Characterization of multiphoton laser scanning device optical parameters for image restoration

Abstract

Fluorescent nanobeads embedded in agarose and skin biopsies were used to optically characterize spatial and temporal resolution of multiphoton laser scanning devices (MPLSD). Optical sections based on two-photon excited bead fluorescence have been performed at various sample depths. Three-dimensional reconstruction of the image stacks allowed determination of the point spread function. Using calculated point spread functions to apply deconvolution procedures (e.g. Huygens software), the visualization and hence the interpretation of intradermal structures, such as extracellular matrix components in 150 µm tissue depth, was improved.

Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy

“…Possibly due to the great difficulty in inserting fluorescent nanoparticles inside the skin due to the barrier properties of the stratum corneum (SC), the early trial to measure PSF in skin was based on imaging beads placed on the top of skin samples of different thickness. This experimental setup, however, could not yield true PSF in the different skin layers [12]. Later, Guldbrand et al [13,14] measured the PSF of fluorescent nanoparticles in human skin, inserting them inside the skin specimen either by injection [13] or passive diffusion into the tissue after tape-stripping the SC [14].…”