Two-photon microscopy basics
In a nut shell, two-photon microscopy allows making high definition images of living cells by using the low-energy infra-red laser light that penetrates deeper into a living animal’s brain, skin or other organs without damaging them. This technology reveals exquisite details by visualizing individual cells (and even sub-cellular organelles) in their natural undisturbed environment. Since two-photon microscopy is non-invasive, it allows re-examining one and the same experimental animal (and precisely the same cellular ensemble) over and over again, days or weeks later. This yields an unmatched wealth of information on disease progression and drug action dynamics and greatly increases statistical significance of the results.
In more technical terms, two-photon microscopy (also known as Multiphoton Excitation Laser Scanning Microscopy) is based on the effect of simultaneous absorption of two infra-red photons by a fluorophore (usually, a fluorescent protein, synthetic dye or a tissue's autofluorescent component). Non-linear summation of two infra-red photons' energy results in excitation of fluorophore, which emits a photon in the visible spectrum. Since the likelihood of coincident absorption of two photons is strictly limited in space, visible photons are emitted only by those fluorescent molecules that are located exactly in the focal point. The emitted photons are then collected by sensitive photomultipliers while the pulsed infra-red laser scans point-by-point through the plane of interest (and plane by plane, stacking two-dimensional XY images into a Z-stack by shifting focus in the vertical direction). Finally, dedicated computer software combines the data into a sharply focused 2D image and, eventually, a 3D volumetric image is reconstructed.