one quarter the fluorescence photons excited by the original pulse (the two-photon fluorescence process is proportional to square of excitation power), but there are twice as many of pulses, so without a change in the laser output power, this system creates half as much total excitation. On the other hand, by increasing the laser power, one can induce more total excitation and greater signal without crossing damage thresholds or saturating chromophores. This is true since one may double laser output to render each new pulse as powerful as the original pulse but there remain twice as many pulses (as long as the interpulse interval is sufficient to allow molecules of fluorophore to physically relax, this has not been a problem in practice, given that fluorescence lifetime of typical dyes is in 0.5–20 ns range). This approach of changing the properties of the laser beam itself may be combined with other methods discussed below.

Enhanced scanning methods
We and others have concentrated on optimizing the scanning aspects of two-photon microscopy. Traditionally “frames” in a time lapse movie were created by scanning the focused laser across the microscopic field in a series of parallel lines. The scanning occurred by control of the angle of the laser path using galvanometer mirrors which pivot about fixed axes. Resonant mirror systems have gained some favor since they allow for very high speed (up to several KHz) scanning in one axis for faster scanning of each line in the image (Tsien, 1995). This technology is based on galvanometer mirrors that resonantly sweep back-and-forth at a fixed frequency when activated and can produce very nice full frame scanning results (Rochefort et al., 2009), though it does not allow for flexibility in how the scanners are used, restricting at least one axis to linear back-and-forth scanning. Another fast scanning method utilizes acousto-optical devices which control the beam path not with mirrors but with crystals whose refractive properties are very quickly altered by varying sound waves passing through them (Goldstein et al., 1990). These devices allow for flexible fast scanning and can be optimized to produce very useful results (Otsu et al., 2008), but have the limitation that only a narrow range of angles can be scanned without degradation of image quality.

Random access scanning
A different strategy is to only scan the points of interest rather than all of the pixels in a frame (Nikolenko et al., 2007). In this method the galvanometer mirror is commanded to jump from one point of interest (i.e., cell body) to another in series until all points of interest are sampled and then repeat the sampling cycle – each cycle representing a “frame”. With relatively small numbers of points of interest this can allow for very fast scans, though the method slows down significantly with larger numbers of points. This method has been optimized in recent work wherein the momentum of the scan mirrors was taken into account such that scanning either occurs with maximal optimized acceleration and deceleration between points (Lillis et al., 2008) or as a single smooth movement at constant speed passing through all of the necessary points of interest without stopping (Gobel and Helmchen, 2007), thereby improving the efficiency of the method yet more. These authors later expanded this method into the third dimension using a Z-scanning objective adaptor (Gobel et al., 2007).