Multi-Dimensional Laser Confocal Microscopy on Live Cells in Submicroliter Volumes Using Glass Capillaries

Imaging live cells using laser confocal microscopy requires the use of complex and rather cumbersome incubation chamber systems in order to maintain the correct physiological conditions. The volume of these chambers is in the range of a few hundred microliters. Here we present an easy and convenient alternative in the form of glass capillaries that accommodate volumes of 0.2–10 microliters. The capillaries can be loaded with both suspension and adherent cells. The loaded capillaries are taped on microscope slides and submerged into the immersion oil that covers the objective. The correct temperature is maintained using a thermostat-controlled objective heater. We demonstrate that using microlens enhanced ­rotating Nipkow disc based confocal illumination, in combination with cold CCD cameras, maximum resolution multicolor time lapse fluorescence images can be obtained from live cells. The images obtained are free from disturbing optical distortions. Imaging in submicroliter volumes allows for fluorescence visualization of very rare cell types isolated using flow or affinity sorting or obtained by fine needle biopsies.