ZEISS Lightsheet 7

Mouse kidney cleared with iDISCO protocol and imaged in ethyl cinnamate with ZEISS Lightsheet 7 detection optics 5× / 0.16 foc and Clr 20× / 1.0 nd = 1.53 (insert). The mouse was perfused with DyLight 594 conjugated tomato lectin to visualize vasculature and glomeruli (red). In green: auto-fluorescence to visualize tissue anatomy. 3D whole organ imaging and computational image analysis of glomerular size and number helps to gain a better understanding of the mechanisms of diverse kidney diseases, e.g. diabetic nephropathy. Processed with arivis Vision4D^® on ACQUIFER HIVE.

3D Data set of a P10 mouse trachea displaying the anatomical organization of mechanosensory nerve fibers. Staining: DAPI, Collagen IV (Alexa 488 antibody), sensorial fibers (reporter strain expressing tdTomato, Alexa 555 antibody), neurofilament protein NF200 (myelinated s nerve fibers, Alexa 647 antibody).

The sample was cleared in PEGASOS (Jing et al:, 2018, Cell Research) imaged in BB-PEG at RI of 1.54 with 5× / 0.16 foc detection optics and Cl r 20× / 1.0 nd = 1.53 respectively. 5× magnification data set: pixel scaling 0.61 × 0.61 × 1.63 micron, 3×3 tiles, Zoom 1.5×, 1230 z-sections, volume 2.57 × 2.58 × 2 mm 20× magnification data set: pixel scaling 0.23 μm × 0.23 × 0.58 micron, 1×5 tiles, Zoom 1.0×, 4206 z-sections, volume 2.0 × 0.45 × 1.82 mm.

Salamanders have the remarkable capability to regenerate their limbs and spinal cords. Molecular genetics tools allow to identify the stem cells responsible for this complex regeneration, and the injury-responsive signals that initiate their proliferation.  This axolotl forearm has been cleared in ethyl cinnamate (Masselink, W. et al. Development 146, (2019)) and imaged with 5× / 0.16 foc detection optics at a refractive index of 1.57. The multi-tile data set was aligned, fused and rendered with ZEN imaging software and arivis Vision4D® software on an ACQUIFER HIVE data platform.

Imaging entire cells in the human brain is a task close to impossible, due to the tremendously sophisticated morphology of neurons and their spread throughout the entire organ. Organoids allow the recapitulation of the human brain to a certain extent, including the production of neurons from neuronal stem cell cultures. With ECi clearing, neuronal morphology can be studied from the local to global level, which opens up fascinating possibilities for the study of neuronal morphology in 3D.

35 day old neuronal organoids sparsely labeled with GFP/tdtomato (3% GFP and 3% tdtomato) imaged with Clr 20× / 1.0 nd = 1.53 objective.
Pixel scaling: 222 × 222 × 567 nm.
Image volume: 1.66 × 0.66 × 1.6 mm.

Imaging intact lymphoid organs in 3D allows to analyze and quantify the immune response to viral infection. T cells were transferred into wildtype host mice prior to harvest. The node was cleaned, fixed and cleared using Ce3D (Li et al. PNAS 163, 2017) prior to imaging at RI = 1.49 (ph 7) with a 5× / 0.16 detection optics (volume 2.5 × 2.5 × 1.6 mm). The image shows GFP labelled native CD8+ T cells (yellow), B cell follicles are stained using B220 (cyan) and the CD31 vasculature network (magenta).

A C57 BL6J mouse was perfused with PBS and 4% PFA. The brain was stained perfusing Cell-Tracker™ CM-DiI Dye – a lipid dye to label the vasculature membranes. The sample was cleared using iDISCO+ protocol, equilibrated in ethyl cinnamate as final RIMS. It was then imaged in ethyl cinnamate at RI = 1.565 with detection optics Fluar 2.5× / 0.12 in a Translucence Mesoscale Imaging Chamber.

The high-resolution insert image on the right was acquired with Clr Plan-Neofluar 20× / 1.0 Corr nd = 1.53. Image volume is 13.1 × 13.1 × 6 mm at a pixel resolution of 1.83 × 1.83 × 6.77 μm. It was acquired in about 40 minutes in 4×4 tiles, 866 z-sections. Data volume is 93 GB.  Data processed with ZEN imaging software and arivis Vision4D^®  on an ACQUIFER HIVE data platform.

PV-tdtomato mouse brain was cleared using CLARITY protocol with final imaging done in EasyIndex at a refractive index of RI = 1.46.

Parvalbumin-Cre yielding expression of tdtomato - Parvalbumin is expressed in a population of interneurons throughout the brain and in Purkinje cells in the cerebellum. The whole brain data set was acquired on ZEISS Lightsheet 7 with detection optics 5× / 0.16 foc. Image volume is 11 × 20 × 8.8 mm at a pixel resolution of 0.91 × 0.91 × 5.35 μm (12028 × 22149 × 1621 voxels). It was acquired in 6×10 tiles, 1621 z-sections. Data volume is 1.2 TB (805 GB after stitching). Data was processed with ZEN imaging software and arivis Vision4D^® on an ACQUIFER HIVE data platform.