Harshini Mukundan

The successful isolation of mycolactone in a laboratory or from a clinical sample relies on proper handling and storage of the toxin. Mycolactone is a light-sensitive and an amphiphilic toxin produced by Mycobacterium ulcerans. The biochemistry of the toxin makes it unstable in aqueous matrices such as blood, which causes it to self-aggregate or present in complex with carrier molecules. This biochemistry also impacts the use of the toxin in vitro, in that it tends to aggregate and stick to substrates in an aqueous environment, which alters its physiological presentation and limits its availability in a sample. Glass materials (i.e., tubes, vials, syringes, plates) should be used when possible to avoid loss of mycolactone sticking to plastic surfaces. Dark containers such as amber vials or aluminum-foil wrapped tubes should be used to avoid photodegradation of the toxin upon exposure to light. Sample storage in organic solvents is ideal for mycolactone stability and recovery; however, this is not always amenable as multiple diagnostic assays might be performed on a single sample (such as PCR or ELISA). In these cases, samples can be stored in an aqueous solution containing a small amount of detergent to enhance recovery of the toxin, and in order to avoid aggregation. Therefore, the downstream manipulations should be carefully considered prior to sample collection and storage. Here we present considerations for the optimal handling and storage of mycolactone in order to obtain quality yield of the toxin for various research and diagnostic applications.

Video-based techniques for structural dynamics have shown great potential for identifying full-field, high-resolution modal properties. One significant advantage of these techniques is that they lend themselves to being applied to structures at very small length scales such as MEMS devices and living cells. These small structures typically will have resonant frequencies greater than 1 Khz, thus requiring the use of high-speed photography to capture their dynamics without aliasing. High speed photography generally requires the structure-under-test (e.g. living cell) to be exposed to high levels of illumination. It is well-known that exposing delicate structures such as living cells to these high levels of light energy can result in damage to their structural integrity. It is therefore desirable to develop techniques to minimize the amount of illumination that is required to capture the modal properties of interest. This is particularly important given that the mechanical properties of living cells have recently been found to be of interest to the biomedical community. For example, it is known that changes in cell stiffness are correlated with grade of metastasis in cancer cells. Compressive sensing techniques could help mitigate this problem, particularly in fluorescence microscopy applications where cells are illuminated using a laser light source. Compressive sampling would allow for the cells to be exposed to the laser light with a significantly lower duty cycle, thus resulting in less damage to the cells. As a result the structural dynamics of the cells can be measured at increasingly high frequencies yielding new information about cellular material properties that can be coupled with biochemical cues to yield new therapeutic strategies. Furthermore, video-based techniques would benefit from the reductions in memory, bandwidth and computational requirements normally associated with compressive sampling. In this work we present a technique that intimately combines solutions to the blind-source separation problem for video-based, high-resolution operational modal analysis with compressive sampling.

Traditionally, performing an experimental modal analysis of a building/structure required instrumenting the structure with a spatially distributed array of accelerometers or strain gages. Alternatively, a laser doppler vibrometer would have to be scanned across the structure of interest in a sequential manner to measure structural response. Recently, researchers at LANL developed a technology that combines the theory of structural dynamics with computer vision that provides the capability to characterize structural dynamics at very high spatial density using only an imager. With this newfound success at the macro-scale, we have exploited this novel technology to a whole new scale- to studying the basic structure of life itself, the human cell. We hypothesize that this new technology and novel application will provide a significantly better understanding of how stiffness and mass distribution changes in a cell as it undergoes epithelial-mesenchymal transition, and in identifying its associated EMC biochemical cues, highlight potential therapeutic targets. For the first time it should be possible to measure the high-resolution mode shapes of cells; given that all cells undergoing cancer metastasis experience a breakdown in the cytoskeleton, this work will enable groundbreaking advances in various fields including medicine and structural dynamics. It is imperative to highlight, that we are only beginning to understand the relationship between biophysical properties of cells and their potential to regulate tumorigenesis and motility, which is commonly known as metastasis. This knowledge could be used to provide verification and validation of finite element models of cellular structure. This work will represent the first time that expertise in experimental structural dynamics will be brought to bear on the problem of characterizing the structural dynamics of cells at high spatial resolution, which is novel and unique on its own. When successful, this new technology could be used to couple the biophysical cues associated with other detrimental human pathologies.