Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull–meninges connections

Abstract

Analysis of entire transparent rodent bodies after clearing could provide holistic biological information in health and disease, but reliable imaging and quantification of fluorescent protein signals deep inside the tissues remained a challenge. Here, we developed vDISCO, a pressure driven, nanobody based whole-body immunolabeling technology to enhance the signal of fluorescent proteins by up to two orders of magnitude. This allowed us to image and quantify subcellular details through bones, skin and highly autofluorescent tissues of intact transparent mice. For the first time, we visualized whole-body neuronal projections in adult mice. We assessed CNS trauma effects in the whole-body and found degeneration of peripheral nerve terminals in the torso. Furthermore, vDISCO revealed short vascular connections between skull marrow and brain meninges, which were filled with immune cells upon stroke. Thus, our new approach enables unbiased comprehensive studies of the interactions between the nervous system and the rest of the body.

Cellular and Molecular Probing of Intact Transparent Human Organs

“…3D-F). To enhance the signal of EGFP in centimeters sized tissue, we used anti-GFP nanobodies conjugated with bright Atto dyes [14]. We demonstrated that the 3D distribution of pancreatic beta cells as single cells or groups of cells within the islets of Langerhans could be readily assessed by our new approach enabling quantification of islet volume and demonstration of Table S2).…”

“…After imaging with light-sheet microscopy, areas of interest from the cleared specimens, such as human brain and human kidney, were dissected and imaged with an inverted laser-scanning confocal microscopy (Zeiss, LSM 880) using Zen 2 software (v.10.0.4.910; Carl Zeiss AG). Before imaging, samples were mounted by placing them onto the glass surface of a 35 mm glass-bottom petri dishes (MatTek, P35G-0-14-C) and adding a few drops of BABB to make sure that the imaging region was immersed in BABB [14]. The imaging was done using a 40x oil-immersion objective lens (Zeiss, ECPlan-NeoFluar × 40/1.30 oil DIC M27, 1.3 NA, WD = 0.21 mm).…”

“…In the last decade, emerging optical tissue clearing methods have enabled fast 3D histology on transparent specimens avoiding major pitfalls of standard histology, especially tissue sectioning [7,8]. Furthermore, new deep tissue labeling methods were developed in combination with clearing methods to better phenotype whole rodent organs and human embryos [9][10][11][12][13][14][15]. Progress in optical tissue clearing firstly allowed clearing of increasingly larger rodent samples (up to whole adult rodent bodies) [16][17][18][19], then adaptation of light-sheet microscopy systems to manage imaging of whole transparent rodent bodies [14,18,20].…”

Use of high-refractive index hydrogels and tissue clearing for large biological sample imaging

“…However, change in size does not result in tissue disruption in the final cleared product and has minimal impact on outcomes 3,14,17 . In fact, increasing tissue volume can be used to increase resolution 18,19 , whereas decreasing the volume can be advantageous for large samples [20][21][22] . In addition to the impact of clearing protocols on tissue size, the chosen method can also alter the mechanical properties of the sample.…”

Alzheimer disease and Apolipoprotein E4: meningeal brain lymphatics point to new clues in pathogenesis

“…These findings suggest that dysfunctional clearance of macromolecules from the brain by the meningeal lymphatic vessels ─ and the recently deciphered molecular anatomical connections between meninges and skull (Cai et al, 2019) ─ will add to the complexity of meningeal architecture and its role in the decline of cognitive function associated with age and neurodegenerative diseases such as Alzheimer's disease (AD) (De Mesquita, Louveau, et al, 2018).…”