Surface plasmon resonance microscopy
[2] The heterogeneity of the refractive index of the metallic surface imparts high contrast images, caused by the shift in the resonance angle.
[4] SPRM is used to characterize surfaces such as self-assembled monolayers, multilayer films, metal nanoparticles, oligonucleotide arrays, and binding and reduction reactions.
[10] Since polaritons are highly sensitive to small changes in the refractive index of the metallic material,[11] it can be used as a biosensing tool that does not require labeling.
In simple words, SPRI technology is an advanced version of classical SPR analysis, where the sample is monitored without label through the use of a CCD camera.
The SPRI technology with the aid of CCD camera gives advantage of recording the sensograms and SPR images, and simultaneously analyzes hundreds of interactions.
[25] Radiation of different wavelengths (green, red and blue) was converted into surface plasmon polaritons, through the interaction of the photons at the metal/dielectric interface.
[citation needed] Figure 4 shows the leakage light captured by a color CCD camera, of the green, red and blue photons in gold (a) and silver (b) films.
[26] The metallic film is capable of absorbing light due to the coherent oscillation of the conduction band electrons induced by the interaction with an electromagnetic field.
This modification causes resonance angle shifting as in the example shown in Figure 10, from θ1 to θ2 due to the change on the surface plasmon propagation constant.
In the past decade, SPR sensing has been demonstrated to be an exceedingly powerful technique and used quite extensively in the research and development of materials, biochemistry and pharmaceutical sciences.
[49] The SPRM instrument works with the combination of the following main components: source light (typically He-Ne laser), that further travels through a prism that is attached to a glass side, coated with a thin metal film (typically gold or silver), where the light beam reflects at the gold/solution interface at an angle greater than the critical angle.
Although the above-mentioned components are some important for SPRM, additional accessories such as polarizers, filters, beam expanders, focusing lenses, rotating stage, etc., similar to several imaging methods are installed and used in the instrumentation for an effective microscopic technique as demanded by the application.
Depending on the applications, and to optimize the imaging technique, the researchers modify this basic instrumentation with some design changes that even include altering the source beam.
[citation needed] SPRM is a useful technique for measuring concentration of biomolecules in the solution, detection of binding molecules and real time monitoring of molecular interactions.
It has been the challenging thing to investigate the involvement of membrane proteins in disease biomarkers and therapeutic targets and its binding kinetics with their ligands.
Atomic force microscopy (AFM) is an excellent method for obtaining high spatial resolution images of membrane proteins,[59] but it might not be helpful to investigate its binding kinetics.
SPR microscopy (SPRM) makes possible to simultaneously optical and fluorescence imaging of the same sample, which prove to get the advantages of both label-based and label-free detection methods in the single setup.
Mismatching of bases in the DNA sequence leads to the number of lethal diseases like lynch syndrome which has high risk of colon cancer.
First G-G mismatch pair is stabilized by attaching it with the ligand, naphthyridine dimer, through hydrogen bonding which make the hairpin structures in double stranded DNA on gold surface.
These results demonstrate that SPR imaging is a promising tool for monitoring single base mismatches and screen out the hybridized molecules.
Based on these results, SPR imaging technique can be opted as diagnostic tool for studying the antibody interactions to protein arrays.
Coupling of SPRM with MALDI-mass spectrometer (SUPRA-MS) enables the multiplex quantification of binding and molecular characterization on the basis of different masses.
To study the aptamer-protein interaction, first oligonucleotides are grafted through formation of thiol Self Assembling Monolayer (SAM) on gold substrate using piezoelectric dispensing system.
Target oligonucleotides having a primary amine group at their 59th end are conjugated to HS-C (11)-NHS in phosphate buffer solution at pH 8.0 for one hour at room temperature.
Surface phase dissociation constant KDsurf (3.84 ± 0.68) is obtained by fitting Langmuir adsorption isotherm on equilibrium signals.
[citation needed] The difference in SPRi image can gives us information regarding the presence of binding and specificity but not suitable for quantification of free protein in case of multiple affinity sites.
Change in SPRi response on the reaction sites is provided by the capturing of kinetic curves and real time images from the CCD camera.
[citation needed] For in vitro studies, Polyamidoamine (PAMAM) dendrimers with amino- and carboxylic-acid external reactive shells are considered as sensing phase.
PAMAM-containing sensors are fixed on the SPRi analysis platform and then exposed to experimental fluids in the flow cell as shown in Figure 21.
SPRM is not adapted to sense the origin and nature of mass change but it detects the modification of refractive index due to mineral precipitation.
