Two-photon singlet oxygen microscopy: the challenges of working with single cells

Area of Science:

• Optics and Photonics
• Biophysics
• Chemical Physics

Background:

• Singlet molecular oxygen (O2(a1Δg)) plays crucial roles in photodynamic therapy and cellular signaling.
• Previous methods for generating O2(a1Δg) often lack the spatial precision required for single-cell analysis.
• Nonlinear optical techniques offer potential for high-resolution excitation and detection.

Purpose of the Study:

• To develop and characterize a microscope capable of producing and detecting singlet molecular oxygen (O2(a1Δg)) in a femtoliter focal volume.
• To investigate the advantages of nonlinear two-photon excitation for spatial resolution in O2(a1Δg) generation.
• To assess the feasibility of applying this technique for time-resolved O2(a1Δg) detection at the single-cell level.

Main Methods:

• Utilizing a nonlinear two-photon photosensitized process to generate O2(a1Δg) within a femtoliter focal volume.
• Detecting the 1270 nm phosphorescence of O2(a1Δg) using photon counting.
• Characterizing the microscope's performance with bulk solutions of photosensitizers.
• Comparing nonlinear excitation with linear one-photon excitation in biological cells.

Main Results:

• Demonstrated the production of O2(a1Δg) via nonlinear two-photon excitation in a localized focal volume.
• Confirmed the 1270 nm phosphorescence emission from the generated O2(a1Δg) population.
• Identified distinct advantages in spatial resolution offered by nonlinear excitation compared to linear methods.
• Observed challenges in applying nonlinear optical techniques for O2(a1Δg) generation and detection in single biological cells.

Conclusions:

• The developed two-photon O2(a1Δg) microscope shows promise for high-resolution studies.
• Nonlinear excitation provides superior spatial resolution for O2(a1Δg) generation.
• Further research is needed to overcome challenges in applying this technique for time-resolved single-cell O2(a1Δg) analysis.