Direct 3D Sampling of the Embryonic Mouse Head: Layer-wise Nanosecond Infrared Laser (NIRL) Ablation from Scalp to Cortex for Spatially Resolved Proteomics

Abstract

Common workflows in bottom-up proteomics require homogenization of tissue samples to gain access to the biomolecules within the cells. The homogenized tissue samples often contain many different cell types, thereby representing an average of the natural proteome composition, and rare cell types are not sufficiently represented. To overcome this problem, small-volume sampling and spatial resolution are needed to maintain a better representation of the sample composition and their proteome signatures. Using nanosecond infrared laser ablation, the region of interest can be targeted in a three-dimensional (3D) fashion, whereby the spatial information is maintained during the simultaneous process of sampling and homogenization. In this study, we ablated 40 μm thick consecutive layers directly from the scalp through the cortex of embryonic mouse heads and analyzed them by subsequent bottom-up proteomics. Extra- and intracranial ablated layers showed distinct proteome profiles comprising expected cell-specific proteins. Additionally, known cortex markers like SOX2, KI67, NESTIN, and MAP2 showed a layer-specific spatial protein abundance distribution. We propose potential new marker proteins for cortex layers, such as MTA1 and NMRAL1. The obtained data confirm that the new 3D tissue sampling and homogenization method is well suited for investigating the spatial proteome signature of tissue samples in a layerwise manner. Characterization of the proteome composition of embryonic skin and bone structures, meninges, and cortex lamination in situ enables a better understanding of molecular mechanisms of development during embryogenesis and disease pathogenesis.

Multimodal image-guided laser ablation system for precise 3D tissue sampling and subsequent analysis of biomolecules for cancer research

“…Another study demonstrates direct 3D laser sampling of the mouse head into the cerebral cortex to investigate the spatial distribution of marker protein abundances for brain tumour characterization. 30 In an unpublished study, focusing on minimizing our sampling abilities, we were able differentiate murine organs with proteomics (LC-MS/MS) with amounts of less than 1 nL, demonstrating reproducibility. Furthermore, we performed laser sampling on a longitudinally sectioned murine kidney using lines of individual laser shots, each analyzed separately.…”

“…The optical path for the laser ablation with a nanosecond infrared laser (NIRL), tuned to a wavelength of 2940 nm (Opolette SE 2731, Opotek, LLC, Carlsbad, CA, USA), is set up similar as in our previous works. 27,28,30 The NIRL beam's divergence and profile are corrected by a focusing lens in combination with two cylindrical lenses of different focal lengths. The collimated and rounded beam is guided passes several mirrors and then overlayed with a 650 nm pilot laser for adjustment and aiming purposes.…”

“…17342650), which is placed at a short distance (<1 mm) over the sample (as described in Ref. 30), but mounted in a motorized xy-stage (U-780.DLS PILine ® , Physikinstrumente (PI), Karlsruhe, Germany) with a motion controller (C-867.2U2 PILine ® , Physikinstrumente (PI), Karlsruhe, Germany).…”

“…20 After these findings, several studies have been published, investigating the potential of this method in termns of simultaneous tissue sampling and homogenization for subsequent quantitative bottom-up proteomics. The most important studies used infrared lasers with pulse widths in the ps-, 21,22 ns- [23][24][25][26][27][28][29][30] and ms-regime, 22,31 but only with a picosecond infrared laser (PIRL) and nanosecond infrared laser (NIRL) no significant heating occurred, damaging neighboring structures. Since the tissue is softly transformed into an aerosol, also other biomolecules, besides the mentioned proteins, such as DNA, 33 mRNA, 34 lipids 35,36 or other metabolites remain intact and can be analyzed with sufficient yields.…”

Co-activation of LIN28A and CTNNB1 disturbs cortical neuronal migration and pia mater integrity

“…As LIN28A and CTNNB1 are both transcriptional and translational regulators, we aimed to investigate the resulting protein level using mass spectrometry-based proteome data with high spatial resolution. Therefore, we used the nanosecond infrared laser (NIRL) nano-volume sampling system (Hahn et al, 2021;Voß et al, 2022) and ablated consecutive cortical layers directly from the skin surface into the cerebral cortex, as described previously (Navolić et al, 2023). We ablated nine consecutive layers at E14.5 and 18 consecutive layers at E18.5 of the CTRL condition in biological replicates (n = 4) and the mouse models GL, GB and GBL (n = 3) (Fig.…”