Characteristics of Fluorescence Emission Excited by Grating-Coupled Surface Plasmons
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Dye molecules placed on metallic gratings can experience an enhanced electromagnetic field if illuminated under surface plasmon excitation conditions, a situation that can be employed for sensor applications. The fluorescence emission in this situation exhibits a characteristic emission polarization and geometry given by the fluorophore/grating interaction. We present experiments visualizing the full shape of the emission profiles and demonstrate how they can be manipulated by means of the grating constant. The excitation and emission processes taking place on the grating surface are characterized by polarization sensitive measurements.
KeywordsSurface plasmon resonance Surface plasmon field-enhanced fluorescence spectroscopy Grating-coupled surface plasmon spectroscopy Chromophore Imaging
Surface plasmon resonance (SPR) spectroscopy  has matured into a versatile method for the quantitative characterization of thin films and interfaces . Recent reviews have demonstrated that there is still an enormous interest in further developing the fundamentals of this optical tool [3, 4, 5] and to describe new directions for various fields of applications [6, 7, 8].
The classical excitation of surface plasmons at a metal-dielectric interface by light using a glass or quartz prism was first described by Otto . However, the alternative technique, introduced by Kretschmann , turned out to be a lot more versatile for widespread applications. The latter method uses a thin film, typically of a noble metal (Au or Ag) evaporated onto the coupling prism, with the surface plasmon mode being excited through the prism at the opposite metal/dielectric interface. Both approaches operate with photons in the total internal reflection geometry and thus meet the needs to match the energy and the momentum between photons and surface plasmons at resonant excitation.
Alternatively, the use of a surface corrugation of the metal surface, i.e., a grating structure, for the required momentum matching offers a number of advantages : (i) by the appropriate choice of the grating constant Λ = 2π/L, with L being the grating periodicity, one can choose the angle of incidence for any wavelength at will ; (ii) similar considerations apply for the recording of scattered surface plasmons, resulting, e.g., from a Raman scattering process, that populate energetically lower-lying (Stokes shifted) plasmon modes. These modes then out-couple at a particular emission angle, again governed by the dispersion relation ; (iii) the equivalent holds for the use of surface plasmon modes for the excitation of the fluorescence of chromophores within the evanescent field of the mode ; (iv) for practical applications, certain optical material parameters, e.g., the thickness of the Au layer evaporated onto the prism in the Kretschmann configuration, need to be very well controlled. This can impose severe challenges for the fabrication process. Gratings are much more forgiving in that in most cases, the conformal deposition of a Au or Ag coating onto the substrate that carries the surface corrugation can easily vary in thickness by a factor of two; (v) the momentum matching by a lead Fourier component of a periodic surface corrugation, together with nano-structures that allow for the simultaneous excitation of localized surface plasmon modes (and combination excitations of propagating and localized modes), currently offers the largest field enhancements that can be applied for surface plasmon-based sensor platforms .
Gratings were mounted inside a home built flow cell connected to a peristaltic pump on one of the arms of two rotary stages (Model 414A00 two-circle goniometer with M-BF-810-12 stepper motors, HUBER Diffraktionstechnik). By rotating the sample, the angle of incidence θ could be changed. The second arm carried a photodiode (PD). The PD (BPW 34 B silicon photodiode, Siemens) recorded the photocurrent generated by the light reflected from the grating for reflectivity measurements.
The fluorescence detection capability was provided by a cooled CCD camera (CoolSNAPHQ Monochrome, Photometrics) positioned with its optical axis oriented parallel to the surface normal of the grating and above its center. The fluorescence emission was turned into a parallel pencil of rays first and then shrunk in solid angle by a Kepler beam expander to fit the sensitive area of the CCD using a paraxial series of three lenses between sample and camera. The light had to pass through two interference filters (670FS10-25 interference filter, transmission maximum 67% at 673 nm, LOT Oriel) matched to the fluorescence wavelength and, for certain experiments, also a polarizer before it reached the detector. Images obtained with the CCD camera are rather two-dimensional angular spectra (a position on the CCD image relates to the angle under which the ray has left the grating) than images in the strict sense (a position in the object plane corresponds to a position in the image plane). Additionally, a photomultiplier tube (PMT) was used with an identical interference filter. With the CCD camera removed, the PMT (H6240-01 photon-counting unit, Hamamatsu) could be mounted on the second goniometer arm instead of the PD. It was used to measure the fluorescence intensity as a function of azimuthal angle in order to calibrate the horizontal axis of the CCD chip.
The surface functionalization of the grating substrates was built sequentially. Immobilization of each layer except the first was monitored by kinetic SPR measurements. The minimum position of the reflectivity curve taken after the addition of the probe sequence gives the angle of incidence for resonant excitation. The sample is then kept fixed at this angle in the following set of experiments. Next, the fluorescently labeled target sequence was injected and cycled for 5 min to guarantee a saturated sensor surface. After this, the flow cell was rinsed with PBS buffer in order to remove free fluorophores. The PD was exchanged against the PMT (with the CCD camera removed in order to free up the required space) to scan the (azimuthal) emission angle relative to the grating normal. Snap shots of the solid angle of emission were taken after having mounted the CCD camera assembly with its optical axis perpendicular to the grating surface.
Results and Discussion
Emission Profile Versus Grating Constant
Changing the grating constant affects the peak emission angle, because a different grating vector is introduced to the momentum-matching condition in reciprocal space. The curve obtained from a sample with a grating constant of Λ = 474.4 nm features lobes that are located closer to each other than for the sample with a higher grating constant. At the same time, the width of the peaks is much smaller. By picking the right grating constant, the emission angles can be engineered in a way that is convenient for a given device geometry. Unfortunately, the fluorescence intensities generated by both samples could not be compared in a quantitative way. Samples with a grating constant of Λ = 474.7 nm only became available much later in this work. At that time, certain properties of the experimental setup, like beam collimation and thus laser power, had been changed.
The two curves in Fig. 6 are characterized by an apparent asymmetry of the two emission peaks which is an artifact caused by bleaching of immobilized dye molecules and not a consequence of a grating corrugation asymmetry or another anisotropy effect. Typically, curves like those shown required a measurement time (and hence exposure to the laser beam) of 5 to 10 min, depending on the desired angular coverage. Reversal of the scan direction reversed the asymmetry.
Shape of the Fluorescence Emission Peaks
Polarization Sensitive Measurements
Conclusion and Outlook
The enhanced electromagnetic field in grating-coupled surface plasmon excitation has been used in this work to excite fluorophores. The fluorescence emitted from a dye-coated grating was found to be strongly directional. The emission geometry was recorded by solid angle imaging. It appears as a double hump symmetric to the surface normal of the sample. We demonstrate how the shape of the emission lobes can be manipulated by varying the grating constant. It is further shown by means of polarization-dependent excitation and detection that this shape is the result of the excited fluorophores, relaxing via intermediate red-shifted surface plasmons, which decay radiatively via the grating. This process of double-grating interaction is favored over other techniques employing, e.g., prism coupling, and achieves double enhancement.
We have examined fundamental properties of grating-coupled SPFS targeted towards its application for biosensing. In future publications, we will demonstrate the in situ fluorescence detection of analyte immobilization, quantify the impact of emission from fluorophores in the bulk on the limit of detection, and describe strategies to compensate this bulk signal contribution.
The authors like to thank B. Menges and J. Dostalek for many enlightening discussions.
Open access funding provided by University of Natural Resources and Life Sciences Vienna (BOKU).
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