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What is 2-photon microscopy, and how is it used to measure population neural activity?

Jan 23, 2016


2-photon microscopy (2PM) is an optical technique for high-resolution, 3-dimensional, functional in vivo-imaging of neural populations. 2PM excites fluorescent dye molecules with targeted laser beams. It is based on the effect of 2-photon absorption. 2-photon absorption is the process by which an electron is excited to a higher energy level by the simultaneous (i.e. within 1 femto-second) absorption of two photons of energy x, rather than one of energy 2x. It was theoretically described by physicist Maria Göppert-Mayer in 1931 but not observed until the 1960s and not used in biology until the late 1980s. The following quote from Winfried Denk, the father of 2PM, is illustrative:

In bright sunlight, a molecule of rhodamine B, an excellent 1- or 2-photon absorber, absorbs a photon through a 1-photon process about once a second, a photon pair by 2-photon absorption every ten million years; no 3-photon absorption is expected throughout the entire age of the universe.

Achieving an appropriately high photon concentration thus requires powerful and pulse-capable lasers. The great advantage of this method is that, since the photon energies are lower, tissue is damaged less and dyes are saved from bleaching; since the wavelengths are longer, greater depths can be reached without scattering. Concretely, 2PM can measure fluorescence changes in cortical layers up to a depth of up to 1mm.

In neuroscience, 2PM is used primarily with fluorescent dyes that bind calcium. The fluorescent dye molecules are excited by 2-photon pulses and the resulting light emissions are measured by a fluorescence microscope. Importantly, the intensity and wavelength of the emitted light (fluorescence) depends on whether or not the dye has bound calcium. Therefore, the excitation-induced change in the emission intensity can be used to measure changes in local calcium concentration. Local calcium concentrations can be measured as a proxy for neural activity because during an action potential, calcium flows into the cell, so changes in calcium concentration can be used as a more-or-less rough proxy for firing. This method works best for single, temporally-isolated spikes or to track the general flow of activity with little regard for precise firing rates or spike times.

In a single frame of population monitoring, the focal point of the laser is rapidly moved from cell to cell (by mirrors, acoustic deflection, or holographic projection), locking fluorescence values over time to points in space and so to neuronal calcium levels. Multiple frames are collected (at frame rates from ~1Hz to 1 kHz, depending on the size of the population under consideration), creating fluorescence time-series. Sophisticated statistical algorithms exist to convert these values into spike trains, with varying success.


  • Denk W and Svoboda K, 1997. Photon upmanship: Why multiphoton imaging is more than a gimmick. Neuron 18, 351-7. (The paper that launched a thousand scopes)

  • Helmchen F, 2009. Two-photon functional imaging of neuronal activity; Chapter in In Vivo Optical Imaging of Brain Function. Boca Ration: CRC Press. (A methods-textbook chapter with a solid review of 2PM in relation to other forms of microscopy. Requires a smidgen more than high-school physics).