L.R. van den Doel

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 µm with a microscope‐based system and 100 µm for an embedded photodiode system. The photodiode system also provides a detection limit of 2.4 µm for ATP/luciferase bioluminescence.

Quantitative evaluation of light microscopes based on image processing techniques

In this note we will present methods based on image processing techniques to evaluate the performance of light microscopes. These procedures are applied to three different ‘high-end’ light microscopes. Tests are carried out to measure the homogeneity of the illumination system. From these tests it follows that Kohler illuminated images can have an exceedingly high amount of shading. Another result found from the illumination calibration is that traditional lamp housings are not designed to make fine-tuning easy. Next, the (automated) stage is considered. Several tests are performed to measure the stage motion in the xy-plane and in the axial direction to address accuracy, precision, and hysteresis effects. Finally, the entire electro-optical system is characterized by measuring the optical transfer function (OTF) at wavelengths 400 nm, 500 nm, 600 nm, and 700 nm. The results of these experiments show that there is a consistent deviation from the theoretical OTF at wavelengths around 400 nm. The final conclusion is that modern light microscopes perform better than their five-to-ten-year-old predecessors.

Nanometer-scale height measurements in micromachined picoliter vials based on interference fringe analysis

Micromachined picoliter vials in silicon dioxide with a typical depth of 6.0 /spl mu/m are filled with a liquid sample. Epi-illuminated microscopic imaging during evaporation of the liquid shows dynamic fringe patterns. These fringe patterns are caused by interference between the direct part and the reflected part of an incident plane wave (reflected from the bottom of the vial). The optical path difference (OPD) between the direct and the reflected wave 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 monitoring of the liquid meniscus as a function of time during evaporation. On the other hand, when there are objects on the bottom of the vial, the height of these objects are observed as phase jumps in the fringes proportional to their height. First, this paper presents the underlying optical model. Secondly, an image processing method is described to retrieve the meniscus profile from the interference pattern. This algorithm is based on estimating the wrapped (relative) phase of the fringe pattern in the recorded images. Finally, this algorithm is applied to measure height differences on the bottom in other micromachined vials with a precision of about five nanometer.