Invited review article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy.
We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.
Carriles
Ramón
R
Department of Photonics, Centro de Investigaciones en Optica, León, Mexico.
Schafer
Dawn N
DN
Sheetz
Kraig E
KE
Field
Jeffrey J
JJ
Cisek
Richard
R
Barzda
Virginijus
V
Sylvester
Anne W
AW
Squier
Jeffrey A
JA
D000042:Absorption; D000465:Algorithms; D000818:Animals; D005453:Fluorescence; D006801:Humans; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D008962:Models, Theoretical; D017711:Nonlinear Dynamics; D055095:Optics and Photonics / Q000295:instrumentation / Q000379:methods; D038761:Photobleaching; D017785:Photons; D013997:Time Factors