High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging

Abstract

Understanding the structure-function relationships between diverse cell types in a complex organ environment requires detailed in situ reconstruction of cell-associated molecular properties in the context of 3D, macro-scale tissue architecture. We recently developed clearing-enhanced 3D (Ce3D), a simple and effective method for tissue clearing that achieves excellent transparency; preserves cell morphology, tissue architecture, and reporter molecule fluorescence; and is robustly compatible with direct immunolabeling. These characteristics permit high-quality multiplex fluorescence microscopy of large tissue volumes, as well as image analysis using advanced platforms such as volumetric histocytometry, collectively allowing quantitative characterization of cells with respect to their spatial positioning within tissues on the basis of phenotypic and functional markers. Ce3D clearing is fast, achieving robust transparency of most tissues within 24 h, albeit still necessitating additional time for staining, imaging, and analysis (1-2 weeks). Here, we provide detailed procedures for tissue clearing using Ce3D, including optimized workflows for tissue processing and staining, as well as treatment of difficult-to-clear organs such as the brain. We also describe a new procedure for RNA detection in Ce3D-treated tissues, as well as provide additional details for the volumetric histocytometry data processing steps. Finally, we discuss limitations and work-around strategies for improving antibody-based tissue immunolabeling, fluorophore multiplexing, large-volume microscopy, and computational analysis of large image datasets. Together, these detailed procedures and solutions for high-resolution volumetric microscopy with Ce3D enable quantitative visualization of cells and tissues at a high level of detail, allowing exploration of cellular spatial relationships in a variety of tissue settings.

Figure 1.. The flowchart of Ce3D clearing, imaging and analysis

The flowchart demonstrates the individual steps (brackets) and their approximate timing requirements for the Ce3D clearing, imaging and Histocytometry analysis protocol, as denoted by numbers in parentheses and the time guidelines below. Tissues are first perfused [Step 1(A)i] and fixed by either PLP or BD fixative at 4°C [Steps 1(A)ii-v]. Tissues are next blocked and permeabilized at 37°C to enhance antibody penetration and minimize nonspecific antibody binding [Step 1(A)vi]. Tissues are then stained with directly conjugated antibodies with the total incubation time depending on the size of the tissue [Steps 1(A)vii-x]. The Ce3D solution is made during the blocking/staining steps and the RI is checked at room temperature (RT). Tissues are next cleared at RT [Step 1(A)xi-xiiii] and imaged using confocal [Step 2], two-photon or light-sheet microscopy. After image acquisition, spectral unmixing (compensation) is conducted to remove spillover [Steps 3, 4]. Deconvolution is also used to enhance the spatial allocation of imaged signal [Step 5]. Segmentation of individual cells is finally performed [Step 8] to extract data for Cellular gating [Step 12] and 3D Histocytometry [Step 13] for quantitative analysis of cellular position within tissues.

Figure 2.. Clearing, Mounting and Microscope Setup

A) To achieve tissue clearing, the samples are perfused (left vs. middle) and cleared using the Ce3D solution (right). Scale bar − 1 mm. B) Samples are next mounted on either a slide or in a petri dish based on sample volume and the type of utilized objective and microscope. C) Inverted or upright microscope setups can be used for sample imaging.

Figure 3.. Volumetric Ce3D microscopy of diverse organs.

A-B) Small intestine: the spatial relations of lymphatic vessels, nerves and immune cells inside villi and basal part of small intestine were revealed by their corresponding markers: EpCAM (blue), Lyve-1 (yellow), Beta-tubulin III (green), CD3 (magenta), KLRG-1 (green) and CD11c-YFP (yellow). Quality of individual cell imaging is shown in a zoom-in inset. Scale bars in A: main − 200 μm; inset − 40 μm; Scale bars in B: main − 100 μm; inset − 30 μm. C) The tubular and glomerular structures of kidneys were visualized with CD31 (magenta) staining and autofluorescence (green). Scale bar − 400 μm. D) Actin-DsRed and CD11c-YFP were used to visualize kidney tubular structure and the localization of immune cells. Individual cells are shown in a zoom-in inset. Scale bars: main − 200 μm; inset − 50 μm. E) 3D reconstruction of the whole lymph node labeled with B220 (cyan), CD4 (magenta), CD35 (yellow), ERTR-7 (blue) and a genetically-encoded fluorescent protein, CD11c-YFP (green). Scale bar − 300 μm. F) A single virtual z-slice view of the lymph node (from E). Individual cells were revealed in inset 1 (B cell zone) and inset 2 (T cell zone). Scale bars: main − 100 μm; inset − 30 μm. G) Liver macrophages were visualized using a genetically-encoded fluorescent protein reporter, LysM-tdTomato (red), with respect to the vascular bed revealed with Collagen IV (green) staining. Scale bar − 60 μm. H) The 3D organization of ducts in the mammary gland was revealed by GATA3 antibody (red). Immune cells were revealed by genetically-encoded fluorescent proteins CD11c-YFP and CX3CR1-GFP in the same channel (green), while blood vessels were revealed using autofluorescence signal (blue). Scale bars: main − 400 μm; inset − 60 μm. I) The spatial relationships between astrocytes (GFAP, yellow) and blood vessels (CD31, green) were revealed in the 3D brain slice. Scale bar − 100 μm. J) 3mm deep image stack of a lung was acquired by using two-photon microscope and the motCORR dipping objective to reveal the bronchial airways with Cytokeratin (cyan) staining. Scale bar − 300 μm. Individual virtual Z-slices demonstrate the quality of the obtained signal throughout the Z stack. Scale bars − 50 μm in a, b, c, d. All tissues were isolated from mice maintained at an Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at NIAID. All procedures were approved by the NIAID Animal Care and Use Committee (NIH).

Figure 4. 3D in situ hybridization with immunostaining

A) The flowchart of steps involved in 3D in situ hybridization and Ce3D tissue clearing. Step numbers referring to the procedure steps as detailed in Box 1 are indicated in brackets. B) The standard control slide from ACD was used to compare the signal density with (right) and without (left) Ce3D treatment using the probes of three housekeeping gene (EPIB, green; FOLR2A, yellow; Ubiquitin, magenta). Data demonstrate that Ce3D tissue treatment does not lead to noticeable loss of mRNA signal. Scale bar, 5μm. C) Volumetric in situ hybridization for the Ubiquitin mRNA (magenta) was combined with antibody staining to detect lymphatic channels (Lyve-1, yellow) and intestinal epithelium (EpCAM, blue). Total volumetric reconstruction of the imaged slice (Z depth = 350μm, scale bar − 50μm, left), as well as a single virtual Z-slice view (right, scale bar = 15 μm) is shown. D) Volumetric imaging of a LN stained by the combination of in situ hybridization (CD8, yellow) and immunofluorescence (CD8 and B220, magenta and green, respectively). Scale bars: main − 15 μm; inset − 5 μm. All tissues were isolated from mice maintained at an Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at NIAID. All procedures were approved by the NIAID Animal Care and Use Committee (NIH).

Figure 5.. Volumetric Histocytometry example workflow

250um fixed sections of steady-state murine lymph nodes were stained with antibodies against various lymphocyte populations (CD3, CD4, CD8, B220, CD45), Ce3D cleared, imaged with confocal microscopy (40x objective), deconvolved, and corrected for depth attenuation. A) Volumetric reconstruction of the 3D image in Imaris. Scale bar – 200 μm. B) Image processing steps for cell segmentation. All cell membrane signals were normalized with respect to each other and summed together using Imaris Channel Arithmetics XTension. The Sum channel was next inverted, smoothed and corrected for contrast and gamma to enhance separation between cells. An optional enhancement step (bottom panels) was also performed by generating 2D skeletons on the Sum channel in ImageJ. The skeletons were next used for Boolean gating of the Inverse Sum channel outside of the skeleton signal using Channel Arithmetics. C) The enhanced Inverse Sum channel was used to generate cell surface objects in Imaris. D) Data on all cell objects was exported into Excel, concatenated into a single CSV file, and imported into FlowJo for hierarchical population gating and analysis (top panels). Positional visualization of the gated cells, presented as density distributions, was also performed in FlowJo (bottom panels). E) Cell object gating and visualization was also performed in Imaris using Object Filters and gating thresholds from (D). All tissues were isolated from mice maintained at an Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at NIAID. All procedures were approved by the NIAID Animal Care and Use Committee (NIH).