An epifluorescence microscopic surface balance to observe monolayers at the air-water interface

Nag, Kaushik (1991) An epifluorescence microscopic surface balance to observe monolayers at the air-water interface. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (21MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

The details of the design and construction of an epifluorescence microscopic surface balance and some preliminary observations on lipid monomolecular layers performed using this balance are discussed. The balance consists of a Teflon trough with a computer - controlled movable barrier mounted on a vibration reducing platform. The balance is equipped with an epifluorescence microscopic attachment by which visual observation of fluorescent probes in the monolayer is possible. Barrier movement can be utilized to give monolayer compression and expansion velocity from 20 mm²/sec up to 600 mm²/sec. A Wilhelmy dipping plate connected to a force transducer is used to measure surface tension in the monolayer during compression and expansion. The epifluorescence microscope is coupled to a charge couple device in tandem with a microchannel plate which permits observation of the low light level fluorescence from the monolayer. Images of the monolayer under compression were visualized, stored, digitized and processed using a video unit and operator interactive software. -- Using the balance preliminary studies of phase behaviour have been performed for monolayers of dipalmitoyl phosphatidylcholine (DPPC) and other lipids. The phase changes were observed by incorporating 1 mol% of a fluorescent probe, NBD-PC, into the lipid forming the monolayer. This probe had no appreciable effect on the isotherms and surface tension properties of DPPC in monolayers. DPPC was observed to undergo a number of phase transitions when compressed in monolayers. The liquid - expanded to liquid - condensed phase transition was visualized as the formation of dark patches (domains) from a fluorescent background. The domains increased in average size from 100 μ² to 1500 μ² in a nonlinear manner as a function of area occupied per molecule of the DPPC, when monolayers were compressed at a rate of 20 mm²/sec. The size, shape and growth of these domains were quantitatively characterized and found to be dependent on compression rates. At a fast speed of compression (4 A²/molecule/sec) the domains were smaller in size, more irregularly shaped, and unimodally distributed. At a slow compression speed (0.13 A²/molecule/sec) the domains were larger in size and more regular in shape. The shapes of the domains were found to change dramatically when a small amount (2 mol %) of cholesterol was incorporated in the monolayer. No domain formation was observed in monolayers for lipids with one or two unsaturated chains.

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