two-photon microscopy

Two-photon microscopy is a way of taking sharp, detailed pictures deep inside living brain tissue, in an animal that is still alive and even awake. It relies on fluorescence: the tissue is tagged with a special dye or protein that glows when light strikes it. Normally one packet of light (one photon) carries enough energy to make the dye glow. Here the trick is the opposite — the microscope uses gentler, lower-energy infrared light, where it takes two photons arriving at the very same instant to add up to enough energy to make the dye glow even once.

Because two photons hitting together is so unlikely, the glow happens only at the single tiny spot where the laser is squeezed to its tightest focus, and nowhere along the path leading to it. (This dependence on light being concentrated, rather than just present, is what is meant by calling the effect nonlinear.) That self-confining glow gives two big rewards. First, the long-wavelength infrared light slips through tissue without scattering as much as ordinary light, so it reaches far below the surface — letting researchers see cells hundreds of micrometers down, where older microscopes only see a blur. Second, since the rest of the sample is never lit up, the delicate living cells suffer far less light damage and can be watched for hours.

These strengths make two-photon microscopy a workhorse for studying the brain at work in a living body — what scientists call in vivo, meaning inside the intact organism rather than in a dish. A common use is watching individual neurons fire in real time by following calcium signals, flashes of brightness from a glow that flares each time a cell becomes active.