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

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 labeled 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 or serial blockface scanning electron microscopy, 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 femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities.

Preliminary results from the first monochromated and aberration corrected 200-kV field-emission scanning transmission electron microscope.

Experimental results from the first monochromated and aberration-corrected scanning transmission electron microscope operated at 200 kV are described. The formation of an electron probe with a diameter of less than 0.2 nm at an energy width significantly under 0.3 eV and its planned application to the chemical analysis of nanometer-scale structures in materials science are described. Both energy and spatial resolution will benefit from this: The monochromator improves the energy resolution for studies of energy loss near edge structures. The Cs corrector allows formation of either a smaller probe for a given beam current or yields, at fixed probe size, an enhanced beam current density using a larger condenser aperture. We also point out another advantage of the combination of both components: Increasing the convergence angle by using larger condenser apertures in an aberration-corrected instrument will enlarge the undesirable chromatic focus spread. This in turn influences spatial resolution. The effect of polychromatic probe tails is proportional to the product of convergence angle, chromatic aberration constant, and energy spread. It can thus be compensated for in our new instrument by decreasing the energy width by the same factor as the beam convergence is increased to form a more intense probe. An alternative in future developments might be hardware correction of the chromatic aberration, which could eliminate the chromatic probe spread completely.