Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium

Area of Science:

• Biomedical Optics
• Microscopy
• Biophotonics

Background:

• Two-photon fluorescence microscopy (TPFM) is valuable for deep-tissue biological and biomedical imaging due to near-infrared photon penetration.
• Characterizing TPFM parameters in turbid media is crucial for its effective application as a 3D research technique.
• Scattering in biological tissues can degrade image quality, necessitating an understanding of its impact on TPFM.

Purpose of the Study:

• To investigate the impact of scattering on two-photon point spread functions (PSFs) in various turbid media.
• To characterize the performance of TPFM objectives in mimicking tissue environments.
• To determine the depth limits at which scattering significantly affects TPFM imaging.

Main Methods:

• Utilized gel phantoms with fluorescent microspheres and Liposyn III to simulate turbid biological tissue.
• Performed a comprehensive characterization of two-photon PSFs using water and oil immersion objectives.
• Evaluated PSFs in samples with varying concentrations of Liposyn III (0% to 2%).

Main Results:

• The presence of scatterers (up to 2% Liposyn III) did not significantly degrade the PSF widths of the objectives.
• PSF width remained largely unaffected up to imaging depths of approximately 100 micrometers for oil immersion objectives.
• PSF width remained largely unaffected up to imaging depths of approximately 200 micrometers for water immersion objectives.

Conclusions:

• Two-photon microscopy maintains good imaging resolution in moderately scattering media within specific depth ranges.
• The findings support the utility of TPFM for deep-tissue imaging applications in biology and medicine.
• Further studies can explore higher scattering concentrations and different tissue models.