A microfluidic device and computational platform for high-throughput live imaging of gene expression.

To fully describe gene expression dynamics requires the ability to quantitatively capture expression in individual cells over time. Automated systems for acquiring and analyzing real-time images are needed to obtain unbiased data across many samples and conditions. We developed a microfluidics device, the RootArray, in which 64 Arabidopsis thaliana seedlings can be grown and their roots imaged by confocal microscopy over several days without manual intervention. To achieve high throughput, we decoupled acquisition from analysis. In the acquisition phase, we obtain images at low resolution and segment to identify regions of interest. Coordinates are communicated to the microscope to record the regions of interest at high resolution. In the analysis phase, we reconstruct three-dimensional objects from stitched high-resolution images and extract quantitative measurements from a virtual medial section of the root. We tracked hundreds of roots to capture detailed expression patterns of 12 transgenic reporter lines under different conditions.

Note: curve fit models for atomic force microscopy cantilever calibration in water.

Atomic force microscopy stiffness calibrations performed on commercial instruments using the thermal noise method on the same cantilever in both air and water can vary by as much as 20% when a simple harmonic oscillator model and white noise are used in curve fitting. In this note, several fitting strategies are described that reduce this difference to about 11%.

Electrosprayed core-shell microspheres for protein delivery.

This communication describes a single-step electrospraying technique that generates core-shell microspheres (CSMs) with encapsulated protein as the core and an amphiphilic biodegradable polymer as the shell. The protein release profiles of the electrosprayed CSMs showed steady release kinetics over 3 weeks without a significant initial burst.

Polymeric particle formation through electrospraying at low atmospheric pressure.

Electrospraying is a simple and versatile technique capable of producing polymeric particles. However, most investigations carried out thus far have been performed at ambient atmospheric pressure without studying the influences of pressure on the formation of polymeric particles. Here, we report our investigation on the effects of varying the pressure and the solution concentration on the microstructures of electrosprayed polymeric particles. Pressures are varied from ambient atmospheric pressure to 380 mmHg below ambient pressure, and solution concentrations are varied over a range of 3-7 w/v %. By varying these parameters, we manipulated the rate of solvent evaporation and the solidification of the electrosprayed particles. The results show that changes to the pressure had significant effects on the microstructure and morphology of poly(epsilon-caprolactone) (PCL) particles. The average particle size became larger as the chamber pressure decreased. At a solution concentration of 5 w/v % and a pressure 150 mmHg below ambient pressure, uniform and spherical PCL particles were generated. Electrospun fibers were formed when a solution concentration of 7 w/v % was used. The developed technique can be applied to prepare polymeric drug delivery carriers though a low-pressure-assisted spray-drying method, and is particularly suitable for fabricating delivery microspheres encapsulated with temperature-sensitive drugs and biomolecules.

A method for atomic force microscopy cantilever stiffness calibration under heavy fluid loading.

This work presents a method for force calibration of rectangular atomic force microscopy (AFM) microcantilevers under heavy fluid loading. Theoretical modeling of the thermal response of microcantilevers is discussed including a fluid-structure interaction model of the cantilever-fluid system that incorporates the results of the fluctuation-dissipation theorem. This model is curve fit to the measured thermal response of a cantilever in de-ionized water and a cost function is used to quantify the difference between the theoretical model and measured data. The curve fit is performed in a way that restricts the search space to parameters that reflect heavy fluid loading conditions. The resulting fitting parameters are used to calibrate the cantilever. For comparison, cantilevers are calibrated using Sader's method in air and the thermal noise method in both air and water. For a set of eight cantilevers ranging in stiffness from 0.050 to 5.8 N/m, the maximum difference between Sader's calibration performed in air and the new method performed in water was 9.4%. A set of three cantilevers that violate the aspect ratio assumption associated with the fluid loading model (length-to-width ratios less than 3.5) ranged in stiffness from 0.85 to 4.7 N/m and yielded differences as high as 17.8%.