Search

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.

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 mm, 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. © 2001 Optical Society of America OCIS codes: 100.2650, 120.2830.

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.

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.