Research On Surface Plasmon Resonance Imaging Based On Microobjective

Surface plasmon resonance(SPR)is a physical optics phenomenon that is induced by the interaction of light-wave with electron of metals.Surface plasmon wave(SPW)is generated by exciting free electrons on metal surface utilizing the evanescent wave caused by total reflection.The wave vector of SPW and evanescent wave are identical at a certain incident angle or wavelength of light,thus resonance occur leading to intense absorption of incident light and emergence of minima on reflectivity,besides,the refractive index variation of analyte adjacent to metal surface have an impact on the position(resonant angle or wavelength)of resonant peak.Owing to the sensors based on SPR technique have an advantage of high sensitivity,fast measurement,less consumption of sample and label-free detection of biological sample without damaged,surface plasmon resonance sensing technique has been used broadly in surface topography,analysis of biochemical and immune,drug screening and environmental monitoring,and has become an optical technique for detection of surface structure on the interface of metal/dielectric.Commonly,SPR sensors perform single-channel detection for sample,single-channel detection cannot satisfy requirement of high throughput and large-scale analysis for living substances which is necessary in rapid-developing life sciences.Surface plasmon resonance imaging technique combining surface plasmon resonance with image technology based on CCD camera is capable of monitoring resonant datum of single and multiple points,exhibiting image of sample in real-time,which has a profound significance in life sciences.In this dissertation,a high NA microobjective serves as a coupling element,the theory and application of surface plasmon resonance imaging is investigated,the details are as follows:1.Establishment of surface plasmon resonance imaging system based on amicroobjective with high NA.The theory of surface plasmon resonance is investigated.Theoretical model of surface plasmon resonance is established,and the influence of physical factors on surface plasmon resonance such as the refractive index of coupling element,material and thickness of metal film is analyzed.Combining He-Ne laser,CCD camera and other optical mounts,surface plasmon resonance imaging system based on microobjective is built up,in which a high NA microobjective serves as coupling element and imaging chip is fabricated by evaporating gold film on substrate.Surface plasmon resonance is excited by converging 632.8 nm light upon an imaging chip using the microobjective,imaging information of back focal plane and sample are collected by CCD camera,respectively.Detection of the refractive index of the samples.In the experiment,a microscope objective serves as a coupling element,and surface plasmon resonance is excited using 632.8 nm lights from a He-Ne laser converging on the interface of sample and gold film.The intensity distribution at the back focal plane of the objective lens was captured by a CCD camera,and then processed in a computer to display.The refractive indexes of air,water and ethanol are measured by using the apparatus,and the experimental results are agree well with those of theoretical.2.Measurement of refractive index of sample by using the imaging system.A new method for measuring refractive index of medium using surface plasmon resonance imaging apparatus is presented,the method does not involve rotating or moving components compare to conventional SPR apparatus for angular modulation,and is able to monitor the image on back focal plane,read out and process SPR data in real-time avoiding inaccuracy introduced by mechanical rotation.Total reflection is employed to calibrate the angle associated with the back focal plane of microobjective,thus,quantitative measurement of refractive index for medium can be operated utilizing the image on back focal plane.Three common gas or liquid are used as medium to be measured,and surface plasmon resonance datum of threemedium are extracted from images on back focal plane,respectively,by which the relationship between refractive index and resonant angle are obtained,thus the refractive index of each medium is determined.3.Research of surface plasmon resonance imaging based on slit scanning.A novel SPR imaging approach for sample based on slit scanning is presented.Owing to the incident angle of light beam can be restricted and adjusted by a slit,image of sample corresponding to some certain incident angle related to the position of the slit can be obtained,thus images with different contrast gradient appear at different incident angles.Angular modulation within some certain angle range for sample is implemented via slit scanning,besides images vary with incident angle can be collected.In the experiment,the image of Si N grating on gold film is acquired.Among a series of sample images corresponding to different incident angles,an image with highest contrast exists and appears at a appropriate incident angle.4.Acquisition of the SPR angular spectra of sample.A method for acquiring SPR angular spectra of sample is presented.230 images of sample which vary with the incident angles are collected by CCD camera via slit scanning.Surface plasmon resonance curve of each pixel was extracted from the 230 images of sample.Two-dimensional and three-dimensional angular spectra of SPR are reconstructed utilizing the resonant angles extracted from the surface plasmon resonance curves.Due to the angular spectra represents the differences of resonant angles corresponding to each point of sample,tiny differences of refractive index in sample are able to be observed more distinctly.This method eliminates the influence of instable reflectance on imaging,and improves the contrast gradient and definition of image.This method is no more observing microstructure of analyte from the aspect of reflected light but from the aspect of refractive index which will be a microscopy with great potential.