Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium.
In recent years, fluorescence microscopy based on two-photon excitation has become a popular tool for biological and biomedical imaging. Among its advantages is the enhanced depth penetration permitted by fluorescence excitation with the near-infrared photons, which is particularly attractive for deep-tissue imaging. To fully utilize two-photon fluorescence microscopy as a three-dimensional research technique in biology and medicine, it is important to characterize the two-photon imaging parameters in a turbid medium. We investigated the two-photon point spread functions (PSFs) in a number of scattering samples. Gel samples containing 0.1-microm fluorescent microspheres and Liposyn III were used as phantoms mimicking the turbid environment often found in tissue. A full characterization of the two-photon PSFs of a water and oil immersion objective was completed in samples composed of 0, 0.25, 0.5, 1, and 2% Liposyn III. Our results show that up to depths of about 100 (oil) and 200 microm (water), the presence of scatterers (up to 2% Liposyn III) does not appreciably degrade the PSF widths of the objectives.
Dong
Chen-Yuan
CY
National Taiwan University, Microscopic Biophysics Laboratory, Department of Physics, Taipei 106, Taiwan.
Koenig
Karsten
K
So
Peter
P
D003198:Computer Simulation; D003238:Connective Tissue / Q000502:physiology* / Q000648:ultrastructure*; D005782:Gels; D007090:Image Interpretation, Computer-Assisted / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D008863:Microspheres; D008954:Models, Biological; D009391:Nephelometry and Turbidimetry / Q000295:instrumentation / Q000379:methods*; D010316:Particle Size; D019047:Phantoms, Imaging; D012542:Scattering, Radiation; D012685:Sepharose; D041622:Tomography, Optical / Q000379:methods*
D005782:Gels; D012685:Sepharose