| Popis: |
In transmission-through-dye (TTD) microscopy, a nontoxic and cell-impermeant dye Acid Blue 9 is added to the cell culture medium, and the sample is placed in a shallow gap that is just slightly deeper than the cells. In the simplest case, such a gap can be formed between a coverslip and a slide kept apart by a small amount of grease. Alternatively, an attachment to a condenser with a horizontal watertight window at the bottom can be built for an inverted microscope; it is lowered into a Petri dish to bring the window close to the cells, thus creating a temporary narrow gap between the cells and the window. As the cells displace the dye, the depth of the absorbing layer becomes complementary to cell thickness; when such a sample is imaged in transmitted light at the wavelength of maximal dye absorption (630 nm), thicker cells appear brighter. By applying logarithmic transformation to the image and correcting it for the background level, the image contrast can be quantitatively converted to cell thickness and volume [1]. The accuracy of measurements by TTD imaging has been verified using spherical beads and confocal scanning. Dead cells with leaky plasma membranes are darker than the background and are easily distinguishable from intact cells. TTD can be realized on any widefield microscope, with only a single bandpass filter added anywhere in the optical path, or on a laser scanning microscope in transmission mode. It is well suited for prolonged time-lapse experiments because the cells are not exposed to high-intensity illumination and the results are not affected by possible instability in the light source. TTD is also compatible with fluorescence imaging, making possible simultaneous measurements of the cell volume and various fluorescent markers [2]. We are currently using this method to investigate cell volume changes during apoptosis and the osmotic properties of normal and apoptotic cells. |
