- Autor
- Seitenbereich
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011-019
- Zusammenfsg.
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Transmitted light and fluorescence microscopy often suffers from the limited Depth of Field (DOF) of microscope objectives. For this reason, plant cells can in most cases not completely be visualized in a single image at suitable magnification. In case of fluorescence signals, this problem can to some extent be circumvented by the use of confocal laser scanning microscopy (CLSM) which is able to remove disturbing light that is out of focus, thus leading to a clear image within the focal plane. Several of such confocal images can then be combined to provide a virtual image pretending a greater DOF. However, this trick is not possible for transmitted bright field microscopy, not even if CLSM operating in the imaging mode is applied. Instead, stacking software packages, which are based on algorithms capable of recognizing those parts of an image that are in focus, might provide an alternative approach to overcome this problem. Here we show that such software packages can be used to assemble mathematically the focal information of a z-stack of arbitrary microscope pictures into a virtual single image. This virtual image shows an enhanced DOF irrespective of whether the original data are epifluorescence or transmitted light microscopy images of plant cells and if they were obtained by conventional microscopy or CLSM. This method thus provides an affordable alternative to confocal image analysis and a meaningful tool in plant cell imaging. Furthermore, this method is even suitable to visualize organelle movement within a single image.
