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 Köhler 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.