Real-time luminescence microspectroscopy monitoring of singlet oxygen in individual cells

Abstract

A new setup for direct microspectroscopic monitoring of singlet oxygen (¹O₂) has been developed in our laboratory using a novel near-infrared sensitive InGaAs 2D-array detector. An imaging spectrograph has been inserted in front of the 2D-array detector, which allows us to acquire spectral images where one dimension is spatial and the other is spectral. The work presents a detailed examination of sensitivity and noise characteristics of the setup and its ability to detect ¹O₂. The ¹O₂ phosphorescence-based images and near-infrared luminescence spectral images recorded from single TMPyP-containing fibroblast cells reflecting spectral changes during irradiation are demonstrated. The introduction of spectral images addresses the issue of a potential spectral overlap of ¹O₂ phosphorescence with near-infrared-extended luminescence of the photosensitizer and provides a powerful tool for distinguishing and separating them, which can be applied to any photosensitizer manifesting near-infrared luminescence.