A simple flash and freeze system for cryogenic time-resolved electron microscopy.

As the resolution revolution in CryoEM expands to encompass all manner of macromolecular complexes, an important new frontier is the implementation of cryogenic time resolved EM (cryoTREM). Biological macromolecular complexes are dynamic systems that undergo conformational changes on timescales from microseconds to minutes. Understanding the dynamic nature of biological changes is critical to understanding function. To realize the full potential of CryoEM, time resolved methods will be integral in coupling static structures to dynamic functions. Here, we present an LED-based photo-flash system as a core part of the sample preparation phase in CryoTREM. The plug-and-play system has a wide range of operational parameters, is low cost and ensures uniform irradiation and minimal heating of the sample prior to plunge freezing. The complete design including electronics and optics, manufacturing, control strategies and operating procedures are discussed for the Thermo Scientific™ Vitrobot and Leica™ EM GP2 plunge freezers. Possible adverse heating effects on the biological sample are also addressed through theoretical as well as experimental studies.

Flattening of Diluted Species Profile via Passive Geometry in a Microfluidic Device.

In recent years, microfluidic devices have become an important tool for use in lab-on-a-chip processes, including drug screening and delivery, bio-chemical reactions, sample preparation and analysis, chemotaxis, and separations. In many such processes, a flat cross-sectional concentration profile with uniform flow velocity across the channel is desired to achieve controlled and precise solute transport. This is often accommodated by the use of electroosmotic flow, however, it is not an ideal for many applications, particularly biomicrofluidics. Meanwhile, pressure-driven systems generally exhibit a parabolic cross-sectional concentration profile through a channel. We draw inspiration from finite element fluid dynamics simulations to design and fabricate a practical solution to achieving a flat solute concentration profile in a two-dimensional (2D) microfluidic channel. The channel possesses geometric features to passively flatten the solute profile before entering the defined region of interest in the microfluidic channel. An obviously flat solute profile across the channel is demonstrated in both simulation and experiment. This technology readily lends itself to many microfluidic applications which require controlled solute transport in pressure driven systems.

Electrocoalescence based serial dilution of microfluidic droplets.

Dilution of microfluidic droplets where the concentration of a reagent is incrementally varied is a key operation in drop-based biological analysis. Here, we present an electrocoalescence based dilution scheme for droplets based on merging between moving and parked drops. We study the effects of fluidic and electrical parameters on the dilution process. Highly consistent coalescence and fine resolution in dilution factor are achieved with an AC signal as low as 10 V even though the electrodes are separated from the fluidic channel by insulator. We find that the amount of material exchange between the droplets per coalescence event is high for low capillary number. We also observe different types of coalescence depending on the flow and electrical parameters and discuss their influence on the rate of dilution. Overall, we find the key parameter governing the rate of dilution is the duration of coalescence between the moving and parked drop. The proposed design is simple incorporating the channel electrodes in the same layer as that of the fluidic channels. Our approach allows on-demand and controlled dilution of droplets and is simple enough to be useful for assays that require serial dilutions. The approach can also be useful for applications where there is a need to replace or wash fluid from stored drops.

Droplet sensing by measuring the capacitance between coplanar electrodes in a digital microfluidic system.

In this paper, we report a novel method of droplet sensing in a two-plate digital microfluidic system (DMS) based on coplanar capacitance measurement. The total capacitance between the two adjacent electrodes on the lower plate depends on the position of the droplet. Both numerical and experimental results show that the capacitance is maximal at the midpoint between two electrodes. The value of maximum capacitance increases with the volume of the droplet. Further, the measured capacitance is a function of the gaps between the electrodes as well as the plates. This new method of droplet sensing adds to the functionality of DMSs by allowing single plate measurement.

Droplet position control in digital microfluidic systems.

Research on so called digital microfluidic systems (DMS) capable of manipulating individual microdroplets on a cell-based structure has enormously increased in the past few years, mainly due to the demand of the technology-dependent biomedical applications. Significant research in this area has been related to the simulation and modeling of droplet motion, demonstration of different drop actuation techniques on laboratory-scale prototypes, and droplet routing and scheduling for more efficient assay procedures. This paper introduces the basics of the control analysis and design of a DMS, which is a relatively unexplored area in digital microfluidics. This paper starts with a discussion on a simplified dynamic model of droplet motion in a planar array of cells, and continues with more complicated dynamic models that are necessary to realize the structure of an appropriate closed-loop control system for the DMS. The control analysis and design includes both the transient and steady-state responses of the DMS under external driving forces. The proposed control analysis and design approach is implemented into SIMULINK models to demonstrate the performance of the DMS through simulation using the system parameters previously reported in the literature.