UC Berkeley

There are innumerable systems of interest in medicine and biological research for which the fundamental imaging technology exists to examine the entities of interest, but no sufficiently robust and efficient technique for isolating and preparing the sample has been devised. The lack of sample preparation methodology often serves as a barrier to healthcare access and scientific discovery. Here, I describe three instances of such barriers and the progress made to overcome them.

First, I discuss the poor access to medical diagnostics in endemic regions for human schistosomiasis and other neglected enteric and urinary helminthic diseases. These pathogens have a profound impact on morbidity and mortality in the regions they affect, despite the existence of safe and effective therapeutics. In wealthy regions, detecting the causative pathogens has been successfully performed via microscopy in medical laboratories for a century. However, in regions with poor access to laboratory equipment like pipettes, centrifuges, and microscopes, the available diagnostic technologies have poor sensitivity and specificity, requiring drug administration decisions to be made at a population level.

To address the need for a point-of-care test that can diagnose schistosomiasis on an individual basis, I present a novel, inexpensive, specialized capillary which accepts urine from a syringe and in a single step, captures schistosome eggs from solution and spreads them in a monolayer. The monolayer can then be imaged using an inexpensive mobile phone microscope, and schistosome eggs can be identified and counted using a machine vision algorithm running on the phone’s processor. In this way, with minimal training, users can take urine from a patient and receive a quantitative diagnosis for Schistosoma Haematobium infection in under a minute, for less than one US dollar. I present our assessment of the new capillary for capturing parasite eggs and the new algorithm for detecting eggs, both in the lab with reconstituted samples, and in the hands of clinical personnel in field studies of school children in Ghana and Côte D’Ivoire. Because of the promising results of these studies, we have invested in infrastructure to mass produce the diagnostic, with the ultimate goal of providing rapid diagnostic capabilities to the entire region affected by S. Haematobium.

Next, I address a barrier to imaging which manifests within the Fletcher Lab and other groups who are interested in probing the biophysical characteristics and interactions of transmembrane proteins. For many transmembrane proteins of interest, it is difficult to decouple the behavior of the specific protein from the vast complexity of a living cell. In other words, the noise due to other cellular processes drowns out the signal of the molecules of interest under a given condition chosen by the researcher. The desire to limit this noise leads many experimentalists to pursue in vitro reconstitution of transmembrane proteins in an artificial lipid bilayer, such as a black lipid membrane or a supported lipid bilayer, as a platform for their experiments. In these systems, the composition of lipids, proteins, and other biomolecules can be controlled. One such system, which is homeomorphic with the plasma membrane of a cell, is a giant unilamellar vesicle (GUV). Past results from our lab demonstrated the ability to form GUVs with a great deal of control over contents of the lumen using a microfluidic jet. Here, I present a new technique for forming GUVs by replacing the microfluidic jet with an ultrasonic transducer, which preserves the loading capabilities of the microfluidic jet and confers additional potential advantages over the state of the art.

In the new system, a black lipid membrane is formed, with the desired luminal solution of the GUV on one side and the extravesicular solution on the other. This membrane is placed such that it is orthogonal to the axis and incident to the focal point of a high-intensity focused ultrasound transducer. By activating the piezoelectric transducer with a short burst of RF power, the system forms a region of highly concentrated Reynolds stress near the focal point and the resulting acoustic streaming results in a tightly focused jet of moving liquid. This jet deforms the planar membrane until a GUV pinches off, filled with the solution on the proximal side. Compared to the microfluidic jet, this method eliminates the complications associated with bringing a capillary into close proximity with the planar lipid bilayer and potential clogging of capillaries with small diameters. The acoustic jetting technique is also more amenable to encapsulating micron-scale particles, small unilamellar vesicles, and viscous solutions. I discuss some of the operating conditions for the acoustic transducers and other elements in the system, and their effects on the resulting GUVs, and I review practical limitations to this technique that may prevent its wide adoption except in niche applications.

Finally, I address a barrier to sample preparation at the cutting edge of biological imaging: transmission electron cryo-microscopy (cryo-EM). Cryo-EM requires a thin sample of biomolecules encased in vitreous ice, through which electrons can be transmitted. As the imaging instrumentation has improved significantly in recent years, the inability to consistently form thin uniform samples of biomolecules has become a bottleneck in the cryo-EM workflow. Currently, the vast majority samples are prepared by pipetting a volume of aqueous sample onto a TEM grid, pressing a piece of filter paper against the droplet to wick off the majority of the liquid, and plunging the grid and remaining solution into liquid ethane to freeze it. This blotting technique is effective, but often results in regions of thick ice therefore poor electron transmission, dried areas of the grid, and significant protein denaturation at the air-water interface.

To improve our understanding of the fluidic phenomena at play during the blotting technique, and to inform the development of new techniques, I examine the liquid lubrication layer during and after blotting. I found that the capillary pressures within the absorbent filter paper cause air to enter the lubrication layer in a tortuous and unrepeatable fashion while the filter paper is still in contact with the sample. This propagation of air allows proteins to denature at the newly formed interfaces and creates dewetted regions on the surface which serve as boundaries to prevent the complete relaxation of the water surface when the filter paper is removed. The resulting lubrication layer therefore has varying thickness which can persist to the ice stage. With these insights in mind, we propose directions for the development of cryo-EM sample preparation techniques which avoid the irreproducibility and sample damage inherent in filter paper blotting.