Abstract
We present volumetric imaging and computational techniques to quantify neuronal and myelin architecture with intrinsic scattering contrast. Using spectral / Fourier domain Optical Coherence Microscopy (OCM) and software focus-tracking we validate imaging of neuronal cytoarchitecture and demonstrate quantification in the rodent cortex in vivo. Additionally, by ex vivo imaging in conjunction with optical clearing techniques, we demonstrate that intrinsic scattering contrast is preserved in the brain, even after sacrifice and fixation. We volumetrically image cytoarchitecture and myeloarchitecture ex vivo across the entire depth of the rodent cortex. Cellular-level imaging up to the working distance of our objective (~3 mm) is demonstrated ex vivo. Architectonic features show the expected laminar characteristics; moreover, changes in contrast after the application of acetic acid suggest that entire neuronal cell bodies are responsible for the "negative contrast" present in the images. Clearing and imaging techniques that preserve tissue architectural integrity have the potential to enable non-invasive studies of the brain during development, disease, and remodeling, even in samples where exogenous labeling is impractical.
Highlights
Label-free volumetric optical microscopy has many advantages over conventional histology and light microscopy, including sample preservation and the capability to image endogenous contrast
When Optical Coherence Microscopy (OCM) cell counts were compared with Two-photon microscopy (TPM) neuron and astrocyte counts, the OCM cell counts agreed better with neuron counts than astrocyte counts (Fig. 2(d))
We attribute the drop in neuron and astrocyte densities measured by TPM beyond 250 μm to vessel shadowing and the limited penetration depth of TPM
Summary
Label-free volumetric optical microscopy has many advantages over conventional histology and light microscopy, including sample preservation and the capability to image endogenous contrast. Several label-free microscopic techniques have been used to image intrinsic contrast in thick brain tissues. Third-harmonic generation [3] and coherent anti-Stokes Raman scattering microscopy [4] have been demonstrated to image myelin based on its structural and biochemical properties, respectively. Third-harmonic generation visualizes neuronal cell bodies due to lack of structural phase matching [5]. Elastic backscattering techniques such as Optical Coherence Microscopy (OCM) and confocal reflectance microscopy provide contrast comparable to third-harmonic generation in brain tissue, depicting both neuronal cell bodies and myelinated axons [6,7,8,9]. The birefringent properties of the myelin sheath can be interrogated with polarization-sensitive imaging to provide additional contrast [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
