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Vanderbilt Biophotonics Center

Vanderbilt Biophotonics Center
  2. Research
  3. Optical Imaging
  4. Multiphoton Microscopy

Multiphoton Microscopy

Multiphoton Microscopy

Multiphoton TissueMultiphoton Microscopy is a three-dimensional fluorescence imaging technique that is useful for investigating optically thick samples such as tissue.  When two photons are simultaneously absorbed by a fluorophore (each with half the energy of the molecule's absorption band), the emission is the same as that in the single-photon case.  The probability of a two-photon absorption event is much smaller than single-photon absorption, so only fluorophores in the focal volume of the laser are excited (this is where photon density is highest).   However, single-photon excitation occurs both above and below the plane of focus.  Thus, multiphoton excitation allows for depth-resolved imaging of fluorophores in biological samples.  The near infrared wavelengths used to excite organic fluorophores in multiphoton imaging are also less damaging and can penetrate deeper into tissue than the visible wavelengths used in single photon imaging.  The difference between single- and multi-photon imaging of tissue is apparent in the image above, where the degraded contrast in the confocal image is due to the collection of light from out-of-focus depths.

References

MC Skala, KM Riching, A Gendron-Fitzpatrick, KW Eliceiri, and N Ramanujam.  In vivo multiphoton microscopy of metabolic oxidation-reduction states and fluorescence lifetimes in normal and pre-cancerous epithelia.  Proceedings of the National Academy of Sciences USA 104:(49); 19494-19499, (2007).

MC Skala, KM Riching, DK Bird, A Gendron-Fitzpatrick, KW Eliceiri, PJ Keely, and N Ramanujam.  In vivo multiphoton fluorescence lifetime imaging of free and protein-bound NADH in normal and pre-cancerous epithelia. Journal of Biomedical Optics, 12:(2); 024014 (2007)

MC Skala, JM Squirrell, KM Vrotsos, JC Eickhoff, A Gendron-Fitzpatrick, KW Eliceiri, and N Ramanujam. Multiphoton microscopy of endogenous fluorescence differentiates normal, pre-cancerous and cancerous squamous epithelial tissues. Cancer Research 65:(4); 1180-1186 (2005).

M Skala and N Ramanujam. Multiphoton Redox Ratio Imaging for Metabolic Monitoring in vivo .Methods Mol Biol 594; 155-162 (2010).