Dual-axis confocal configuration for depth sensitive fluorescence spectroscopy

The dual-axis confocal (DAC) configuration provides a high axial resolution, long working distance (WD), and large dynamic range. These properties can reveal depth-resolved fluorescence spectra. We present a depth sensitive fluorescence spectroscopy based on the DAC configuration. The system enables high axial resolution of 3.23 µm and a long WD of 3.73 mm compared to that of 4.68 µm and 2.1 mm for comparable single-axis confocal configurations, respectively. Besides, a DAC configuration also offers a superior dynamic range and rejection of out-of-focus scattered light based on the principle of Huygens–Fresnel integrals. Additionally, to locate the target layer, the collection path of the DAC configuration will be used as the other illumination path, forming a dual-axis illumination configuration. These beam paths are used to locate the target layer using a white light imaging system with a commercial low numerical aperture objective. A multi-layer fluorescence phantom of Barrett’s esophagus containing fluorescein isothiocyanate and Alexa Fluor 514 was used to verify the principle of depth-resolved fluorescence spectroscopy. The results show that the DAC configuration can collect fluorescence spectra from microscopic regions with high axial resolution.