Two photon excitation

Two photon excitation can have advantages for imaging thicker samples. Rather than exciting the fluorophore with one photon, the excitation is produced by two lower energy photons. Only in a specific region is the excitation light focused with sufficient intensity to cause fluorescence.

duke lmcf 1 photon vs 2 photon excitation

Two photon excitation has a number of advantages:

  • The longer wavelength excitation penetrates further into the sample
  • Sometimes this means of excitation is less damaging
  • Photobleaching or uncaging is possible with fine z-axis resolution
  • Some fluorophores are only efficiently excited with 2 photon

A two photon excitation microscope doesn't need to use a pinhole. Because the sample is only excited in a very specific plane we know where the light has come from so all the light can be imaged and excellent z-axis resolution still be obtained.

1photon vs 2 photon confocal systems

The other big difference between our 2 photon and single photon systems is the laser used: 2 photon excitation requires a pulsed laser to enable excitation via the two step process (the half-way state is very short lived so the second photon needs to act very soon after the first). We have a femtosecond pulsed laser for doing this, and it cost a frightening amount of money.