Abstract
Optical microscopy is a valuable tool for in vivo monitoring of biological structures and functions because of its non-invasiveness. However, imaging deep into biological tissues is challenging due to the scattering and absorption of light. Previous research has shown that 1300 nm and 1700 nm are the two best wavelength windows for deep brain imaging. Here, we combined long-wavelength illumination of ~1700 nm with reflectance confocal microscopy and achieved an imaging depth of ~1.3 mm with ~1-micrometer spatial resolution in adult mouse brains, which is 3-4 times deeper than that of conventional confocal microscopy using visible wavelength. We showed that the method can be added to any laser-scanning microscopy with simple and low-cost sources and detectors, such as continuous-wave diode lasers and InGaAs photodiodes. The long-wavelength, reflectance confocal imaging we demonstrated is label-free, and requires low illumination power. Furthermore, the imaging system is simple and low-cost, potentially creating new opportunities for biomedical research and clinical applications.
Highlights
Noninvasive imaging deep into scattering biological tissues with high spatial resolution is important for biomedical research and clinical applications
We demonstrate in vivo long wavelength reflectance confocal microscopy (LW-RCM) for mouse brain imaging at more than 1.2-mm depth with illumination wavelength at ~1.7 μm, imaging through the entire neocortex and the external capsule, and reaching the subcortical region of the mouse brain
We demonstrated that long-wavelength reflectance confocal microscopy (LWRCM) is a simple, robust, and low-cost technique for label-free, high-resolution in vivo imaging of the mouse brain down to ~1.3 mm depth
Summary
Noninvasive imaging deep into scattering biological tissues with high spatial resolution is important for biomedical research and clinical applications. We demonstrate in vivo long wavelength reflectance confocal microscopy (LW-RCM) for mouse brain imaging at more than 1.2-mm depth with illumination wavelength at ~1.7 μm, imaging through the entire neocortex and the external capsule, and reaching the subcortical region of the mouse brain. The long wavelength confocal microscopy demonstrated in this paper is simple and robust; and achieves high spatial resolution (~1 μm) at depth comparable to MPM and OCT, which is 3 to 4 times deeper than previously reported imaging depth of reflectance confocal imaging [16]. The demonstrated method is promising for biomedical research and clinical applications where label-free, high spatial resolution imaging deep within tissue is required
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