Fluorescence microscopy interfacing

Figure 9.29 Membrane formation by meteoritic amphiphilic compounds (courtesy of David Deamer). A sample of the Murchison meteorite was extracted with the chloroform-methanol-water solvent described by Deamer and Pashley, 1989. Amphiphilic compounds were isolated chromatographically on thin-layer chromatography plates (fraction 1), and a small aliquot ( 1 p,g) was dried on a glass microscope slide. Alkaline carbonate buffer (15 p,l, 10 mM, pH 9.0) was added to the dried sample, followed by a cover slip, and the interaction of the aqueous phase with the sample was followed by phase-contrast and fluorescence microscopy, (a) The sample-buffer interface was 1 min. The aqueous phase penetrated the viscous sample, causing spherical structures to appear at the interface and fall away into the medium, (b) After 30 min, large numbers of vesicular structures are produced as the buffer further penetrates the sample, (c) The vesicular nature of the structures in (b) is clearly demonstrated by fluorescence microscopy. Original magnification in (a) is x 160 in (b) and (c) x 400.

The other important technique for the study of films at the air/water interface which has recently been introduced is fluorescent microscopy. This technique was introduced by von Tscharner and McConnell [90] and Mohwald [91, 92]. It depends on the fact that certain amphiphilic fluorescent dyes become incorporated into islands of the surface active material under study. Furthermore, where two phases of the surface active material coexist, the dye can often be chosen so that it segregates preferentially into one phase. A shallow Teflon trough is employed with a water immersion objective incorporated into the bottom. The depth of water is adjusted so that the objective focuses on the water surface. The layer of material at the air/water interface is illuminated by a xenon lamp. The fluorescent light so generated passes via the objective and suitable filters to an image-intensified video camera and the image is displayed on a television screen. In some versions of this technique the fluoresence is viewed from above. Most of the pioneering work in this field was devoted to the study of phospholipids, a topic to which we will return. Recently this technique has been applied to the study of pen-tadecanoic acid and this work will be considered here as it relates directly to other papers discussed in this section. 

Fig. 12. The figure at the left is the schematic illustration of laser induced total internal reflection fluorescence microscopy for the single molecule detection at the liquid-liquid interface. Abbreviations ND ND filter, 1/2 1/2 plate, M mirror, L lens, C microcell, O objective (60 x), F band path filter, P pinhole, APD avalanche photodiode detector. The figure at the right shows the composition of the microcell.

Fig. 13. Single molecule detection of Dil at the dodecane-water interface by fluorescence microscopy (left). Short photon burst in the SDS systems and (right) long burst in the DMPC systems.

Numerous techniques have been employed to examine the monolayer structure of phospholipids at the air/water interface including surface tension, fluorescence, neutron and X-ray reflection, and IR and Raman spectroscopy. In contrast, very few techniques are suitable to examine monolayers at the oil/water interface. Surface tension and fluorescence microscopy [46-48] have shed some light on these buried monolayers, but most other surface techniques are hampered because of effects from the bulk liquids. Since VSFS is insensitive to the bulk, it is an excellent technique for probing these monolayers. 

The first observation of the single molecule detection at the liquid interface has been recently accomplished by means of the TIR fluorescence microscopy [13]. Figure 10.5 shows the optical arrangement of the measurement method and the microcell used in the study. 

FIGURE 10.5. The left figure shows the schematic illustration of laser-induced fluorescence microscopy under the total internal reflection for the detection of single Dil molecules at the dodecane-water interface. Abbreviations ND, ND filter XJ2, kl2 plate M, mirror L, lens C, microcell containing dodecane and aqueous phases O, objective (60 x ) F, bandpath filter P, pinhole APD, avalanche photodiode detector. The right portion of the figure shows the composition of the tnicrocell. 

K.J. Stine and C.M. Knobler, Fluorescence Microscopy A Tool for Studying the Physical Chemistry of Interfaces, Ultramicroscopy 47 (1992) 23. (Review short introduction to fluorescence and fluorophores basic instrumentation for fluorescence microscopy and extensions to study dynamics and resonance energy transfer, confocal scanning microscopy results obtained with Langmuir monolayers.)... 

In the present work, we study ice crystal growth in AFGP solutions with phase contrast and fluorescence microscopies in a 1-directional growth apparatus. With fluorescence microscopy we have directly visualized the protein dynamics at the interface of a growing ice crystal. Contrary to previous understandings, the proteins become incorporated into veins and not directly into the crystal matrix." This indicates that the proteins only weakly adsorb to the interface. Under slower growth conditions no veins are... 

Early studies on monolayers of chiral molecules like 2-hydroxyalkanes, amphiphilic amino acids, 2-methylhexacosanoic acid esters, and hydroxy-hexadecanoic acid and its esters have been reviewed. The interesting question about monolayers of chiral molecules is whether the parameters which can be determined and the phase transitions are different for pure enantiomers and racemates. For components of biomembranes like phosphatidylcholines 10 this appears not to be the case," but for synthetic compounds like iV-(a-methylbenzyl-stearamide) 11 specific interactions between the molecules of the enantiomers are observed (Chart 2). ° In recent years, advanced techniques have been developed to probe the order in monolayers at the air-water interface, including surface X-ray diffraction, and microscopic techniques, viz. fluorescence microscopy, and Brewster angle microscopy (BAM). The X-ray diffraction technique has been used to identify homochiral and heterochiral two-dimensional domains in mono-layers of racemic amphiphilic amino acids on subphases containing glycine. Fluorescence microscopy requires the introduction in the monolayer of a small... 

Spiral structures can also arise in monolayers of achiral molecules, as in the study by polarized laser excitation fluorescence microscopy of chiral defects in pentade-canoic acid. Using a rigid achiral molecule with a series of chiral centers (14), separation of chiral domains in the racemate has been observed by atomic force microscopy (AFM) on monolayers transferred from the air-water interface to... 

Total internal reflection fluorescence microscopy (TIRFM) is a promising alternative approach to low background fluorescence imaging [68], For excitation of molecules on a surface or within a thin slice of the sample, an evanescent optical field is used traveling along the interface between a medium with a high refractive index n, (typically a quartz glass prism) and... 

As a consequence, researchers from different disciplines of the life sciences ask for efficient and sensitive techniques to characterize protein binding to and release from natural and artificial membranes. Native biological membranes are often substituted by artificial lipid bilayers bearing only a limifed number of components and rendering the experiment more simple, which permits the extraction of real quantitative information from binding experiments. Adsorption and desorption are characterized by rate constants that reflect the interaction potential between the protein and the membrane interface. Rate constants of adsorption and desorption can be quantified by means of sensitive optical techniques such as surface plasmon resonance spectroscopy (SPR), ellipsometry (ELL), reflection interference spectroscopy (RIfS), and total internal reflection fluorescence microscopy (TIRE), as well as acoustic/mechanical devices such as the quartz crystal microbalance (QCM)... 

Herron, J. N., Muller, W., Paudler, M., Riegler, H., Ringsdorf, H., Sici, P. A. (1992). Specific recognition-induced self-assembly of a biotin lipid/streptavidin/Fab fragment triple layer at the air/water interface Ellipsometric and fluorescence microscopy investigations, Langmuir, 8 1413. 

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