Phase-Contrast Light Microscopy of Living Cells Cultured in Small Volumes

Phase contrast, one of the most commonly used contrasting techniques in light microscopy, has crucial drawbacks when used to image cells in small culture wells due to the formation of a meniscus at the airwater interface. In this study we demonstrate how it is possible to perform phasecontrast microscopy on living cells in volumes of less than 30 µl. Adherent cells were cultured in the 25 µl channels of microslides, fluorescently stained, and observed by phase-contrast and fluorescence microscopy. 95% of the cells were accessible by phase contrast in the channels, whereas in the 96-well plates only 1% of the culture area showed satisfactory contrast. In order to explain this finding we have analysed the optical basis of phase-contrast microscopy to elucidate the disturbing influence of a meniscus on the optical light path.