Phase contrast reflectance confocal brain imaging at 1650nm.

Abstract

The imaging depth of microscopy techniques is limited by the ability of light to penetrate biological tissue. Recent research has addressed this limitation by combining a reflectance confocal microscope with the NIR-II (or shortwave infrared) spectrum. This approach offers significant imaging depth, is straightforward in design, and remains cost-effective. However, the imaging system, which relies on intrinsic signals, could benefit from adjustments in its optical design and post-processing methods to differentiate cortical cells, such as neurons and small blood vessels. We implemented a phase contrast detection scheme to a reflectance confocal microscope using NIR-II spectral range as illumination. We analyzed the features retrieved in the images while testing the imaging depth. Moreover, we introduce an acquisition method for distinguishing dynamic signals from the background, allowing the creation of vascular maps similar to those produced by optical coherence tomography. The phase contrast implementation is successful to retrieve deep images in the cortex up to using a cranial window. Vascular maps were retrieved at similar cortical depth and the possibility of combining multiple images can provide a vessel network. Phase contrast reflectance confocal microscopy can improve the outlining of cortical cell bodies. With the presented framework, angiograms can be retrieved from the dynamic signal in the biological tissue. Our work presents an optical implementation and analysis techniques from a former microscope design.