We present the fabrication of specialized nanotools, termed nanoscalpels, and their application for nanolithography and nanomechanical manipulation of biological objects. Fabricated nanoscalpels have the shape of a thin blade with the controlled thickness of 20-30 nm and width of 100-200 nm. They were fabricated using electron beam induced deposition at the apex of atomic force microscope probes and are hard enough for a single cut to penetrate a approximately 45 nm thick gold layer; and thus can be used for making narrow electrode gaps required for fabrication of nanoelectronic devices. As an atomic force microscope-based technique the nanoscalpel provides simultaneous control of the applied cutting force and the depth of the cut. Using mammalian cells as an example, we demonstrated their ability to make narrow incisions and measurements of local elastic and inelastic characteristics of a cell, making nanoscalpels also useful as a nanosurgical tool in cell biology. Therefore, we believe that the nanoscalpel could serve as an important tool for nanofabrication and nanosurgery on biological objects.

https://iopscience.iop.org/article/10.1088/0957-4484/20/44/445302/pdf

An atomic force microscope nanoscalpel for nanolithography and biological applications

A combined scanning electron microscope and focussed ion beam instrument is suitable for micro- and nanopatterning, cross-sectioning and subsequent imaging, of specimens at room temperature as well as under cryo conditions. In order to reveal internal details, samples are conventionally milled with the ion beam positioned perpendicular to the sample surface. Using this approach certain limitations are frequently encountered, e.g. accumulation of redeposited material, shadowing effects, image distortion and a limited imaging area. Here we show an approach in which samples are pre-trimmed using a microtome to obtain a sample block face that is parallel to the ion beam. This new grazing incidence geometry eliminates the need for removal of bulk material with the ion beam and enables immediate fine polishing of a pre-selected area of interest. Many of the limitations previously described are avoided and in addition milling time is reduced, whilst creating larger cross-sectional areas. Another advantage is that electron imaging can be accomplished by tilting the sample surface perpendicular to the electron beam, providing a geometrically undistorted image. The proposed approach is suitable for materials that can be microtomed, both in ambient and cryogenic conditions, and proves to be of particular benefit for biological and food samples.

Focussed ion beam milling at grazing incidence angles.

Bacterial cells constantly face complex environmental changes in their natural habitats. While steady-state cell size correlates with nutrient-determined growth rate, it remains unclear how cells regulate their morphology during rapid environmental changes. Here, we systematically quantified cellular dimensions throughout passage cycles of stationary-phase cells diluted into fresh medium and grown back to saturation, and found that cells exhibit characteristic dynamics in surface area to volume ratio (SA/V). SA/V dynamics were conserved across many genetic/chemical perturbations, as well as across species and growth temperatures. We developed a model with a single fitting parameter, the time delay between surface and volume synthesis, that quantitatively explained our SA/V observations, and showed that the time delay was indeed due to differential expression of volume and surface-related genes. The first division after dilution occurred at a tightly controlled SA/V, a previously unrecognized size-control mechanism highlighting the relevance of SA/V. Finally, our time-delay model successfully predicted the quantitative changes in SA/V dynamics due to altered surface area synthesis rates or time delays from translation inhibition. Our minimal model thus provides insight into how cells regulate their morphologies through differential regulation of surface area and volume synthesis and potentiates deep understanding of the connections between growth rate and cell shape in complex environments.

/pdf/precise-regulation-of-the-relative-rates-of-surface-area-and-48brnvxhvx.pdf

Precise regulation of the relative rates of surface area and volume synthesis in dynamic environments

The neuronal basis of goal-directed behavior requires interaction of multiple separated brain regions. How subcortical regions and their interactions with brain-wide activity are involved in action selection is less understood. We have investigated this question by developing an assay based on whole-brain volumetric calcium imaging using light-field microscopy combined with an operant-conditioning task in larval zebrafish. We find global and recurring dynamics of brain states to exhibit pre-motor bifurcations towards mutually exclusive decision outcomes which arises from a spatially distributed network. Within this network the cerebellum shows a particularly strong pre-motor activity, predictive of both the timing and outcome of behavior up to ~10 seconds before movement initiation. Furthermore, on the single-trial level, decision directions can be inferred from the difference neuroactivity between the ipsilateral and contralateral hemispheres, while the decision time can be quantitatively predicted by the rate of bi-hemispheric population ramping activity. Our results point towards a cognitive role of the cerebellum and its importance in motor planning.

/pdf/cerebellar-neurodynamics-during-motor-planning-predict-3sv4k4fhe9.pdf

Cerebellar neurodynamics during motor planning predict decision timing and outcome on single-trial level

For cross-sectional observations of biological samples by electron microscopy, TEM is usually used. However, the sample preparation for TEM observations requires laborious procedures and takes from several hours to a day or more [1]. The sample preparation for SEM observations is less laborious, thus SEM is used more and more for cross-sectional observations. As a sectioning technique for biological samples, focused ion beam (FIB) has received much attention [2], because it has been used in material science and industry fields to obtain accurately and precisely sectioned samples. But, because of the high beam energy, ion-beam induced damage is more severe in the biological field, and precautions are vitally necessary. We developed an alternative technique for sectioning of cells, which is based on lowenergy Ar ion-beam milling, and is easy and rapid. Here, we report our method using a cultured cell (HEK293T) as a sample.

Application of Ar Ion Beam Milling on Sectioning of Cells for SEM Observations

Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Applications of Focused Ion Beam (FIB) On Yeast Cell and SARS Virus

The focused ion beam (FIB) is a relatively novel technique to biomedical electron microscopy as it open up new means for the observations and examinations of a wide range of biomedical and biological materials. The focused ion beam, or FIB tool has been utilized mainly in the fields of material sciences and industry. The (FIB) uses high-energy gallium ions to precisely and accurately section or mill samples. Lately FIB method have been used to prepare biological samples such as yeast cells and virus particles. Yeast cells Schwanniomyces occidentalis S. occidentalis were prepared by vacuum sucking them into cellulose tubing, plunge freezing them in liquid nitrogen, followed by chemical fixation in glutaraldehye and postfixed with osmium tetroxide, dehydrated in a series of ascending alcohol concentration up to absolute alcohol, then freeze dried overnight. In the case of SARS virus, the tissue culture containing virus particles was chemically fixed with glutaraldehyde, dehydrated in ascending order of alcohol concentrations and then freeze dried.

/pdf/applications-of-focused-ion-beam-fib-on-yeast-cell-sars-2bqrzk4wca.pdf

Applications of Focused Ion Beam (FIB) on Yeast Cell & SARS Virus

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Applications of Focused Ion Beam (FIB) On Yeast Cell and SARS Virus

Applications of Focused Ion Beam (FIB) on Yeast Cell & SARS Virus