Fig. 1: Introduction to TPE microscopy.
From: More than double the fun with two-photon excitation microscopy
a Hypothetical excitation (solid blue and orange lines) and emission spectra (solid green line) of a fluorophore in one-photon and two-photon excitation (one-photon and two-photon excitation maxima indicated as blue and orange dashed lines, respectively) and typical emission collection (green bars, bottom). b Simplified Jablonski diagram showing the ground state (S0), first excited state (S1), triplet state (T1) and vibrational states (thin lines). Absorption of one or two photons of the right energy excites the molecule and allows for fluorescence and phosphorescence (return to ground state) after energy dissipation through vibrational states. Inter system crossing (ICS) can take the molecule into a long-lived dark-state. From the excited states molecules can react further by photo-bleaching (loss of fluorescence). The bottom panel outlines approximate time-scales for the processes shown in the Jablonski diagram. c Principles of one-photon and two-photon excitation and emission at the focal plane and out-of-focus. One-photon absorption increases linearly with incident laser light whereas two-photon absorption increases non-linearly (quadratically) with incident laser light (left panels). In TPE microscopy, this allows for fluorescence excitation only at the focal spot. In one-photon excitation, this highlights the increased photo-bleaching due to out-of-focus excitation (gray). Further this explains the necessity for an aperture in standard confocal detection (d). d Scheme of a typical detection in one- and two-photon excitation experiments: in one-photon configuration, a pinhole rejects out-of-focus light whereas in TPE microscopy fluorescence only originates in the focal plane, thus additional scattered photons can be collected, increasing detection efficiency. e Scheme of a typical TPE laser scanning microscope. Depicted are beam path, TPE laser properties, non-descanned detection, and digital image reconstruction. For comparison with conventional confocal LSM, the position of the descanned detection (before the galvo mirror) is indicated by a green dashed line (meaning in descanned detection the main dichroic mirror and the detectors would be moved to this position).
