Diffusion of microspheres in sealed and open microarrays.

In several experiments, we study the diffusion of microspheres with different radii in microarrays filled with a variety of aqueous solutions of ethylene glycol. We study diffusion in open and closed (sealed) microarrays. In sealed nanoliter wells, the tracers show pure diffusion, whereas in open reactors, a radial outward-directed evaporation-induced liquid flow is superimposed onto the diffusion. In general, one of the following quantities can be calculated if the others are known: the temperature, the viscosity of the medium, the radius of the microbeads, or the diffusion constant. The estimated diffusion constants in closed microarrays are in good agreement with theoretical predictions based on the Brownian motion. We monitor the motion of the microbeads under a microscope and extract their paths in time from the digital recordings. Ambiguous paths due to the crossing of two trajectories can be detected. We show that low microsphere concentrations or high viscosities do not hamper a robust estimation of the diffusion parameters.

Monitoring enzymatic reactions in nanolitre wells.

We have developed a laboratory-on-a-chip microarray system based on nanolitre-capacity wells etched in silicon. We have devised methods for dispensing reagents as well as samples, for preventing evaporation, for embedding electronics in each well to measure fluid volume per well in real-time, and for monitoring the fluorescence associated with the production or consumption of NADH in enzyme-catalysed reactions. Such reactions can be found in the glycolytic pathway of yeast. We describe the design, construction and testing of our laboratory-on-a-chip. We also describe the use of these chips to measure both fluorescence (such as that evidenced in NADH) as well as bioluminescence (such as evidenced in ATP assays). We show that our detection limit for NADH fluorescence is 5 micro m with a microscope-based system and 100 micro m for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 micro m for ATP/luciferase bioluminescence.

Ring formation in nanoliter cups: quantitative measurements of flow in micromachined wells.

Drying of DNA spots on microarrays and spilled coffee yields ringlike stains, because the outward flow transports dissolved particles to the border. Contact line pinning and diffusion limited evaporation of a liquid sample are the two necessary conditions to induce an outward directed liquid flow during evaporation. In this paper we present quantitative measurements of this flow field visualized by microspheres which are injected into a liquid sample in circular wells with a radius of 100-150 micro m and a depth of 6 micro m. The motion, including Brownian motion, of these microspheres with a radius of 0.25 micro m is recorded using digital fluorescence microscopy. Our analysis, using optic flow, does not require object identification, nor tracking of the individual objects. The spatiotemporal measurement space is sparsely filled at only those space/time positions where a microsphere is present. A confidence measure is computed indicating the presence of microspheres in this measurement space. The circular well shape allows us to transform the sparse measurement space into a denser, averaged radial velocity field. In this transformation we "interpolate" the radial velocity between values with a high confidence, which results in quantitative measurements of this outward flow field during the complete time interval of the evaporation process and at all radial positions in the circular wells. This allows for a quantitative validation of the elegant theory of ring formation.

Temporal phase-unwrapping algorithm for dynamic interference pattern analysis in interference-contrast microscopy.

A temporal phase-unwrapping algorithm has been developed for the analysis of dynamic interference patterns generated with interference-contrast microscopy in micromachined picoliter vials. These vials are etched in silicon dioxide, have a typical depth of 6 mum, and are filled with a liquid sample. In this kind of microscopy, fringe patterns are observed at the air-liquid interface. These fringe patterns are caused by interference between the directly reflected part of an incident plane wave and the part of that wave that is reflected on the bottom of the vial. The optical path difference (OPD) between both parts is proportional to the distance to the reflecting bottom of the vial. Evaporation decreases the OPD at the meniscus level and causes alternating constructive and destructive interference of the incident light, resulting in an interferogram. Imaging of the space-varying OPD yields a fringe pattern in which the isophotes correspond to isoheight curves of the meniscus. When the bottom is flat, the interference pattern allows for monitoring of the meniscus as a function of time during evaporation. However, when there are objects on the bottom of the vial, the heights of these objects are observed as phase jumps in the fringes proportional to their heights. First, we present a classical electromagnetic description of interference-contrast microscopy. Second, a temporal phase-unwrapping algorithm is described that retrieves the meniscus profile from the interference pattern. Finally, this algorithm is applied to measure height differences of objects on the bottom in other micromachined vials with a precision of ~5 nm.