Microfluidic Cryo-TEM Method

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Brief Description: Transmission electron microscopy (TEM) is a high resolution imaging technique whereby a small wavelength, electron beam is transmitted through an ultra-thin specimen such that the electron beam interacts with the specimen as it passes through to create an image of fine detail.  This image is then magnified and focused onto a visualization device, e.g., layer of photographic film, or detected by a sensor/CCD camera.  TEM can elucidate a single column of atoms, and therefore, is a salient visual analysis technique/tool in a range of scientific fields, biological (cryo-TEM) and physical.  TEM sample preparation is complex and specific to the specimen material and information desired.  It requires a highly controlled, miniaturized environment, precision, accuracy, and lack of contamination. With cryo-TEM, the specimen slice, tens to hundreds of nanometers thick, is placed on a typical mesh, metal grid with other attributes, devices and conditions to stabilize the specimen-containing sample, and then the sample is flash frozen just before image capture. Many generic techniques have been used for sample preparation of the required ultra thin specimen sections.  However, during normal deposition of a dilute sample [with specimen] onto the support grid, a droplet is generally created that is too large and thick for use, thus the sample must be blotted with filter paper.  This blotting introduces high shear to the specimen and significantly increases the time required in readying the sample, both of which limit the ability to examine certain specimens and the formation of particular natural structures over time.  In conventional cryo-TEM, the blotting procedure can deform the molecular structure of the specimen of interest.  To improve sample preparation, and therefore, the resultant image and analysis over current techniques, the issues of high shear and time need to be addressed. The invention offered here overcomes current state-of-the-art limitations.  The innovation is a microfluidic cryo-TEM method and system that automates sample preparation.  The automated system deposits and controls sample droplet size, via microcapillaries, to eliminate the need for a blotting step, and it uses a very low shear rate for removal of excess sample fluid.  Sample preparation is accelerated overall, and flow rates and residence times in the capillary microchannels allow direct visualization of previously unobserved structures such as aggregates formed within the 10–100 millisecond time scales.  System operation/automation is monitored and carefully controlled via computer to optimize the timing of all events, allowing very accurate knowledge of microstructure age at time of freezing – a unique window for the examination of the temporal evolution of aggregate morphologies.  Additionally, an optimized microfluidic setup offers a novel opportunity to rapidly screen the short and long time scale formation of nanomaterials, soft colloids and vesicles. The broader market is scientific/biomedical R&D, with market segments in academic/non-profit, industrial, and government/military basic research, commercial drug discovery and development, environmental and/or defense laboratories.  Applications are many in microscopic visualization and analysis of: single molecules, molecular surface structures and topography, and/or spatial relationships; microstructures/morphology for characterization of bio/nanomaterials; crystal structure, defects, phases and composition; binding site locations in drug discovery and development, e.g., drug to target, ligand to receptor, etc., to potentially describe molecular mechanism-of-action; characterize environmental impact/agents on structures/materials;  biodefense agents and/or toxins; unknown molecular samples from disaster sites or space; among many other scenarios in which microstructure characterization is desired to identify and inform about physical and chemical properties of a substance. Information: US patent 9,312,095 is issued (04/12/2016) US patent 9,355,813 is issued (05/31/2016)