Optical Multilayer Interference Tomography Compatible with Tape-Based Serial SEM for Mesoscale Neuroanatomy

Abstract

Understanding the complexity of animals' behaviors and intellect relies on decoding the underlying structure and wiring diagram of their brains. Various imaging techniques have been developed to tackle this challenge, yet they still have limitations in mapping all cells in a brain region at mesoscale level. Here we propose an optical multilayer interference tomography (OMLIT) method with serial sectioning capability for mesoscale neuroanatomy. With OMLIT, we can distinguish and reconstruct all cell bodies, large branches of dendrites and myelinated axons, and the vasculature in the region of interest, enabling fast mapping of cell density and classification of certain cell types. Furthermore, using the same form of ultrathin sample section library, our method offers seamless compatibility with tape-based serial scanning electron microscopy, providing a correlative approach to reconstruct both mesoscale and microscale brain atlases in the same tissue.

A Convenient All-Cell Optical Imaging Method Compatible with Serial SEM for Brain Mapping

“…The images were normalized, and equivalent areas were selected within both the cell body and surrounding tissue to calculate the average brightness, which was used to determine the contrast [46]. For each OMLIT configuration with specific coating thickness and section thickness, 50 pairs of cells and surrounding tissue areas were evaluated to calculate the average contrast.…”

“…In order to investigate the optimal OMLIT configuration for the best imaging results, we employed a matrix laboratory (MATLAB) program that describes the reflection and interference phenomena within a multilayer structure for the optical model [46], simulating the light intensity reflected from the cell and its surrounding tissue. During the simulation, it was assumed that both the incident and exit media were lossless dielectrics, and that all layers within the multilayer structure exhibited nonmagnetic properties.…”

“…Specifically, two 5-week-old male Shank3B +/− mice were anesthetized using a combination of Zoletil (55 mg/kg) and xylazine (5 mg/kg). They then underwent transcardial perfusion, with an initial infusion of 30 mL of artificial cerebrospinal fluid, followed by an injection of 30 mL of fixative solution to preserve the fine structure of the biological tissue [46]. The brain was carefully removed from the skull, and a cortical tissue block approximately 2 × 2 × 2 mm 3 in size was excised.…”

“…Optical multilayer interference tomography (OMLIT) was recently reported [46] and showed its capability as an imaging technique for generating mesoscale and microscale brain maps within a single sample. However, the method was easily limited by imperfect coating processes, which yielded metal layers with structured and scratched surfaces, causing confusion and contamination in the images of cells.…”

“…Our method combines large-scale tomography with a high-resolution serial imaging method, achieving low-cost and high-throughput mesoscale brain mapping. Compared to current configurations of OMLIT [46], we omit the step of coating on the collection tape to address issues such as uneven surfaces resulting from non-uniform atomic deposition, fractures caused by poor ductility of metal, and peeling and warping due to inadequate bonding of metal coating, as well as problems arising from contamination and scratching of the metal coating surface. The removal of this step resulted in fewer artificial defects as well as reduced costs since there is no need for an expensive coating instrument and metal targets.…”