Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences

Abstract

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently-labelled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods such as synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) or serial blockface scanning electron microscopy (SBF-SEM) might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here we showcase the utility of a novel femtosecond laser device to prepare microscopic samples (1) of an optimised geometry for synchrotron X-ray microscopy as well as (2) for subsequent volume electron microscopy applications, embedded in a wider correlative multimodal imaging workflow (Fig. 1).

Characterization of human trabecular bone across multiple length scales: A correlative approach combining X-ray tomography with LaserFIB and plasma FIB-SEM

“…This fs laser has been traditionally employed in studying metallic materials 55,56 , but has recently attracted growing interest for biological materials processing in clinical and biomedical research 26,[57][58][59] . The increased use and application of the fs laser are largely attributable to its ultrashort laser pulse duration, which results in limited penetration depth of the thermal diffusion zone in the material 60 .…”

“…The laser processing occurs in an airlock, separate from the Crossbeam FIB-SEM chamber, so as to avoid contamination from laser ablation debris. Site-specific laser processing, with accuracy on the scale of a few micrometers, is enabled by SEM stage position registration of the specimen ROIs and also fiducial aperture locations on the laser specimen holder, followed by moving the laser specimen holder into the airlock and then laser scanning registration of the fiducial apertures 26 . This process allows the accurate positional placement of the SEM ROI images into the laser CAD software such that laser processing can be precisely positioned over the specimen ROIs.…”

“…Deerinck et al explored the potential of using the fs laser for large volume machining and electron microscopy imaging of mouse colon tissue 61 . In another study, Bosch et al demonstrated a workflow to extract multiple pillar-shaped micrometer-sized samples from a resin-embedded mouse olfactory bulb glomerulus 26 , a result that highlighted the advantage of using the fs laser in biological sample preparation compared to other conventional methods such as ultramicrotomy 62 . In the current study, the fs laser was used for the first time for site-specific human trabecular bone sample preparation guided by 3D correlative microscopy.…”