Development of multimode fluorescence correlation microscopy suitablefor high throughout screening and applications in plant signaltransduction research (biological aspects).
11 / 1999 - unknown
The central objective is the development of a real multimode fluorescence correlation microscope (FCM). Implementation of novel technological developments in opto-electronics accurate sample positioning, piezo-mechanical focusing and insertion of multiple exication and detection ports results in a confocal microscope which combines different modalities: (cross-) correlation spectroscopy (diffucion mapping of fluorescent molecules, measurement of concentrations and interactions), quantitative time-laps digital image microscopy (tranching fluorescent molecules in cells during their life cycle) and spectral imaging microspcopy (to characterise emission spectra of microscopic objects). One application of FCM in (bio)technology is the use as a device to screen large numbers of samples. Present advances in biotechnology, biomedicine and chemistry require equipment to screen large numbers of samples both accurately and rapidly, particularly in those situations where biomolecular interactions are studied. FCM allows the creening of such interactions in minute (submicroliter) volumes using 'singel molecule' sensitivity. We will investigate this by screening antibodies, genetically fused to the green fluorescent protein (GFP) of the jellyfish Aequorea victoria, arising from selections of large antibody phage display libraries for binding capapcities. A second application involves the use of the FCM as a research instrument in biology. We will investigate this by studying some molecular events in the sedenetary nematode-plant interaction. Sedentary nematodes are endoparasites inducing feeding cells in the roots of host plants on which they fully rely to complete their lige-cycle. We have chosen this model system because one of the morphological characteristics of these cells is the lack of a central vacuole allowing detailed studies on processes in and around the nucleus. A typical feature of this intimate relationship is the trigering of the cell cycle nematode feeding cells, which never show cell division. Using CFP under control of cell cycle promotors we will measure the buildup and trafficking of this protein. By fusing a red fluorescent and wild type GFP to, respectively, the cell cycle proteins cyc1At (a cyclin) and cdc2a ( a cyclin dependent) kinase we will study their interaction. In a next step the kinase activity of cdc2a will be investigated using a fluorescent substrate. In a final stage we will study the activity of auxin. This plant hormone is one of the key regulators of plant growth and development and as such affects the cell cycle. We recently obtained evidence that this is also true for nematode feeding cell development. Fluorescent auxin analogues will be used to investigate where and how it binds to cells and when the cell cycle is entered as a consequence of this action. Summarising, we will obtain a real multimode FCM useful in both large number screening and biological research. The prior will result in a screening system required in the biotechnological, biomedical, chemical, food and personal health care industries in their search for functional biomolecules. With respect to the latter aspect the model systems used will result in important background knowledge for plant breeding programs.