Surface plasmons fluorescence microscopy

SURFACE PLASMON FLUORESCENCE TECHNIQUES 7. SURFACE PLASMON FLUORESCENCE MICROSCOPY... 

Figure 18. Series of time-lapse surface plasmon fluorescence microscopy images taken during hybridization (a) and dissociation (b) of the target T3, to the surface-attached probes PI - P3 (left column of 3 spots PI (MMI to T3), middle column P3 (MMO), right column P2 (MM2 to T3).

Surface plasmon fluorescence spectroscopy and microscopy are very young techniques. However, the results obtained so far are very promising and hold great potential both for fundamental studies as well as for practical applications, e.g., in sensor development. The obtainable signal-to-noise levels, as well as, the documented lower limit of detection are very encouraging. 

There are several other techniques Uke the fluorescent dye displacement assays, footprinting, Fourier transform infrared spectroscopy. X-ray crystallography, electron microscopy, confocal microscopy, atomic force microscopy, surface plasmon resonance etc used for hgand-DNA interactions that are not discussed here. 

Liebermann T, Knoll W (2003) Parallel multispot detection of target hybridization to surface-bound probe oligonucleotides of different base mismatch by surface-plasmon field-enhanced fluorescence microscopy. Langmuir 9 1567-1572... 

Apart from optical microscopy, there are some other optical techniques which are truly surface sensitive and have found widespread use. Examples are ellipsometry (see Section 9.4.1), total internal reflection fluorescence (TIRF) [316], and surface plasmon resonance techniques [348],... 

Are any of these structures typical of those that would be observed in a pure amphiphile The role played by the probe, which is essential to the fluorescence method, is not completely clear. It has been argued that the formation of dendritic structures in phospholipids is the result of constitutional supercooling, a mechanism that depends on the differential solubility of an impurity between two phases. This may not be the case similar patterns have been observed in LB films by surface-plasmon microscopy, for which no probe is added. The foam structures at the LE-G transition have also been attributed by some to the presence of the probe, but foams have also been observed in monolayers composed solely of a labeled amphiphile. 

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)... 

With some detection technologies compounds can be screened in microarrays instead of plate wells. This requires that either the small molecule or the target be attached to a solid support, usually a glass slide, as shown in Figure 6.9. The other potential binding partner, which can be labeled (say with a fluorescent dye if fluorescence microscopy is used) or unlabeled (if surface plasmon resonance detection, discussed later, is to be used), is then exposed to the shde and interactions are detected. 

X-ray photoelectron spectroscopy Surface plasmon resonance Quartz crystal microbalance Waveguide interfaometry (Spectroscopic) eUipsometry Fluorescence spectroscopy and microscopy (including immunofluorescence, total internal reflection fluorescence)... 

Biomolecule detectors incorporate a biorecognition device capable of selectively recognizing the analyte of interest in connection with a signal transducer and a suitable output device. Transduction methods include a variety of optical (surface plasmon resonance [SPR], fluorescence), electrochemical (voltammetry, impedance, field effect), mechanical (cantilever, surface probe microscopy), and mass-based systems (quartz crystal microgravimetry [QCM], mass spectrometry). Selection of the appropriate transduction system is partially determined by the nature of information sought (quantitative or qualitative), the analyte (concentration, molecular weight), the sample size, and assay timeline. 

Figure 17. Schematic experimental setup for surface-plasmon and surface-plasmon field-enhanced fluorescence microscopy in the Kretschmann configuration.

The absorption spectra of silver nanorods deposited on glass substrates are shown in Figure 47. Silver nanorods display two distinct surface plasmon peaks transverse and longitudinal, which appear at 420 and 650 nm, respectively. In our experiments, the longitudinal surface plasmon peak shifted and increased in absorbance as more nanorods are deposited on the surface of the substrates. In parallel to these measurements, we have observed an increase in the size of the nanorods (by Atomic Force Microscopy, data not shown). In order to compare the extent of enhancement of fluorescence with respect to the extent of loading of silver nanorods deposited on the surface, we have arbitrarily chosen the value of absorbance at 650 nm as a means of loading of the nanorods on the surface. This is because the 650 nm is solely attributed to the longitudinal absorbance of the nanorods. 

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