Surface plasmon coupled fluorescence in the ultraviolet and visible spectral regions using zinc thin films.

The use of zinc thin films deposited onto glass supports for surface plasmon coupled fluorescence (SPCF) over a broad 200 nm wavelength range is demonstrated. Fresnel calculations performed in the ultraviolet and visible spectral range are predicted to generate surface plasmon modes in 30 nm zinc thin films. In this spectral range, the extent of coupling of light to zinc thin films was shown to be significant as compared to similar aluminum, gold, and silver thin films. The experimental demonstration of SPCF using 30 nm zinc thin films in the ultraviolet and visible spectral regions was undertaken using three different fluorophores 2-AP, POPOP, and FITC, respectively. Surface plasmon coupled fluorescence from zinc thin films was p-polarized and highly directional with lambda max conferred at an angle of 58, 68, and 60 degrees for FITC, POPOP, and 2-AP, respectively. s-Polarized emission from zinc thin films was negligible for all fluorophores except for a sample spin coated from a 10% PVA solution, which resulted in significant s-polarized emission due to the generation of waveguide modes. The experimental results are consistent with reflectivity curves that are theoretically predicted using Fresnel calculations. Given the growing use and utility of plasmon-enhanced fluorescence in the analytical and biological sciences, our findings will serve as a useful tool for workers in the ultraviolet and visible spectral regions.

Metal-Enhanced Excimer (P-type) Fluorescence.

In this letter, we report the first observation of metal-enhanced excimer (P-type) fluorescence from pyrene. Pyrene in close proximity to silver island films (SIFs) shows enhanced pyrene excimer emission with a approximately 2.5-fold higher emission intensity observed from SIFs and 5x10(-3) M pyrene, as compared to a quartz control sample containing no silver nanoparticles. Our findings suggest two complementary methods for the enhancement: i) surface plasmons can radiate coupled monomer and excimer fluorescence efficiently, and ii) enhanced absorption (enhanced electric field) further facilitates enhanced emission. This observation is helpful in our laboratory's continued goal to develop a unified plasmon-luminophore description.

Metal-enhanced e-type fluorescence.

In this letter, we report metal-enhanced e-type fluorescence. Eosin in close proximity to silver island films (SiFs) shows enhanced e-type fluorescence with an approximately two-fold higher intensity observed from SiFs, as compared to a control sample. Our findings suggest two complementary mechanisms for the enhancement: surface plasmons can radiate e-type delayed fluorescence efficiently and enhanced absorption also facilitates enhanced emission from both S(1) and T(1) states. This observation is helpful in our understanding not only for studying the interactions between plasmons and fluorophores but also for our laboratories continued efforts to develop a unified plasmon-lumophore description.

Extraction and detection of DNA from Bacillus anthracis spores and the vegetative cells within 1 min.

The use of a combination of low-cost technologies to both extract and detect anthrax DNA from spores and vegetative cells in two steps within 1 min is described. In a cavity, microwave energy is highly focused using thin-film aluminum "bow-tie" structures, to extract DNA from whole spores within 20 s. The detection of the released DNA, from less than 1000 vegetative cells, without additional preprocessing steps is accomplished in an additional 30 s by employing the microwave-accelerated metal-enhanced fluorescence technique. The new platform technology presented here is a highly attractive alternative method for DNA extraction and the fast detection of gram-positive bacteria and potentially other pathogenic species and cells as well.

Spectrally resolved fluorescence correlation spectroscopy based on global analysis.

Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.

Plasmonic engineering of singlet oxygen generation.

In this article, we report metal-enhanced singlet oxygen generation (ME(1)O2). We demonstrate a direct relationship between the singlet oxygen yield of a common photosensitizer (Rose Bengal) and the theoretical electric field enhancement or enhanced absorption of the photosensitizer in proximity to metallic nanoparticles. Using a series of photosensitizers, sandwiched between silver island films (SiFs), we report that the extent of singlet oxygen enhancement is inversely proportional to the free space singlet oxygen quantum yield. By modifying plasmon coupling parameters, such as nanoparticle size and shape, fluorophore/particle distance, and the excitation wavelength of the coupling photosensitizer, we can readily tune singlet oxygen yields for applications in singlet oxygen-based clinical therapy.

Microwave-accelerated surface plasmon-coupled directional luminescence 2: a platform technology for ultra fast and sensitive target DNA detection in whole blood.

The application of Microwave-Accelerated Surface Plasmon-Coupled Luminescence (MA-SPCL) to fast and sensitive DNA hybridization assays in buffer and whole blood is presented. In this regard, a model DNA hybridization assay whereby a fluorophore-labeled target ssDNA specific to human immunodeficiency, Hepatitis C (Hep C), is probed by an anchor probe immobilized on thin gold films, is driven to completion within 1 min with microwave heating, as compared to an identical assay completed in approximately 4 h at room temperature. Finite-Difference Time-Domain calculations show that gold disks are preferentially heated around the edges creating a temperature gradient along the disks, which in turn results in the larger influx of complementary DNA towards anchor probe-modified surface. Thermal images of the assay platform during microwave heating also provide additional information on the microwave heating pattern in the microwave cavity. Finally, the effects of low power microwave heating on the ability of DNA to re-hybridize with the complimentary target on the surface gold films, which allows the multiple re-use of the gold films, is demonstrated. The MA-SPCL technique offers an alternative approach to current DNA based detection technologies, especially when speed and sensitivity are required, such as in the identification of DNA or even RNA-based diseases using whole blood samples that affect human health.

Metal-Enhanced Fluorescence from Nanoparticulate Zinc Films.

A detailed study of metal-enhanced fluorescence (MEF) from fluorophores in the blue-to- red spectral region placed in close proximity to thermally evaporated zinc nanostructured films is reported. The zinc nanostructured films were deposited onto glass microscope slides as individual particles and were 1-10 nm in height and 20-100 nm in width, as characterized by Atomic Force Microscopy. The surface plasmon resonance peak of the zinc nanostructured films was approximately 400 nm. Finite-difference time-domain calculations for single and multiple nanostructures organized in a staggered fashion on a solid support predict, as expected, that the electric fields are concentrated both around and between the nanostructures. Additionally, Mie scattering calculations show that the absorption and scattering components of the extinction spectrum are dominant in the UV and visible spectral ranges, respectively. Enhanced fluorescence emission accompanied by no significant changes in excited state lifetimes of fluorophores with emission wavelengths in the visible blue-to-red spectral range near-to zinc nanostructured films were observed, implying that MEF from zinc nanostructured films is mostly due to an electric field enhancement effect.

Three-dimensional tissue cytometer based on high-speed multiphoton microscopy.

Image cytometry technology has been extended to 3D based on high-speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell-cell and cell-extracellular matrix interactions. The imaging system was based on high-speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm(3)/h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/10(5). This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important.

Microwave-accelerated metal-enhanced fluorescence: application to detection of genomic and exosporium anthrax DNA in <30 seconds.

We describe the ultra-fast and sensitive detection of the gene encoding the protective antigen of Bacillus anthracis the causative agent of anthrax. Our approach employs a highly novel platform technology, Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF), which combines the use of Metal-Enhanced Fluorescence to enhance assay sensitivity and focused microwave heating to spatially and kinetically accelerate DNA hybridization. Genomic and exosporium target DNA of Bacillus anthracis spores was detected within a minute in the nanograms per microliter concentration range using low-power focused microwave heating. The MAMEF technology was able to distinguish between B. anthracis and B. cereus, a non-virulent close relative. We believe that this study has set the stage and indeed provides an opportunity for the ultra-fast and specific detection of B. anthracis spores with minimal pre-processing steps using a relatively simple but cost-effective technology that could minimize casualties in the event of another anthrax attack.

Low temperature metal-enhanced fluorescence.

In this short letter, we describe the effects of low temperature on the Metal-Enhanced Fluorescence (MEF) phenomenon. Fluorophores close to Silver Island Films (SiFs) show on average two- to ten-fold enhancements in their fluorescence signatures at room temperature. However, at 77 K, we have observed that MEF is even more pronounced as compared to an identical glass control sample. We also demonstrate that the further enhancements in MEF occur at low temperature over a range of visible wavelengths for different fluorophores, for both SiFs and 20 nm surface deposited gold colloids.

Real-time thermal imaging of microwave accelerated metal-enhanced fluorescence (MAMEF) based assays on sapphire plates.

In this paper, we describe an optical geometry that facilitates our further characterization of the temperature changes above silver island films (SiFs) on sapphire plates, when exposed to microwave radiation. Since sapphire transmits IR, we designed an optical scheme to capture real-time temperature images of a thin water film on sapphire plates with and without SiFs during the application of a short microwave pulse. Using this optical scheme, we can accurately determine the temperature profile of solvents in proximity to metal structures when exposed to microwave irradiation. We believe that this optical scheme will provide us with a basis for further studies in designing metal structures to further improve plasmonic-fluorescence clinical sensing applications, such as those used in microwave accelerated metal-enhanced fluorescence (MAMEF).

Spatial and temporal control of microwave triggered chemiluminescence: a protein detection platform.

We have combined the principles of microwave circuitry and antenna design and our recent work in microwave-triggered metal-enhanced chemiluminescence to now "trigger" chemically and enzyme-catalyzed chemiluminescent reactions with spatial and temporal control. With this technology platform, we achieve spatial and temporal control of enzyme and chemically catalyzed chemiluminescence reactions to achieve more than 500-fold increases in "on-demand" photon flux from chemically catalyzed chemiluminescent reactions. We also report a 6-fold increase in photon flux from HRP-catalyzed assays on disposable coverslips functionalized with HRP and placed proximal to the substrates modified with thin-film aluminum triangle disjointed "bow-tie" structures. In addition, we demonstrate the applicability of this technology to develop multiplexed or high-throughput chemiluminescent assays. We also demonstrate the clinical and biological relevance of this technology platform by affixing aluminum structures in proximity to HRP protein immobilized on nitrocellulose to improve the sensitivity for this model Western blot scheme by 50-fold. We believe analytical applications that rely on enzyme-catalyzed chemiluminescence, such as immunoassays, may greatly benefit from this new platform technology.

Metal-enhanced singlet oxygen generation: a consequence of plasmon enhanced triplet yields.

In this Rapid Communication, we report the first observation of Metal-Enhanced singlet oxygen generation (ME(1)O(2)). Rose Bengal in close proximity to Silver Island Films (SiFs) can generate more singlet oxygen, a three-fold increase observed, as compared to an identical glass control sample but containing no silver. The enhanced absorption of the photo-sensitizer, due to coupling to silver surface plasmons, facilitates enhanced singlet oxygen generation. The singlet oxygen yield can potentially be adjusted by modifying the choice of MEF (Metal-Enhanced Fluorescence) & MEP (Metal Enhance Phosphorescence) parameters, such as distance dependence for plasmon coupling and wavelength emission of the coupling fluorophore. This is a most helpful observation in understanding the interactions between plasmons and lumophores, and this approach may well be of significance for singlet oxygen based clinical therapy.

Microwave-triggered chemiluminescence with planar geometrical aluminum substrates: theory, simulation and experiment.

Previously we combined common practices in protein detection with chemiluminescence, microwave technology, and metal-enhanced chemiluminescence to demonstrate that we can use low power microwaves to substantially increase enzymatic chemiluminescent reaction rates on particulate silvered substrates. We now describe the applicability of continuous aluminum metal substrates to potentially further enhance or "trigger" enzymatic chemiluminescence reactions. Furthermore, our results suggest that the extent of chemiluminescence enhancement for surface and solution based enzyme reactions critically depends on the surface geometry of the aluminum film. In addition, we also use FDTD simulations to model the interactions of the incident microwave radiation with the aluminum geometries used. We demonstrate that the extent of microwave field enhancement for solution and surface based chemiluminescent reactions can be ascribed to "lightning rod" effects that give rise to different electric field distributions for microwaves incident on planar aluminum geometries. With these results, we believe that we can spatially and temporally control the extent of triggered chemiluminescence with low power microwave (Mw) pulses and maximize localized microwave triggered metal-enhanced chemiluminescence (MT-MEC) with optimized planar aluminum geometries. Thus we can potentially further improve the sensitivity of immunoassays with significantly enhanced signal-to-noise ratios.

Fluorescence microscopy in a microwave cavity.

Optical microscopy is a well-established technique that has wide ranging applications for imaging molecular dynamics of biological systems. Typically, these applications rely on external temperature controllers to maintain or change reactions rates of these biological systems. With increasing interest in applying low power microwaves to drive biological and chemical reactions, we have combined optical and microwave based technologies and developed a fluorescence microscope in a microwave cavity. With this instrument, we have found a means to optically image biological systems inside microwave cavities during the application of microwave pulses.

Metal-enhanced phosphorescence: interpretation in terms of triplet-coupled radiating plasmons.

We report our detailed metal-enhanced phosphorescence (MEP) findings using Rose Bengal at low temperature. Silver Island Films (SiFs) in close proximity to Rose Bengal significantly enhance the phosphorescence emission intensity. In this regard, a 5-fold brighter phosphorescence intensity of Rose Bengal was observed from SiFs as compared to a glass control sample at 77 K. In addition, several factors affecting MEP, such as distance dependence and silver film morphology, were also investigated. Our findings suggest that both singlet and triplet states can couple to surface plasmons and enhance both fluorescence and phosphorescence yields. This finding suggests that MEP can be used to promote triplet-based assays, such as those used in photodynamic therapy.

Microwave triggered metal enhanced chemiluminescence: Quantitative protein determination.

We present a new technology that offers a faster alternative to the chemiluminescence-based detection that is used in protein assay platforms today. By combining the use of silver nanostructures with chemiluminescent species, a technique that our laboratories have recently shown can enhance the system photon flux over 50-fold, with the use of low-power microwave heating to additionally accelerate, in essence "trigger", chemiluminescence-based reactions, then both ultrafast and ultrabright chemiluminescence assays can be realized. In addition, the preferential heating of the nanostructures by microwaves affords for microwave triggered metal enhanced chemiluminescence (MT-MEC) to be localized in proximity to the silvered surfaces, alleviating unwanted emission from the distal solution. To demonstrate MT-MEC, we have constructed a model assay sensing platform on both silvered and glass surfaces, where comparison with the identical glass substrate-based assay serves to confirm the significant benefits of using silver nanostructures for metal-enhanced chemiluminescence. Our new model assay technology can detect femtomoles of biotinylated BSA in less than 2 min and can indeed be modified to both detect and quantify a great many other biomolecules as well. As compared to traditional western blot approaches, MT-MEC offers protein quantification, high-sensitivity detection combined with ultrafast assay times, i.e., <2 min.

Microwave-triggered metal-enhanced chemiluminescence (MT-MEC): application to ultra-fast and ultra-sensitive clinical assays.

In this rapid communication we describe a new approach to protein detection with chemiluminescence. By combining common practices in protein detection with chemiluminescence, microwave technology, and metal-enhanced chemiluminescence, we show that we can use low power microwaves to substantially increase enzymatic chemiluminescent reaction rates on metal substrates. As a result, we have found that we can in essence trigger chemiluminescence with low power microwave (Mw) pulses and ultimately, perform on-demand protein detection assays. Using microwave triggered metal-enhanced chemiluminescence (MT-MEC), we not only improve the sensitivity of immunoassays with enhanced signal-to-noise ratios, but we also show that we can accurately quantify protein concentrations by integrating the photon flux for discrete time intervals.