light-sheet fluorescence microscope (lsfm  (Evident Corporation)

Journal: Nature

Article Title: Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage

doi: 10.1038/s41586-023-06899-4

Figure Lengend Snippet: a , Diagram of a sagittal view of dural lymphatics designated upstream lymphatics region #1. b , Light-sheet fluorescence microscopic images showing serial optical sections (numbered) of upstream lymphatics region #1 (white arrowheads) originating near the Prox1 + pituitary gland. These lymphatics course along the cavernous sinus (outlined with blue-dotted lines) and beneath cranial nerve (CN) V (outlined with yellow-dotted lines) en route to the nasopharyngeal lymphatic plexus (NPLP). Anatomical positions are indicated in the lower right corner. Scale bar, 1 mm. Similar findings were obtained from n = 5 mice in three independent experiments. Ant., anterior; Post., posterior; Med., medial; Lat., lateral anatomical position. c , Light-sheet fluorescence microscopic image showing LYVE1-stained (red), blunt-ended Prox1 + /LYVE1 + dural lymphatics (green nuclei) in an enlargement of the region in b section 1 marked by white-dotted line box ( c ) near the Prox1 + pituitary gland (bright green). Scale bar, 100 μm. d , Fluorescence microscopic images of section showing Prox1 + upstream lymphatics region #1 containing FluoSpheres (red arrowheads) along the cavernous sinus. The region of the white dashed-lined box is enlarged in the right panel. Anatomical positions are indicated in the lower left corner. Scale bar, 200 μm. Similar findings were obtained from n = 3 mice in two independent experiments. Ant., anterior; Post., posterior; Med., medial; Lat., lateral anatomical position.

Article Snippet: The samples that underwent tissue clearing and decalcification were imaged using a light-sheet fluorescence microscope (LSFM, Carl Zeiss) with an EC Plan-Neofluar ×5/0.16 lens.

Techniques: Fluorescence, Staining

Journal: Nature

Article Title: Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage

doi: 10.1038/s41586-023-06899-4

Figure Lengend Snippet: a , Diagram of sagittal view of upstream lymphatics region #2. b , Light-sheet fluorescence microscopic images showing serial optical sections (numbered) of lymphatics (outlined with green-dotted lines marked by white arrowheads) along the pterygopalatine artery (PPA, outlined with red-dotted lines) en route to the posterior nasal (green arrows) and nasopharyngeal lymphatic plexuses. A valve (green arrowhead) is located where lymphatics along the PPA join the posterior nasal lymphatic plexus. The white asterisk marks a segment of lymphatic with unknown connections. Anatomical positions are indicated in the upper right corner. Scale bars, 200 μm. Similar findings were obtained from n = 5 mice in three independent experiments. Ant., anterior; Post., posterior; Med., medial; Lat., lateral anatomical position. c , Fluorescence images of sections showing Prox1 + lymphatics in upstream lymphatics region #2 containing FluoSpheres (red arrowheads) along the PPA. Tuft cells of the olfactory epithelium are Prox1 + . Anatomical positions are indicated in the upper right corner. Scale bar, 200 μm. Similar findings were obtained from n = 3 mice in two independent experiments. Ant., anterior; Post., posterior; Med., medial; Lat., lateral anatomical position.

Article Snippet: The samples that underwent tissue clearing and decalcification were imaged using a light-sheet fluorescence microscope (LSFM, Carl Zeiss) with an EC Plan-Neofluar ×5/0.16 lens.

Techniques: Fluorescence

Journal: Frontiers in Immunology

Article Title: ROCKETS – a novel one-for-all toolbox for light sheet microscopy in drug discovery

doi: 10.3389/fimmu.2023.1034032

Figure Lengend Snippet: Modular clearing approach of the ROCKETS processing toolbox allows for simplified sample preparation for LSFM imaging. (A) Overview of presented procedures for processing and simplified clearing of mouse tissues or whole mouse bodies. GITs are processed using the 3D-Swiss Rolls procedure prior to fixation to enable holistic imaging. Other internal organs and tissues can be processed according to size and blood content. Non-perfused large and blood-rich tissues are precleared using the developed preclearing reagent before dehydration. Smaller tissues with less blood content do not require preclearing. All tissues except for the brain and whole mice are dehydrated with ethanol using an automated vacuum tissue processor. Due to its high lipid content, the brain is dehydrated in methanol and additionally delipidated using dichloromethane (MeOH/DCM). Only whole mice require perfusion to ensure timely fixation and decalcification of bones before the preclearing step. All specimens are cleared (RI matching) and imaged in BABB. Indicated times are total processing times from the day of dissection to cleared specimens. (p) = perfusion. (B) Workflow of passive preclearing of non-perfused murine tissues. Fluorescence-labeled molecules are applied in vivo (1) prior to euthanasia, tissue dissection and fixation overnight (2). Fixed specimens are incubated in the ROCKETS preclearing reagent (3) and washed with PBS PC (4) before transfer to vacuum-enhanced dehydration (5) and RI matching with BABB (6). (C) Photographs of mouse tissues at indicated step of preclearing. Specimens are opaque and still contain blood pigments after fixation (2). After preclearing (3) samples are fully decolorized and swollen and become completely transparent after dehydration and RI matching (6). The bottom row shows tissues after dehydration and RI matching without preclearing (immersed in PBS). Particularly blood-rich organs are insufficiently cleared without perfusion or preclearing. Thick squares of the grid = 5 mm. (D) Maximum intensity projections (MIPs) of LSFM images (z = 50 µm) of the tissue’s autofluorescence (545 nm → 595 nm) at the widest diameter of precleared tissues. FR = Female reproductive organs (oviduct and ovary), Sal. glands = Salivary glands. All tissue areas could be imaged entirely without blurring. *brain was not precleared, but dehydrated and delipidated using MeOH and DCM. Scale bars = 1 mm.

Article Snippet: Imaging was conducted using either a light sheet fluorescence microscope (LSFM) Ultramicroscope II ® (UM2, LaVision Biotec, Bielefeld, Germany; now part of Miltenyi Biotec, Bergisch Gladbach, Germany) or LSFM Ultramicroscope Blaze ® (UM Blaze, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Sample Prep, Imaging, Dissection, Fluorescence, Labeling, In Vivo, Incubation

Journal: Frontiers in Immunology

Article Title: ROCKETS – a novel one-for-all toolbox for light sheet microscopy in drug discovery

doi: 10.3389/fimmu.2023.1034032

Figure Lengend Snippet: 3D-Swiss Rolls sample preparation procedure for LSFM imaging enables holistic assessment of the entire GIT. (A) Schematic and (B) photographic representation of the 3D-Swiss Rolls workflow. (1) After euthanasia the lower GIT is disconnected from the body by incisions at the esophagus and rectum and removed entirely. (2) Six specimens are created by cutting as indicated by dashed lines: stomach (STO), three segments of the small intestine (SI 1-3), cecum (CAE) and colon (COL). (3) Each specimen is cleaned by flushing out chyme and feces with PBS PC and then immediately filled with NBF for fixation. (4a) SI and COL segments are cut open along the mesenteric line and (4b) rolled up on wooden sticks to create 3D-Swiss Rolls. (5) The created 3D-Swiss Rolls are then fixed without touching the surfaces of the histology cassette for 14-18 h in NBF at 4°C. After fixation, 3D-Swiss Rolls are unwound and re-rolled on plastic stirring rods for dehydration and clearing (not shown). (C) Surface rendering of LSFM image stacks of the tissue autofluorescence (545 → 595 nm, grey). 3D-Swiss Roll segments of the small intestine (SI1-3) and colon (Col). Stomach (Sto) and cecum (Cae) retained their physiological form. *Proximal end of the organ in 3D-Swiss Rolls. Scale bars = 1 mm.

Article Snippet: Imaging was conducted using either a light sheet fluorescence microscope (LSFM) Ultramicroscope II ® (UM2, LaVision Biotec, Bielefeld, Germany; now part of Miltenyi Biotec, Bergisch Gladbach, Germany) or LSFM Ultramicroscope Blaze ® (UM Blaze, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Sample Prep, Imaging

Journal: Frontiers in Immunology

Article Title: ROCKETS – a novel one-for-all toolbox for light sheet microscopy in drug discovery

doi: 10.3389/fimmu.2023.1034032

Figure Lengend Snippet: Entire mouse body cleared using the ROCKETS whole-mouse procedure and LSFM imaging reveals holistic biodistribution of anti-EpCAM antibody (G8.8R). (A) Mouse body (ventral view) after decalcification, preclearing, dehydration and immersion in BABB shows excellent transparency. Thick squares of the grid = 1 cm. (B) LSFM rendering of the tissue’s autofluorescence (grey) and anti-EpCAM staining (G8.8R in green) as overlay enabled quick localization of antibody disposition and identification of positive tissues. (C-H) LSFM renderings (ventral views) of EpCAM + tissues (G8.8R in green) in situ . M. Gl. = Mammary glands, LN = Lymph node. Scale bars = 2 mm.

Article Snippet: Imaging was conducted using either a light sheet fluorescence microscope (LSFM) Ultramicroscope II ® (UM2, LaVision Biotec, Bielefeld, Germany; now part of Miltenyi Biotec, Bergisch Gladbach, Germany) or LSFM Ultramicroscope Blaze ® (UM Blaze, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Imaging, Staining, In Situ

Journal: Frontiers in Immunology

Article Title: ROCKETS – a novel one-for-all toolbox for light sheet microscopy in drug discovery

doi: 10.3389/fimmu.2023.1034032

Figure Lengend Snippet: 3D renderings of LSFM images show highly heterogeneous binding of anti-EpCAM antibody (G8.8R) between and within organs. EpCAM stainings (G8.8R in green) and tissue anatomy revealed by tissue autofluorescence (grey) in maximum intensity projections (MIPs) of selected positive tissues. EpCAM binding was detected at (but not limited to) previously published sites of EpCAM expression . * Only the first of three segments of the small intestine depicted (corresponding to duodenum and proximal jejunum). Scale bars = 1000 μm.

Article Snippet: Imaging was conducted using either a light sheet fluorescence microscope (LSFM) Ultramicroscope II ® (UM2, LaVision Biotec, Bielefeld, Germany; now part of Miltenyi Biotec, Bergisch Gladbach, Germany) or LSFM Ultramicroscope Blaze ® (UM Blaze, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Binding Assay, Expressing

Journal: Frontiers in Immunology

Article Title: ROCKETS – a novel one-for-all toolbox for light sheet microscopy in drug discovery

doi: 10.3389/fimmu.2023.1034032

Figure Lengend Snippet: 3D renderings and single LSFM images display highly heterogeneous binding of anti-EpCAM antibody G8.8R to the tongue and salivary glands. (A) Dorsal and (B) lateral view of surface renderings of the tongue and associated tissues. Left images depict renderings of the tissue anatomy (grey) and the bound anti-EpCAM antibody (G8.8R in green) as overlay. Right images depict only the antibody signal (green) without anatomical context. (C) Tip of the tongue with positive gustatory fungiform papillae (FungP). (D) Positive sublingual excretory ducts at the tongue bottom. (E) Circumvallate papilla (CiP) and folate papillae (FP). (F) Mucous salivary glands (MSG) and serous salivary glands (SSG), parotid gland (PG) and larynx (LAR). (G) Single digital section of the tongue depicting both mucous (white dashed lines) and serous (orange dashed lines) salivary gland anatomy, (H) bound G8.8R (green) and (I) overlay of both channels. Scale bars = 1 mm (A–F) and 150 μm (G–I) .

Article Snippet: Imaging was conducted using either a light sheet fluorescence microscope (LSFM) Ultramicroscope II ® (UM2, LaVision Biotec, Bielefeld, Germany; now part of Miltenyi Biotec, Bergisch Gladbach, Germany) or LSFM Ultramicroscope Blaze ® (UM Blaze, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: ROCKETS – a novel one-for-all toolbox for light sheet microscopy in drug discovery

doi: 10.3389/fimmu.2023.1034032

Figure Lengend Snippet: 3D renderings and single digital sections reveal EpCAM binding to choroid plexi in the brain. (A) Dorsal MIP of the entire brain anatomy derived from the autofluorescence (grey) and binding of the EpCAM-specific antibody G8.8R (green). (B) Single LSFM image reveals anti-EpCAM antibody binding to choroid plexi of the temporal horn (TH), frontal horn (FH), 3rd ventricle (3V), 4th ventricle (4V, in A) and body (B, central part). (C) Higher magnification image of area indicated in image B displays binding to individual choroid plexus cells. (D) Maximum intensity projections (MIP) and (E, F) surface renderings of the entire frontal horn choroid plexus with bound anti-EpCAM antibody (G8.8R) extending into the ventricular space as indicated in image (A) . Scale bars = 2 mm (A, B) and 100 μm (C–F) .

Article Snippet: Imaging was conducted using either a light sheet fluorescence microscope (LSFM) Ultramicroscope II ® (UM2, LaVision Biotec, Bielefeld, Germany; now part of Miltenyi Biotec, Bergisch Gladbach, Germany) or LSFM Ultramicroscope Blaze ® (UM Blaze, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Binding Assay, Derivative Assay