Introducing the indicator dyes into the target neurons or neuron populations is not trivial and strongly depends on factors, such as animal age, tissue type as well as the signals to be measured (e.g. subcellular signals from dendrites of single cells vs. somatic signals from a cell population). As a consequence, labeling methods range from filling of single cells using microelectrodes to bulk loading with membrane permeable dyes. In the latter case, these dyes are injected into neuronal tissue using a micropipette. Once inside the tissue they pass the cell membrane of neurons and are accumulated within the cells. For brain slices or flat tissues, such as retina, electroporation or ballistic (“dyolistic”) methods are used as well. In addition, the full range of genetically encoded calcium-selective biosensors, expressed via a transgenic approach or using viral vectors, is also compatible with two-photon imaging.
To be able to detect calcium signals, the indicator dye needs to excited, which in a standard fluorescence microscope is usually done with visible light. In this case, one single high-energy photon is sufficient to excite one dye molecule and generate fluorescence (single photon excitation). Alternatively, the same dye molecule can also be excited using two lower energy photons (of approx. double the wavelength). Because such two-photon excitation requires the photons to arrive at the dye molecule almost simultaneously – which makes two-photon events much less likely than single-photon events – a high power, pulsed laser source is required.
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.
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.