MALDI MSI workflow in D. rerio and D. magna. (a) Workflow for the precise analysis of lipid patterns visualizing anatomical features within neuronal and non-neuronal compartments. For sample preprocessing the adult zebrafish was cut cranial to the anal fin. The prepared sample was embedded in carboxymethylcellulose (CMC) and sagittal cryosections were prepared as indicated by the yellow line. Sections were covered with 4-Nitroaniline (pNA) matrix and MALDI MSI experiments were carried out in positive ion mode with 25 µm step size. (b) Workflow for the spatial localization of a command neuron in brain sections. Adult zebrafish were cut cranial to the anal fin and fixated in paraformaldehyde (PFA). After removal of the brain, it was fixated in sucrose and subsequently was embedded in CMC. Coronal cryosections were prepared as indicated by the red line. Brain sections (70 µm) were coated with pNA matrix and MALDI MSI measurements were carried out in positive ion mode with 5 µm step size. Prior to matrix application the sections were investigated by fluorescence microscopy to ensure the presence of the soma of the command neuron. (c) Workflow revealing the spatial distribution of lipids in whole body sections of D. magna. The sample was directly embedded in gelatin solution (8%) and sections (of 18 µm thickness) were prepared and coated with pNA matrix. MALDI MSI experiments (10 µm step size) were performed as described before.

MALDI MS imaging of lipids in neuronal and non-neuronal compartments of D. rerio. (a,e) H&E (hematoxylin and eosin) staining of the sagittal cryosections in adult zebrafish showing detailed structures within the gills’ filaments, eye (retinae, lens), brain (telencephalon, tectum opticum, cerebellum) and liver. (b) RGB Overlay of PC (O-32:0) [M+H]⁺ (red) and PC (30:0) [M+K]⁺ (green). (c,d), (g–i) Positive ion mode MS images. (c) Ion image of PC (O-32:0) [M+H]⁺ showing high intensity in the region of the gills’ filaments (white arrow). (d) Ion image of PC (30:0) [M+K]⁺ visualizing the lens and the retina of the eye (white arrow). (f) RGB Overlay of PC (O-32:0) [M+H]⁺ (red colored), PC (O-36:1) [M+K]⁺ (blue colored) and PC (40:6) [M+H]⁺ (green colored). (g) Ion image of PC (O-32:0) [M+H]⁺ shows distribution with high intensity in non-neuronal tissue, exemplarily shown for muscle tissue (white arrow). (h) Ion image of PC (O-36:1) [M+K]⁺, illustrates the structural differentiation within the tectum opticum and parts of the cerebellum. (i) Ion image of PC (40:6) [M+H]⁺ shows distribution within the brain and eye and presents the liver as relative homogenous tissue (white arrows).

Spatial localization of a central command neuron within tissue sections of zebrafish using MALDI MSI. (a) Schematic representation of a zebrafish brain fixated on a sample holder, with the central command neurons (the pair of Mauthner neurons) shown in green. On the right side the two Mauthner neurons are magnified and the position of two sections (70 µm thickness) are illustrated with dotted lines. (b,c) Positive ion MS images. (b) MS image of PC (36:1) [M+H]⁺ visualizing the axon arrangement before the point where the axons cross the midline. Axons are labeled with green arrows and a schematic illustration of the axon region is shown on the right side of the MS image. (c) MS image of the somata of the two neurons (labeled with green arrows), visualized by the distribution of PC (38:6) [M+H]⁺. (d) Fluoresence microscopy of tissue section from transgenic line Tol056 (that expresses GFP in the neuron) was used to conveniently select appropriate sections for subsequent MSI.

MALDI MSI visualizing the lipid distribution in D. magna. (a) Whole body image of D. magna. (b) RGB Overlay of PC (36:0) [M+Na]⁺ (blue colored), HexCer (41:1;O₂) [M+2Na-H]⁺ (red colored) and SM (38:1;O₂) [M+Na]⁺ (green colored) visualizing different anatomical regions (thoracic legs, egg/embryo, intestine, body wall). (b–d) Positive ion MS images. (c) Ion image of PC (36:0) [M+Na]⁺, highlighting the intestine region (labeled by a white arrow). (d) Ion image of HexCer (41:1;O₂) [M+2N-H]⁺, highlighting the lipid distribution within the egg/embryo (labeled by a white arrow). (e) Ion image of SM (38:1;O₂) [M+Na]⁺, showing the lipid distribution in the surrounding tissue including body wall (labeled by a white arrow) and thoracic legs.