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Radboud universityFaculty of ScienceBiologyHOMEWEB MODULESLight microscopy techniques > Video microscopy

Video microscopy

By combining light microscopy with image enhancement dynamic processes, like organelle movement, can be visualized within living cells.
During the video microscopy course that Prof. dr. I.K. Lichtscheidl (University of Vienna) gave in the framework of a European collaboration program with the RU Nijmegen, special video and light microscopy techniques were demonstrated on tip-growing plant cells (here: Medicago sativa root hairs and Amaryllis pollen tubes). The film sequences shown below are -compressed- real-time views acquired by employing high numerical aperture optics and monochromatic light (a.o. ultraviolet light with quarts optic), and by applying background correction and analog cq digital contrast enhancement.
Technique and object Example
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Bright field without background subtraction
Left: still image. Movie right: germinated pollen grains in which the spindle-shaped generative cell can be discerned, followed by a zoomed view of the movement of organelles in the pollen tube (diameter about 10 micrometer)
Image bright field 46s; real-time
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Video-Enhanced-Contrast (VEC)-bright field
By applying background subtraction in VEC microscopy in conjunction with analog and digital contrast enhancement, the presence and movement of small particles can be determined in more detail, like here orgnalles in the tip of a root hair. Movie right: Chaotic movement is typical for the extreme tip region, whereas directed movement can be seen away from the apex
Image VEC 42s
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Phase-contrast does not only produce contrasted images but also a kind of halo around particles. With this technique it is therefore difficult to identify single organelles in the dense cytoplasm at this pollen tube tip, but the technique works beautifully to image a thin layer of cytoplasm (i.e. a vacuolate cell region) or bacteria
Image phase contrast 38s; real-time:
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Differential Interference Contrast (DIC)
Particles and objects differing in optical density appear with a shadow-effect. Thanks to this property single organelles (for example mitochondria) can be tracked in living cells, like in this pollen tube
Image DIC 32s; real-time
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Dark field
Dark field microscopy is an ideal technique to image tiny particles (e.g. gold-particles for labeling, bacteria, irregularities in the cell wall, silver grains in in situ hybridization, single organelles). In this pollen tube organelles appear as bright objects in a dark background
Image dark field 32s; real-time:
wmv (843 kb)
These views were made with UV light (370, 410, 440nm) that is not suitable for direct eye vision, but can be well detected by a special video camera. With this set-up very small particles, probably Golgi vesicles (about 300 nm in size) that exhibit an extremely rapid chaotic movement, can be observed along the cell membrane in de tip of this pollen tube
Image ultraviolet microscopy 11s; real-time:
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Recommended literature as rtf text or PDF document

These images and movie sequences are protected by the copyright terms of the Radboud Universiteit Nijmegen; in contrast to other vcbio material these views are "intellectual" property of the IECB To the IECB, University of Vienna

Prof. Dr. Irene Lichtscheidl
Cell Physiology and Scientific Film
"IECB = Institute of Ecology and Conservation Biology
University of Vienna (Austria)

last modified: 17 Oct 2011