Exploring mechanical factors in the control of pulmonary surfactant release with optical tweezers

Research project P 14263 Exploring pulmonary surfactant release with optical tweezers Monika RITSCH-MARTE 06.03.2000 We propose to study the mechanisms by which cellular mechanical forces control the release of surfactant in specialized cells in the lung. Insight into these mechanisms is important since normal lung function depends essentially on the presence of a film of pulmonary surfactant which reduces the surface tension. The mechanism of lubrication is exocytosis of lamellar bodies out of alveolar type II (AT II) cells. In our experiments we try to find out whether this kind of exocytosis is stimulated by mechanical stretch of these cells, as e.g. applied during the breathing action. `Dual-beam optical tweezers` (i.e. a later trap for cells with two trapping beams) represent the ideal tool to this end, since they allow to stretch cells in a very controlled way. Simultaneously, exocytosis of single lamellar bodies within the cell can be directly observed using a fluorescence imaging method. Combining optical tweenzers with fluorescence imaging methods for measuring the released surfactant, we expect to establish quantitative relationships between mechanical forces and physiological responses. The investigations will be carried out on single cells. Thus the present project represents an important step towards a better understanding of the relevance of mechanical stretch, as thought to be generated in the lung during inspiration, for release of surfactant.

Optical tweezers can be used to manipulate micrometer-sized dielectric particles. In particular, they can be used to manipulate biological specimens without damage. Within the framework of this project this technique was for the first time used to investigate cells of the lungs` epithel (AT II cells). These cells are part of the alveolus and they secret a material, the so-called surfactant, which - before secretion - is stored in special vesicles. Once secreted to the alveolus, it lowers the surface tension of the lung, thereby facilitating breathing. Accordingly, lack or dysfunction of surfactant is the cause of severe respiratory diseases. In the course of this project we have developed a method enabling the quantitative investigation of the mechanisms determining the release of surfactant. Our results represent an important contribution in the understanding of processes controlling normal secretion of surfactant. Furthermore they might be beneficial to future studies dedicated to the understanding of the respiratory distress syndrome or to the optimization of artificial respiration technology. Optical tweezers exert forces in the pico-Newton regime. Reversely, this can be utilized to measure forces in this range. We have developed an easy-to-use force calibration method which is capable of determining the three dimensional force profil of the trap. The method is based on monitoring of the trapping dynamics. A possible disadvantage of optical tweezers is the limitation of accessible trapping positions to the microscope`s field of view. Furthermore, in optical tweezers only one particle at a time can be trapped. In order to obtain a manipulation tool that overcomes these restrictions we have combined our optical tweezers setup with a fiber- optical trap, which can be moved independently from the optical tweezers. Such an optical "micro-abacus" allows addressing and manipulating of individual particles as well as collective handling of several particles. Interestingly, if the particle size is comparable to the wavelength of the fiber trap, the behaviour of several simultaneously trapped particles strongly depends on the actual size of the particles. We performed a systematical investigation of the self-organization of particles in the trap and explained the phenomenon by a theoretical model.