“A two photon microscope has higher sensitivity than a normal confocal microscope, because it uses two photos instead of one!”  Yes, I can bear witness that this phrase has actually been uttered, and it was not by an undergraduate student.

## No exception to the rule

The condensation of various levels of misunderstandings in this statement is phenomenal- one would be hard pressed to find more mistakes in such a short sentence!  Not knowing how and why a microscope does what it does, is not just a gap in general knowledge. It can lead to lost opportunities for data acquisition- or to misinterpretation of available data. Consequently, it’s essential that research scientists involved in microscopy in some way or the other have a clear understanding of some of the principles behind the function of the imaging apparatus they are using. Two-photon microscopy is no exception to this rule.

## Two into one

In 1931, theoretical physicist Maria Goeppert-Mayer (who later received the Nobel Prize) described the two-photon absorption phenomenon in her doctoral dissertation. She described the simultaneous absorption of two photons of identical or different frequencies, resulting in the excitation of a molecule from one state (usually the ground state) to a higher energy electronic state. Of course, this energy level shift is what happens in one-photon excitation (which is what we use when we excite a fluorophore in normal fluorescence microscopy- see this article for an introduction to fluorescence). However, in the two-photon case, the difference between the lower and upper states of the molecule is equal to the sum of the energies of the two photons, not of a single photon. For this to happen, the absorption of the two photons must be simultaneous.

## Three or more

Two-photon absorption is several orders of magnitude weaker than one-photon absorption, as it depends on the probability of two photons being absorbed at the same time. Unlike one-photon absorption it is a nonlinear process, and the strength of absorption depends on the square of the light intensity. If the excitation is strong enough, three-photon and multi-photon excitation is also possible.

## From theory to image

The effect was demonstrated in practice by Kaiser and Garret in 1963. The prospect of using the phenomenon for microscopy was first introduced in 1978 by Sheppard and Kompfner, and the first practical demonstration took place in 1990, when Denk and colleagues generated two-photon  images of optical sections through chromosomes of live cultured pig kidney cells, stained with a viable DNA stain.

## Intrinsically confocal

This technique is intrinsically confocal: all excitation happens at the focal plane, and all emission comes from the focal plane. More specifically, two-photon fluorescence occurs only at the microscope focal volume- this is the ‘in focus’ volume of a sample which is detected with the confocal microscope. Within the focal volume, the photon density is high- the probability of two-photon excitation occurrence drops exponentially with decreasing intensity outside the focal volume, with 80% of the total excitation happening within this volume. Subsequently, there is no need for pinhole apertures to reject the out-of-focus signal, as such signal practically doesn’t exist.  A typical two-photon excitation point spread function has a full width at half-maximum of 0.3 mm in the radial direction and 0.9 mm in the axial direction when 960 nm excitation light is used, through an objective with a numerical aperture of 1.25.