In vivo label-free confocal imaging of the deep mouse brain with long-wavelength illumination

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 ~1micrometer 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.

Real‐Time In Situ Holographic Optogenetics Confocally Unraveled Sculpting Microscopy

“…HOCUS subsystems can be added to most conventional all‐optical imaging and stimulation systems, and we anticipate it will be routinely used in such experiments. Our optical design was inspired by the back‐scatter descanning arms added to multi‐photon microscopes for observing intrinsic contrast signals from axonal structures, and can be easily modified to also allow their observation.…”

“…Additional improvement can be achieved by removing the background noise arising from residual reflections from the PBS by using a PBS with no residual reflection to unwanted directions, or by adding an additional polarizer to the HOCUS path before the ETL. As well, following Xia et al . the QWP can be replaced by a QWP cover‐slip to further minimize the background reflection signal.…”

“…The optical configuration of our system is presented in Figure b and is based on the concept of confocally descanning the stimulation light's reflection from the tissue, thereby reconstructing its shape and position. Our holographic optogenetics confocally unraveled sculpting (HOCUS) detection module is added onto an existing all‐optical imaging and stimulation system described in detail elsewhere can, in principle, be added to any microscope with 2P optogenetic stimulation capabilities.…”

“…Photons emitted from the sample, due to ballistic reflections from the excitation source or fluorescence, are imaged with a confocal detection system. Previously, this approach has been used for long‐wavelength reflectance imaging, in combination with a fluorescence imaging setup, using the reflections of the excitation light . In our setup, we use reflection of the 2P optogenetic stimulation laser and get additional information about its position, dimensions, and intensity distribution.…”

Uncovering the biology of myelin with optical imaging of the live brain

“…The signal arises specifically from multilayered myelin which provides additional information about myelin compaction which cannot be determined using fluorescence imaging alone. This technique can be used to visualize myelin in the cerebral cortex, spinal cord, and peripheral nerves deep into tissue using various laser wavelengths (Hill et al, ; Hill & Grutzendler, ; Schain et al, ; Xia et al, ). SCoRe requires lower laser intensities compared to confocal or multiphoton fluorescence microscopy.…”