Cells of the bakers yeast (Sacharomyces cerevisiae) are very small eukaryotic single cell organisms, whose genetics has been studied extensively. Many fundamental questions about genetic regulatory pathways have been first established in yeast. For this reason imaging assays with this model organism are of great interest, e.g. to study intracellular transport or localization phenotypes of many gene products.
Nevertheless imaging live yeast expressing proteins tagged with fluorescent proteins poses a big challenge for a number of reasons: Because cells are small, the number of molecules expressed in a cell usually is small, so only very little light is emitted. Also, yeast cells are especially prone to damage by phototoxic effects due to fluorescent illumination. Therefore, care has to be taken to minimize photobleaching and unnecessary illumination when imaging live yeast cells. The short movie shows the movement of the kinetochore bodies in live yeast in a 2 channel time lapse series (GFP and Nomarski/DIC) without any apparent negative effects on cell viability.
Technical details
Yeast cells were suspended in growth medium and mounted on an agar covered slide with a cover slip and observed on an Axioplan 2 imaging upright microscope with a Plan Neofluar 100x/1.30 Oil Iris (DIC III) objective. The slide was screened for cells in DIC contrast mode, cells were focused and brought into the camera field of view. GFP was illuminated using a HBO mercury vapour lamp and the filterset #44. Both aperture diaphragm and field diaphragm of the incident light path were stopped down to minimize light exposure. With these adjustments in place, GFP fluorescence is too weak to be easily observed through the oculars of the microscope.
Imaging was performed using an AxioCam MRm CCD camera. The fully motorized microscope and the camera were controlled by AxioVision 4. The high speed incident light shutter was under direct trigger control by the camera to avoid unnecessary illumination of the sample. A fixed exposure time of 200 msec was used for the GFP channel, a 50 msec exposure was used for the DIC channel. Time lapse imaging was done for about 6 minutes taking a two channel image every 10 seconds.
The following AxioVision modules were used: Multichannel fluorescence and Time lapse.
Only about 10% of the cameras 12 bit dynamic range was used for the fluorescent channel which can be made visible on a computer screen by adjusting AxioVision’s display line of the image histogram to a min/max configuration. This adjustment maps the available pixel intensities in the image to the available 8 bit dynamic range of the computer screen.
After similar contrast adjustment of the DIC channel the time lapse series was exported as a colour merge movie using the AxioVision export function.
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