Understanding Two Photon Absorption
The higher-energy two-photon absorption (S0 – Sn) transitions observed in some fluorescent proteins14 may be very intense, even exceeding the brightness of their 1PA spectra. However, their exact characteristics are still under investigation.
As the name suggests, two photon absorption is a nonlinear process. It involves the conversion of a virtual state into a real excited one by the simultaneous absorption of two photons.
Background
Two-photon absorption is a nonlinear optical process in which simultaneous absorption of two photons of identical or different wavelengths is used to excite a molecule from one energy state to another, typically a higher energy state. This third-order nonlinearity allows imaging of specimens at depths unobtainable with conventional fluorescence or confocal microscopy.
However, there are limitations to this technique. For example, the two-photon induced photobleaching of some fluorophores reduces the image contrast observed when the procedure is compared to conventional confocal imaging in thick preparations (see e.g. Figure 7).
In order to avoid this degradation, the intensity of the laser pulse must be kept below the threshold of two-photon absorption. This requires the use of a pulsed femtosecond laser source. Furthermore, it is necessary to avoid overlapping the time windows of a two-photon excitation sequence with that of the detection system. The latter is a critical factor in achieving high-quality two-photon microscopy images. Using a non-descanned detector significantly increases the collection efficiency and is essential for maximal deep penetration of the excitation light into living tissue.
Spectral Characteristics
Unlike linear absorption, two photon absorption requires simultaneous absorption of photons at different frequencies in order to excite a molecule from its ground state to a higher energy state. As a result, two-photon spectral characteristics are quite different from one-photon absorption (also called one-photon emission or 1PA).
Specifically, 2PA cross sections depend both on the transition dipole moment squared and the local electric field strength within the protein barrel. This makes them more sensitive to changes in the chromophore environment than 1PA spectra, which only vary slightly when |Dm10| change.
Knowing these differences is important for selecting the right mutant for a given application, as it may require the use of non-linear optics techniques with a different excitation wavelength lex than those used for linear fluorescence measurements (for example, to avoid absorption through a band gap). Moreover, this information also helps researchers correct measured 2PA spectra for variations in experimental conditions and chemometrics. For these reasons, 2PA spectral shape and cross section values are best determined using a reference standard sample prepared under identical excitation conditions.
Excitation Wavelengths
Molecular two-photon absorption (2PA) requires simultaneous absorption of photons of different frequencies in order to promote a molecule from its ground state into its excited electronic state. This nonlinear optical process is measured by its peak brightness (Emax) as a function of the laser’s excitation wavelength, which is referred to as the 2PA cross section. Unlike linear absorption, the peak brightness of a dye’s 2PA spectra depends on the laser’s power, pulse duration and spatial and temporal beam profiles, which are highly variable across the tuning range. This variability makes it difficult to obtain reliable pure molecular 2PA cross section values.
To overcome this, two-photon laser scanning microscopy (PLSM) utilizes a tunable pulsed laser to focus the optical input into a region at the microscope focal point where the light is significantly crowded. Consequently, the probability of a photon in this region interacting with the fluorophore in its lowest energy state is several orders of magnitude larger than that in unfocused light.
Excitation Intensity
A free electron cannot absorb a photon because the conditions for conservation of energy and momentum are not satisfied. Only electrons that are bound to atoms can absorb and emit photons.
Two-photon absorption is a third-order nonlinear optical process. It is typically used with pulsed lasers to achieve high intensity excitation.
In order to maximize the signal-to-noise ratio in FCS, one needs to select a suitable dye with adequate two-photon excitation efficiency. Peak brightness spectra of fluorescent proteins can be used to guide this selection.
In this study, peak brightness spectra were experimentally collected for 37 common organic dyes and genetically encoded Ca2+ indicators using a mode-locked Ti:Sapphire laser. The spectral data show that many dyes exhibit strong two-photon excitation and are capable of achieving high peak brightness under this condition. The data also reveal the optimal excitation wavelengths for these probes. Furthermore, the data provide new insight into the correlation between protein structure and two-photon fluorescence.