Earl W. Cornell

An approach to rapid protein crystallization using nanodroplets

An approach that enables up to a two order of magnitude reduction in the amount of protein required and a tenfold reduction in the amount of time required for vapor-diffusion protein crystallization is reported. A prototype high-throughput automated system was used for the production of diffraction-quality crystals for a variety of proteins from a screen of 480 conditions using drop volumes as small as 20 nL. This approach results in a significant reduction in the time and cost of protein structure determination, and allows for larger and more efficient screens of crystallization parameter space. The ability to produce diffraction-quality crystals rapidly with minimal quantities of protein enables high-throughput efforts in structural genomics and structure-based drug discovery.

Automating fruit fly Drosophila embryo injection for high throughput transgenic studies

To decipher and manipulate the 14 000 identified Drosophila genes, there is a need to inject a large number of embryos with transgenes. We have developed an automated instrument for high throughput injection of Drosophila embryos. It was built on an inverted microscope, equipped with a motorized xy stage, autofocus, a charge coupled device camera, and an injection needle mounted on a high speed vertical stage. A novel, micromachined embryo alignment device was developed to facilitate the arrangement of a large number of eggs. The control system included intelligent and dynamic imaging and analysis software and an embryo injection algorithm imitating a human operator. Once the injection needle and embryo slide are loaded, the software automatically images and characterizes each embryo and subsequently injects DNA into all suitable embryos. The ability to program needle flushing and monitor needle status after each injection ensures reliable delivery of biomaterials. Using this instrument, we performed a set of transformation injection experiments. The robot achieved injection speeds and transformation efficiencies comparable to those of a skilled human injector. Because it can be programed to allow injection at various locations in the embryo, such as the anterior pole or along the dorsal or ventral axes, this system is also suitable for injection of general biochemicals, including drugs and RNAi.

High-throughput on-the-fly scanning ultraviolet-visible dual-sphere spectrometer

We have developed an on-the-fly scanning spectrometer operating in the UV-visible and near-infrared that can simultaneously perform transmission and total reflectance measurements at the rate better than 1 sample per second. High throughput optical characterization is important for screening functional materials for a variety of new applications. We demonstrate the utility of the instrument for screening new light absorber materials by measuring the spectral absorbance, which is subsequently used for deriving band gap information through Tauc plot analysis.

Implementation and application of the peak scaling method for temperature measurement in the laser heated diamond anvil cell

A new design for a double-sided high-pressure diamond anvil cell laser heating set-up is described. The prototype is deployed at beamline 12.2.2 of the Advanced Light Source at Lawrence Berkeley National Lab. Our compact design features shortened mechanical lever arms, which results in more stable imaging optics, and thus more user friendly and more reliable temperature measurements based on pyrometry. A modification of the peak scaling method was implemented for pyrometry, including an iterative method to determine the absolute peak temperature, thus allowing for quasi-real time temperature mapping of the actual hotspot within a laser-heated diamond anvil cell without any assumptions on shape, size, and symmetry of the hotspot and without any assumptions to the relationship between fitted temperature and peak temperature. This is important since we show that the relationship between peak temperature and temperature obtained by fitting the Planck function against the thermal emission spectrum averaged over the entire hotspot is not constant but depends on variable fitting parameters (in particular, the size and position of the fitting window). The accuracy of the method is confirmed through measuring melting points of metal wires at ambient pressure. Having absolute temperature maps in real time allows for more differentiated analyses of laser heating experiments. We present such an example of the pressure variations within a heated hotspot of AgI at a loaded base pressure of 3.8 GPa.

High-efficiency microarray printer using fused-silica capillary tube printing pins.

We describe a contact printing approach for microarrays that uses fused-silica capillary tubes with tapered tips for printing pins and a pressure/vacuum system to control pin loading, printing, and cleaning. The printing process is insensitive to variable environmental factors such as humidity, and the small diameter of the pins allows routine printing from 1536 well source plates. Pin load capacity, 0.2 microL in the current system, is adjustable by controlling pin length. More than 2000 spots can be printed per 0.2-microL pin load (<100 pl/spot), and densities of >12,000 spots/cm(2) are readily achievable. Solutions with a wide range of viscosities and chemical properties can be printed. The system can print tens of thousands of different solutions at high speed, due to the ability to use large numbers of pins simultaneously, and can produce a large number of replicate arrays since all of the solution picked up by the pins is available for deposition.

A multichannel gel electrophoresis and continuous fraction collection apparatus for high-throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ∼10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.