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Quantitative Brewster angle microscopy of the surface film of human broncho-alveolar lavage fluid

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Abstract

The morphology, thickness and surface pressure of the surfactant film of broncho-alveoalar lavage (BAL) fluid from patients with sarcoidosis were investigated during spontaneous adsorption of the BAL's surface active material at the air/aqueous buffer interface at 37 °C. The biochemical parameters of the BAL fluid determined were protein (Lowry), total phospholipids (from phosphate after ashing) and the individual phospholipids (HPLC). During the spontaneous adsorption of the pulmonary surfactant the surface pressure increased from initially 26 mN/m to 44 mN/m in the equilibrium state. Simultaneously to the increase of the surface pressure, a continuous increase of the reflectivity signal was observed by quantitative Brewster angle microscopy (BAM). The film thickness is calculated from the reflectivity values using an optical model. The effect of the uncertainty of the refractive index, which has to be estimated, is discussed. The BAM images show the inhomogeneous nature of the surfactant film with three distinct phases of different reflectivity, even at relatively low surface pressures. For the brightest phase, the thickness amounts to approximately 12 nm in the equilibrium state of adsorption. This suggests a multilamellar structure. Additionally, we found visual evidence for an adsorption mechanism involving the spreading of vesicles at the interface, in agreement with published results. Differences in the morphology and thickness of the pulmonary surfactant film reported in the literature are obviously due to the varying experimental conditions and materials. We think that the experimental conditions chosen in our study provide a more realistic view of the structure in the lungs in vivo.

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Notes

  1. An example of a non-photometric measurement is the nulling ellipsometry which measures the rotation angles of optical elements at a signal minimum. In this case the absolute magnitude of the signal is not important

  2. The approximation may be easily derived using (1) the trigonometric relations following from the fact that, at the Brewster angle, reflected and refracted beams are perpendicular, (2) Brewster' s law and (3) a thin film approximation where the thickness of the film is small compared to the wavelength

  3. Special care has to be taken with the background signal of the camera when no light is present, as this "black level" will not be scaled in the same way as the light signal when the exposure time changes

  4. A non-linear response, e.g. at high brightness levels, would result in a deviation from the linear curve

  5. The gray level resolution of the camera is not changed by a factor of 20 in this way. For each setting of the exposure time, G still has 8-bit resolution

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Acknowledgements

This work was supported by a grant from the Bundesministerium für Bildung und Forschung (project number: 13N7283/0).

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Authors and Affiliations

  1. Independent Research Group, Surfactant Adsorption Layers, Max-Planck-Institute of Colloids and Interfaces, Am Mühlberg 1, 14424 , Potsdam-Golm, Germany

    Klaus Winsel, Klaus Lunkenheimer & Katrina Geggel

  2. Nanofilm Technology, Göttingen, Germany

    Dirk Hönig

  3. Department of Pneumonology, Charitè, Medical Clinic, Berlin, Germany

    Christian Witt

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  1. Klaus Winsel
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  2. Dirk Hönig
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  3. Klaus Lunkenheimer
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  4. Katrina Geggel
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  5. Christian Witt
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Correspondence to Klaus Winsel.

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Winsel, K., Hönig, D., Lunkenheimer, K. et al. Quantitative Brewster angle microscopy of the surface film of human broncho-alveolar lavage fluid. Eur Biophys J 32, 544–552 (2003). https://doi.org/10.1007/s00249-003-0290-2

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  • DOI: https://doi.org/10.1007/s00249-003-0290-2

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