The benefits, however, more than outweigh the increase in technical requirements:
- One advantage of using low energy photons is that they are less scattered and absorbed by biological tissue, thus can penetrate deeper into the brain.
- Another advantage arises from the fact that two-photon excitation only happens where the photon density is high enough – that is in a tiny volume in the focus of the microscope’s objective lens.
While single (high energy) photons can excite the dye and generate fluorescence at any unspecified point of their trajectories through the tissue, the low energy photons can excite the dye and generate fluorescence exclusively in the focal volume. Therefore, by using pulsed laser light and focusing it by an objective lens one thus can very precisely determine at which spot inside the tissue excitation will happen and from which, thus, a measure of neuronal activation is taken.
Along the same lines, because with two-photon excitation fluorescence is restricted to a tiny volume, much less overall phototoxicity is generated than with single-photon excitation.
Two photon calcium imaging has been developed in anesthetized animals in vivo but is at the verge to be applied successfully in awake animals that have been trained. Important precondition for imaging in trained animals is the development of calcium dyes that are either non-toxic or are genetically programmed and transfected by virus, and thus allow repetitive imaging.
Retina research and wavelengths
Another field where two-photon imaging has proven to be extremely valuable is retina research. With conventional single-photon microscopy, the wavelengths used for fluorescence excitation of currently available dyes range roughly from 350–700 nm. However, these wavelengths are also efficiently absorbed by retinal photopigments and, thus, strongly excite (and even bleach) photoreceptors. As a result, the light used to generate fluorescence also leads to a saturating light response in the photoreceptors and effectively blinds the retina to visual stimulation. One way out of this dilemma is to make use of the fact that two-photon microscopy employs long wavelengths – up into the infrared range (900 nm and longer) – for dye excitation. Because wavelengths > 900 nm are is very inefficiently absorbed by photoreceptors, it is possible to use two-photon imaging with fluorescence-based tools to study retinal neurons and their response to visual stimuli – presented by a separate visible light stimulator – in the functionally intact (isolated) retina.