Adsorption fluorescence microscopy

Fig. 6 Adsorption of microcapsules onto the (PLL/HA)24/PLL films, (a-c) Confocal fluorescent microscopy images of the capsules exposed to the near-IR light irradiation, (d) CLSM image of the film surface (the film is prepared with PLL-FITC black lines are scratches made by a needle for easier film imaging), (e) Cross-sectional profile of the capsules after step-by-step laser exposure (the sections from top to bottom correspond to the images a-c, respectively), (f) Optical microscopy images of the capsules after light irradiation. Scale bars (a-c, f) 4 pm, (d) 25 pm. Reproduced from [100]...

Measurement of Adsorption and Uptake by Flow Cytometry, Fluorimetry and Confocal Fluorescence Microscopy... 

Fig. 10.11 Adsorption of 3LNPs with PKH26 on SKOV-3 cell membrane at pH 7.4 (a, b) and 6.0 (c, d) at 4°C observed with confocal scanning laser fluorescence microscopy. Differential interference contrast (a, c) and red fluorescence channel (b, d)...

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)... 

The electrochemical systems studied by the fluorescence method are based upon the adsorption of lipid-hke compounds similar to the molecules that make up the cell membrane. The vast literature of methods and a variety of fluoro-phores are available for our use in the study of electrochemical systems. A brief review of the use of fluorescence microscopy in the study of biological systems is presented, because a number of the probes used for staining the biological structures are relevant for the electrochemical work presented. Moreover, the methods used in biological imaging to encourage fluorescence and to improve contrast are relevant for the work on electrode surfaces. 

Fluorescence Microscopy of the Adsorption of DOPC onto an Hg Drop... 

Here, a laser beam totally internally reflects at a sohd/hquid interface, creating an evanescent field, which penetrates only a fraction of the wavelength into the liquid domain. When using planar phosphoHpid bilayer and fluorescently labeled proteins, this method allows the determination of adsorption/desorption rate constants and surface diffusion constants [171—173]. Figure 6.29 shows a representative TIRF-FPR curve for fluorescein-labeled prothrombin bound to planar membranes. In this experiment the experimental conditions are chosen such that the recovery curve is characterized by the prothrombin desorption rate. It should be mentioned that, similar to other applications of fluorescence microscopy, two and three photon absorption might be combined with FRAP in the near future. 

Protein adsorption studies on artificial hip-joint materials have focused mainly on albumin, as this is the most abundant protein in synovial fluid. Studies to evaluate albumin adsorption on ceramics and metals have been carried out using XPS and radiolabeling techniques [10.11] as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and chromatography [12,13]. Fluorescence microscopy has been applied to evaluate albumin adsorption on UHMWPE [6,14]. 

Samples, one half coated with SiOa and the other half with Ti02, were used for quantitative surface analysis after each of the siuface treatment steps (cleaning, self-assembly, and polymer and protein adsorption, section 2). These samples exhibit material contrast on a macroscopic scale and are discussed in section 3.1. Micropat-temed surfaces were subjected to identical siuface modification procedures and characterized qualitatively by imaging ToF-SIMS (section 3.2) and fluorescence microscopy (section 3.3) and were used in the cell experiments (section 3.4). In both types of samples, material contrast (on a macroscopic or microscopic scale, Figure la) is converted into contrast with respect to protein adhesion (Figure Ic) via a series of surface modification steps (self-assembly of DDP, adsorption of PLL-g-PEG section 2). 

Figure 4. Fluorescence microscopy image on Oregon Green labeled streptavidin subjected to the SMAP-treated, 5x5 gw Ti02 in S102 substrate. Streptavidin adsorption can only be observed on the Ti02/DDP spots, while the Si02/PLL-g-PEG remains protein resistant. The inset shows the local distribution of fluorescence of the Oregon Green labeled streptavidin across the surface (in arbitrary units). A contrast of 100 1 was observed.

One of the simplest methods to study adsorption at the oil water interface is to measure the variation of interfacial tension as a function of concentration. If the polymer used for adsorption is monodisperse, then the Gibbs equation (51) may be used to estimate the surface excess. However, if the polymer is polydis-perse, this method will give erroneous values of the surface excess because the larger molecules will tend to adsorb preferentially, and the equation is imable to account for this adsorption behavior. As a result, most of the data available in the literature report the change in the interfacial tension as a function of concentration without attempting to convert it into an adsorbed amoimt. Apart from interfacial tension measurements, other techniques such as total internal reflection fluorescence microscopy (52) and scintillation measurements from radiolabeled polymers (53) have also been used to measure the adsorption at the liquid-liquid interface. 

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