Photonic Crystal Enhanced Fluorescence
Using a photonic crystal structure with an optical resonance that matches the wavelength of a laser used to excite fluorescent molecules, we can excite adsorbed fluorescent-tagged biomolecules and cells to emit more light than they would on an ordinary surface. At the same time, a photonic crystal surface can also have a resonance at the wavelength of light given off by fluorescent molecules, so that emitted photons can be directed toward a microscope objective. The enhanced excitation and extraction phenomena can be used to detect fluorescent-tagged proteins and DNA with greater sensitivity than currently-used methods – enabling advances in gene expression analysis for humans and crops, and the ability to detect disease biomarkers at low concentrations.
Details of the mechanisms for enhancing fluorescence using photonic crystal structures and the detection instrument developed for enhanced fluorescence and label-free imaging are described below. Applications for this approach being developed by the group are also discussed.
Photonic Crystal Enhanced excitation and extraction
Enhancing the output of fluorescent species is highly desirable for a wide range of biological applications including DNA sequencing, gene expression, single molecule detection and high resolution cell imaging. An extensive literature exists on the effects of metals on fluorophores, and on the use of a variety of metal surface configurations or metallic particle that involve plasmonic resonances to enhance fluorescence. A relatively smaller variety of dielectric structures, such as waveguides and optical resonators have been exploited for this purpose.
The Guided-mode resonance (GMR) effect occurring in photonic crystal slabs leads to heightened energy density within the resonator under steady-state operation conditions (via continual reinforcement of the leaky modes by the externally incident illumination), the magnitude of which is directly related to the resonance quality factor. A high resonance Q-factor leads to high intensity near-fields with which fluorophores can interact and fluoresce with greater intensity, due to an enhanced absorption rate. The electric near-fields produced by a device in response to TM polarized incidence are shown in Fig. 1, which shows the electric field intensity (Ez2, Ex2). As is evident, the maximum near-field intensity occurs where the resonant mode is confined, with evanescent tails penetrating both the substrate and superstrate of the device. For the purpose of fluorescence enhancement, these enhanced fields represent greater incident energy density.
Fig 1: Calculated near-electric field intensity profiles for the resonant mode at λ=632.8 nm for TM polarized incidence showing enhancement of both Ez and Ex field components. The color scale associated with each figure represents the intensity of the electric field and is normalized to the unit intensity incident wave.
Example of enhancement in detection sensitivity: Detecting Cy-5 conjugated Streptavidin
Fig 2: Fluorescence scans off (left) and on (right) resonance measured for photonic crystal device, showing the enhanced fluorescence effect. The plot below the figure shows the intensity profile on the sensor surface as a function of position, as marked by the line on the fluorescence scans.
The existence of leaky modes that overlap the fluorescence emission spectrum opens up pathways for the emitted light to escape into free-space. Besides direct emission, the fluorescence can now couple to the overlapping leaky modes and Bragg scatter out of the structure, thereby greatly reducing the amount of light trapped as guided-modes, in comparison to an unpatterned substrate. If the dispersion of these overlapping leaky modes is close to the Г-point band-edge i.e. K|| (magnitude of in-plane wave vector) ~ 0, most of the emitted light will be extracted within small angles with the surfaced normal. More generally, appropriately engineering the leaky dispersion of the PC facilitates the funnelling of guided light into regions of space where it can be easily detected
Fig 4: In our group, we attempt to engineer the band structure of photonic crystals in order exploit both the enhanced excitation and enhanced extraction effects at once, which can result in greatly enhanced signal to noise ratio in the detection of molecular fluorescence:
Detection Instrument for Enhanced – Fluorescence and Label-Free Imaging on Photonic Crystal Surfaces
The goal of this work is to design and demonstrate an imaging instrument optimized for both enhanced fluorescence (EF) and label-free imaging (LF), while overcoming sensitivity and resolution limitations of previously employed instruments. This instrument enables imaging of a photonic crystal surface using the same illumination source and imaging optics for both imaging modalities. With this approach, high resolution and high sensitivity LF and EF imaging may be accurately registered with each other for application in several important areas of life sciences research.
Fig5: Combined enhanced-fluorescence and label-free imaging instrument schematic
Gene expression microarrays
The use of microarrays to determine gene expression levels from biological samples has become an extremely informative procedure for life sciences researchers. These DNA microarrays promise to impact the practice of medicine, particularly in the diagnosis and treatment of cancer. Currently, microarrays are performed on glass microscope slides and are capable of discerning large changes in gene expression between two biological tissues. However, these microarrays are not capable of accurately determining smaller changes on the order of a few to dozens of nucleic acid molecules. By using photonic crystals as the substrate to perform the microarray, we can take advantage of unique nanoscale optical effects to more strongly excite the fluorescent molecules used to tag the nucleic acid molecules. Fluorescence from excited molecules can be further enhanced by altering the direction of emitted light to be more efficiently detected by the microarray instrumentation. The result of this enhancement is an increase in the signal-to-noise ratio of the DNA microarray, which enables more accurate quantification of small changes in amounts of DNA present. Because the photonic crystals we work with can be fabricated over large areas and easily integrated onto microscope slides, we can perform DNA microarray experiments with the same commercial microarray equipment used throughout many labs all over the world. Currently we are collaborating with Professor Lila Vodkin of the Department of Crop Sciences here at UIUC to apply our photonic crystals to soybean genome microarray experiments in order to assess the performance of our photonic crystal substrate.
Fig6: Images of identical DNA microarray experiments performed on a glass slide and a photonic crystal. Both microarrays have the same probe sequences and were exposed to the same soybean nucleic acid sample. The photonic crystal enhanced fluorescence allows for detection of many sequences that cannot be detected on the glass slide
Disease biomarker detection
Human serum contains a complex mix of proteins from many sites in the body that can potentially indicate a range of disease states if the relative concentrations of specific proteins associated with disease show dramatic changes. While concentrations of a single protein in serum may yield enough information to justify that protein’s use as a biomarker, it may be more informative to measure the concentrations of multiple proteins at once to achieve a protein concentration profile, just as gene expression profiles are often generated. Many researchers are exploring the use of the microarray format in immunofluorescent protein detection in order to efficiently measure the serum concentrations of many proteins at once. These sandwich immunofluorescent microarrays are often performed on glass slide substrates, but their performance can be improved by performing them on photonic crystal substrates capable of supporting enhanced fluorescence. The nanoscale optical properties of photonic crystals can increase the excitation of the fluorescent molecules used to tag the assay antibodies, and these properties also help to steer the emitted light towards the detection instrumentation. These effects can help increase the signal-to-noise ratio of immunofluorescent assays, providing increased resolution and lower limits of detection. We are currently characterizing the impact of photonic crystal enhanced fluorescence on the detection of the cytokine Tumor Necrosis Factor-a (TNF-a), which plays a role in inflammation.
Fig7: Spatial line profiles of fluorescence intensities from a TNF-a immunofluorescent microarray on a photonic crystal and on a glass slide. The substrates are compared at a TNF-a concentration of 8 pg/ml, which is near the lower limit of commercial ELISA assays. The photonic crystal has a signal-to-noise ratio that is 8 times that of the glass slide.
- “Combined enhanced fluorescence and label-free biomolecular detection with a photonic crystal surface”, P. C. Mathias, N. Ganesh, L. L. Chan, and B. T. Cunningham, Appl. Opt. 46, 2351-2360, 2007
- “Enhanced fluorescence emission from quantum dots on a photonic crystal surface”
N. Ganesh, W. Zhang, P. C. Mathias, E. Chow, J. A. N. T. Soares, V. Malyarchuk, A. D. Smith & B. T. Cunningham, Nature Nanotechnology 2, 515 – 520, 2007
- “Distance dependence of fluorescence enhancement from photonic crystal surfaces”
N. Ganesh, P. C. Mathias, W. Zhang, and B. T. Cunningham, J. Appl. Phys. 103, 083104, DOI:10.1063/1.2906175, 2008
- “Graded wavelength one-dimensional photonic crystal reveals spectral characteristics of enhanced fluorescence”, P. C. Mathias, N. Ganesh, W. Zhang, and B. T. Cunningham, J. Appl. Phys. 103, 094320, DOI:10.1063/1.2917184, 2008
- “Enhanced Fluorescence on a Photonic Crystal Surface Incorporating Nanorod Structures”, Wei Zhang, Nikhil Ganesh, Patrick C. Mathias and Brian T. Cunningham, , Small, Vol. 4, No. 12, p. 2199-2203, 2008.
- "Photonic crystals with SiO2-Ag "post-cap" nanostructure coatings for surface enhanced Raman spectroscopy," S.-M. Kim, W. Zhang, and B.T. Cunningham, Applied Physics Letters, Vol. 93, p. 143112, DOI: 10.1063/1.2998695, 2008.
- "Leaky-mode assisted fluorescence extraction: Application to fluorescence enhancement biosensors," N. Ganesh, I.D. Block, P.C. Mathias, W. Zhang, E. Chow, V. Malyarchuk, and B.T. Cunningham, Optics Express, Vol. 16, No. 26, p. 21626-21640, 2008.
- "Application of Photonic Crystal Enhanced Fluorescence to a Cytokine Immunoassay," P.C. Mathias, N. Ganesh, and B.T. Cunningham, Analytical Chemistry, Vol. 80, No. 23, p. 9013-9020, 2008.
- "Employing two distinct guided-mode resonances to improve fluorescence enhancement from photonic crystals," P.C. Mathias, H.-Y. Wu, and B.T. Cunningham, Applied Physics Letters, Vol. 95, p. 021111-021113, 2009.
- "Optimizing the spatial resolution of photonic crystal label-free imaging," I.D. Block, P.C. Mathias, S. I. Jones, L. O. Vodkin, and B.T. Cunningham, Applied Optics, 2009.
- "A detection instrument for enhanced fluorescence and label-free imaging on photonic crystal surfaces," I.D. Block, P.C. Mathias, N. Ganesh, S.I. Jones, B.R. Dorvel, V. Chaudhery, L. Vodkin, R. Bashir, and B.T. Cunningham, Optics Express, Vol. 17 Issue 15, pp.13222-13235, 2009.
- "Magnification of photonic crystal fluorescence enhancement via TM resonance excitation and TE resonance extraction on a dielectric nanorod surface," H.-Y Wu, W. Zhang, P.C. Mathias, and B.T. Cunningham, Nanotechnology, Vol. 21, p. 125203-125210, 2010.
- "Improved detection of differentially expressed genes on a DNA microarray by photonic crystal enhanced fluorescence," P.C. Mathias, S. I. Jones, H.-Y. Wu, F. Yang, D.O. Gonzalez, L.O. Vodkin, and B.T. Cunningham, Analytical Chemistry, Vol. 82, No. 16, p. 6854-6861, 2010.
- "Multi-color fluorescence enhancement from a photonic crystal surface," A. Pokhriyal, M. Lu, C. Huang, S.C. Schulz, and B.T. Cunningham, Submitted to Applied Physics Letters, Vol. 97, No. 12, p. 121108-121110, 2010. PMCID: PMC2955725
- "Photobleaching on photonic crystal enhanced fluorescence surfaces," V. Chaudhery, M. Lu, C. S. Huang, S. George, and B. T. Cunningham, Journal of Fluorescence, Published Online November 12, 2010, DOI 10.1007/s10895-010-0760-8.
- "Photonic crystal enhanced fluorescence using a quartz substrate to reduce limits of detection," A. Pokhriyal, M. Lu, V. Chaudhery, C.-S. Huang, S. Schulz, and B.T. Cunningham, Optics Express, Vol. 18, No. 24, p. 24793-24808, 2010.
- "Application of photonic crystal enhanced fluorescence to cancer biomarker microarrays," C.-S. Huang, S. George, M. Lu, V. Chaudhery, R. Tan, R.C. Zangar, and B.T. Cunningham, Analytical Chemistry, Vol. 83, No. 4, p. 1425-1430, 2011 (Featured on Cover).
- "Angle-scanning photonic crystal enhanced fluorescence microscopy," V. Chaudhery, M. Lu, A. Pokhriyal, S. Schulz, and B.T. Cunningham, IEEE Sensors Journal, Accepted, September 2011.
- "Spatially selective photonic crystal enhanced fluorescence and application to background reduction for biomolecule detection assays," V. Chaudhery, M. Lu, C.-S. Huang, A. Pokhriyal, J. Polans, S.C. Schulz and B.T. Cunningham, Optics Express, Vol. 19, No. 23, p. 23327–23340, 2011.
Related Conference Presentations
- "Enhanced fluorescence via photonic crystal slabs incorporating nanorod structures," W. Zhang, N. Ganesh, P.C. Mathias, and B.T. Cunningham, Conference on Lasers and Electro Optics, San Jose, CA, May 2008.
- "Distance dependent amplification of molecular fluorescence via photonic crystal slabs," N. Ganesh, P.C. Mathias, W. Zhang, and B.T. Cunningham, Conference on Lasers and Electro Optics, San Jose, CA, May 2008.
- "Combined Enhanced Fluorescence and Label-Free Biomolecular Sensing with a Two-Dimensional Photonic Crystal." P.C. Mathias, N.Ganesh, and B.T. Cunningham, IEEE LEOS Conference, Orlando, FL, October 2007.
- "Photonic crystal enhanced fluorescence," N. Ganesh and B.T. Cunningham, 2007 CLEO/QELS Conference, Baltimore, MD, May 2007.
- "Resonant enhancement of fluorescence from Quantum Dots on a Photonic Crystal Slab," N. Ganesh, W. Zhang, P.C. Mathias, and B.T. Cunningham, 2007 MRS Fall Meeting, San Francisco, CA, April 2007.