Two-photon Excitation Microscopy Service

Two-photon excitation microscopy, also known as non-linear multiphoton, is a fluorescence imaging technique that presents great advantages in three-dimensional imaging. It is a powerful tool for high-resolution imaging of intact tissue samples such as brain slices, embryos, whole organs, and live animals (intra-vital imaging).

Two-photon excitation microscopy improves the detection of signal photons per excitation event (e.g., photobleaching and phototoxicity), especially when imaging highly scattering environments in depth. The principle of this technique is that two low-energy photons from the same laser cooperate to generate a higher-energy electronic transition in a fluorescent molecule. The second power of the light intensity determines the absorption rate. In a focused laser, the intensity is highest in the focus vicinity and drops off with distance above and below. As a result, fluorophores are excited almost exclusively in a tiny diffraction-limited focal volume (as small as ∼0.1 μm3). Because the excitation occurs only in the focal volume, all fluorescence photons captured by the microscope objective constitute a valid signal.

The benefits of two-photon excitation microscopy include but not limited to:

  • Excitation restricted to a tiny focal volume in thick samples
  • Formation of a high-resolution 3D model
  • Real-time visualization
  • Less perturbing to live samples

Creative Biostructure provides high-quality two-photon excitation microscopy services to our customers worldwide. With our team of experienced scientists, as well as our well-established high-resolution microscopy platform, we promise to work closely with customers to pursue your goals. Please feel free to contact us for further information.


  1. Benninger R K P, Piston D W. Two-photon excitation microscopy for the study of living cells and tissues. Current Protocols in Cell Biology. 2013, 59(1): 4.11. 1-4.11. 24.
  2. Svoboda K, Yasuda R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron. 2006, 50(6): 823-839.
For research use only. Not intended for diagnostic, therapeutic or any clinical use.

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