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We describe an optical system that detects the presence of E. coli bacteria, making use of the bacteria's natural fluorescence properties. The system provides an excitation signal at 365 nm and detects the emission signal, from the bacteria, at approximately 445 nm. The system also allows the intensity of the emitted signal to be compared with a user-programmable threshold. This allows rapid testing of many samples in a laboratory setting. Complete setup and performance details are provided, enabling the experimentalist to tailor the system parameters to other species of microorganisms, which may have fluorescence properties at other wavelengths.

We present a dual-channel mobile lidar system based on laser-induced fluorescence (LIF) for real-time standoff detection and concentration distribution analysis of tryptophan. The system employs an ultraviolet laser excitation source and signal detectors for receiving fluorescence signals within two different wavelength bands. The performed experiments measured tryptophan aerosols at two different standoff distances. Moreover, distilled water and ethanol solutions were also detected for comparison. The results show that the system can detect LIF signals of tryptophan, give early warnings, locate the diffusion sources, and monitor the variation of the aerosol concentration distribution in real time.

We present a fast hyperspectral line-scan fluorescence imaging system to verify the feasibility of quantitative fluorescence authentication of powders. Vanillin, which is restricted for use in milk powder, especially in milk powder for infants, is mixed with milk powder in different mass concentrations (5%, 10%, 30%, 50%, 70%, and 90%). Mixed powders are located on a motorized linear stage. A 405 nm line laser is utilized to excite the fluorescence of the sample. Based on galvo scanning, we can generate a laser line with high spatial resolution and high-intensity density on the samples. An imaging spectrometer with a complementary metal-oxide semiconductor (CMOS) camera as detector is built. The spectral range of the spectrometer is 365-810 nm, with about 1 nm spectral resolution. One snapshot of the CMOS can acquire the fluorescent spatial and spectral information of a line region in 100 ms. By scanning the motorized linear stage, we obtain the fluorescence hypercube of the sample. A 100×1926×1216 hypercube, which covers an area of 15  mm×5  mm, is obtained in 50 s. The imaging speed can be enhanced further by increasing the intensity of the excitation laser and the sensitivity of the area camera. Fully constrained least squares, a linear spectral mixture analysis, is utilized to analyze the hypercube obtained by our homemade imaging spectrometer, thus obtaining the pixel concentration of vanillin in each mixed powder. Linear regression analysis is used for the pixel concentration and mass concentration of vanillin. A linear relationship with coefficient of determination R 2 equal to one is observed, which demonstrates the capability of fluorescence hyperspectral quantitative analysis in powders.

Laser-induced fluorescence (LIF) techniques to analyze atmospheric aerosols are commonly applied for research and human exposure monitoring, but are very expensive or offer poor spectral resolution. Here, we discuss how a recently proposed instrument can acquire resolved fluorescence spectra from many individual particles in a single camera image using four excitation wavelengths matched with common biological fluorophores for particle discrimination at lower cost. We discuss emission intensity calibration and demonstrate spectral differentiation among four species of pollen. These data provide context for how the instrument could be developed for pollen and mold-spore detection or for use by citizen scientists.

The study of metabolic and oxygen states of cells in a tumor in vivo is crucial for understanding of the mechanisms responsible for tumor development and provides background for the relevant tumor's treatment. Here, we show that a specially designed implantable fiber-optic probe provides a promising tool for optical interrogation of metabolic and oxygen states of a tumor in vivo. In our experiments, the excitation light from a ps diode laser source is delivered to the sample through an exchangeable tip via a multimode fiber, and the emission light is transferred to the detector by another multimode fiber. Fluorescence lifetime of a nicotinamid adenine dinucleotide (NAD(P)H) and phosphorescence lifetime of an oxygen sensor based on an iridium (III) complex of enzothienylpyridine (BTPDM1) are explored both in model experiment in solutions and in living mice.

Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.

In this Letter, we demonstrate that fluorescence detection by using surface-bound fluorescent molecules can be enhanced by coupling the fluorescence emission with dually polarized resonant modes of a photonic crystal (PC) substrate. The PC was fabricated through nanoreplica molding by using a plastic substrate. It was designed such that the transverse magnetic (TM)- and transverse electric (TE)-polarized resonance coincided at a specific combination of the incident angle and the illumination wavelength. During excitation, the nonpolarized emission from cyanine-5 was simultaneously coupled with the TE and TM resonant modes and reradiated into the detection instrument, increasing the fluorescence collection efficiency and thus, enhancing the fluorescence detection.

Corrugated metallic surfaces offer means for efficient amplification of fluorescence bioassay signal based on the near field coupling between surface plasmons and fluorophore emitters that are used as labels. This paper discusses the design of such plasmonic structure to enhance the sensitivity of immunoassays with epi-fluorescence readout geometry. In particular, crossed gold grating is theoretically and experimentally investigated for combined increasing of the excitation rate at the fluorophore excitation wavelength and utilizing directional surface plasmon-coupled fluorescence emission. For Alexa Fluor 647 dye, the enhancement factor of around EF = 10 2 was simulated and experimentally measured. When applied to a sandwich interleukin-6 immunoassay, highly surface-selective enhancement reaching a similar value was observed. Besides increasing the measured fluorescence signal associated with the molecular binding events on a surface by two orders of magnitude, the presented approach enables measuring kinetics of the surface reaction that is otherwise masked by strong background signal originating from bulk solution.

We present a new hardware simulator (HS) for characterization, testing and benchmarking of digital correlators used in various optical correlation spectroscopy experiments where the photon statistics is Gaussian and the corresponding time correlation function can have any arbitrary shape. Starting from the HS developed in [Rev. Sci. Instrum. 74, 4273 (2003)], and using the same I/O board (PCI-6534 National Instrument) mounted on a modern PC (Intel Core i7-CPU, 3.07GHz, 12GB RAM), we have realized an instrument capable of delivering continuous streams of TTL pulses over two channels, with a time resolution of Δt = 50ns, up to a maximum count rate of 〈I〉 ∼ 5MHz. Pulse streams, typically detected in dynamic light scattering and diffuse correlation spectroscopy experiments were generated and measured with a commercial hardware correlator obtaining measured correlation functions that match accurately the expected ones.

We present the first demonstration of fluorescence correlation spectroscopy (FCS) using superconducting nanowire single-photon detectors (SSPDs) which are free of afterpulses unlike the avalanche photodiode (APD). Multimode-fiber-coupled SSPDs with high system detection efficiency for visible wavelengths were developed and implemented in the FCS system. We performed FCS measurements for Rhodamine B and 6G as fluorescent samples, and found that autocorrelation functions obtained by the SSPDs showed a noise-free curve in the short correlation time region of sub microseconds where the afterpulse effect was dominant using the APD. The obtained results clearly indicate the advantage of SSPDs for the FCS system.

We developed a UV (355 nm) laser-induced fluorescence (LIF) lidar for monitoring the real-time status of blue-green algae. Since the fluorescence spectrum of blue-green algae excited by 355 nm showed the specific fluorescence at 650 nm, the lidar was designed to be able to detect the 650 nm fluorescence as a surveillance method for the algae. The usefulness was confirmed by observation at Lake Suwa over four years (2005-2008). The detection limit of the LIF lidar was 16.65 mg/L for the blue-green algae, which is the range of concentrations in the safe level set by the World Health Organization.

Conventional surface plasmons (SPs) or Bloch surface waves (BSWs) have a wave vector exceeding that of light in vacuum, and, therefore, the surface plasmon-coupled emission (SPCE) or Bloch surface wave-coupled emission (BSWCE) cannot escape from the corresponding structures. With the aid of a high-refractive-index prism or an oil-immersion objective, the SPCE or BSWCE can be coupled into free space. But the large volumes of the prism and objective are certainly unfavorable for miniaturization of the optical systems or inconvenient for applications such as the optical displays. Here we experimentally demonstrate a new method to extract the SPCE or BSWCE with a subsurface dielectric grating. The experimental results verify that the chip-like substrate with two decorated sides can bring out the directional fluorescence emission in free space. The emitting direction and emitting patterns can be tuned by the period size and dimensionality of the gratings. Our work provides a new strategy to realize free-space directional fluorescence emission at a very low cost and compact configuration, which has potential applications in fluorescence-based sensing, imaging, light-emitting diodes, optical displays, and other near-field optical devices.

We propose a multifocal noncontact setup to perform depth-sensitive fluorescence imaging on a two-layered epithelial tissue model. The combination of a microlens array and a tunable lens enables the depth of the multifocal plane to be conveniently adjusted without any mechanical movement of the imaging lens or the sample. This advantage is particularly desirable in the clinical setting. Results from the phantom study demonstrate that the setup can achieve depth-sensitive color imaging for fluorescence measurements, which is further confirmed by spectral measurements.

We present a fluorescence correlation spectroscopy (FCS) approach to obtain spectral cross-talk free auto- and cross-correlation functions for probes with highly overlapping emission spectra. Confocal microscopes with either a hyperspectral EM-CCD or six-channel PMT array spectral detection were used, followed by a photon filtering correlation approach that results in spectral unmixing. The method is highly sensitive and can distinguish between Atto488 and Oregon Green 488 signals so that auto-correlation curves can be fitted without the need for cross-talk correction. We also applied the approach to the membrane dye Laurdan whose emission is dependent on the lipid order within the bilayer. With fluorescence spectral correlation spectroscopy (FSCS), we could obtain spectral cross-talk free auto- and cross-correlation functions corresponding to Laurdan located in liquid ordered and liquid disordered phases.

Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, flow velocities and concentrations in an imaging mode. Here we extend this technique to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS), which allows to measure molecular interactions in an imaging mode. We present a theoretical framework for SPIM-FCCS fitting models, which is subsequently used to evaluate several test measurements of in-vitro (labeled microspheres, several DNAs and small unilamellar vesicles) and in-vivo samples (dimeric and monomeric dual-color fluorescent proteins, as well as membrane bound proteins). Our method yields the same quantitative results as the well-established confocal FCCS, but in addition provides unmatched statistics and true imaging capabilities.

We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obtaining absolute diffusion coefficient of fast moving fluorescing molecules at nanomolar concentrations, based on fast polarization modulation of the excitation beam by a resonant electro-optical modulator. The first approach rotates every second linearly polarized laser pulse by 90 degrees to obtain independent intensity readout for both foci, similar to original design. The second approach combines polarization modulation of cw laser and fluorescence lifetime correlation spectroscopy (FLCS) like analysis to obtain clean correlation curves for both overlapping foci. We tested our new approaches with different lasers and samples, revealed a need for intensity cross-talk corrections by comparing the methods with each other and discussed experimental artifacts stemming from improper polarization alignment and detector afterpulsing. The advantages of our solutions are that the polarization rotation approach requires just one pulsed laser for each wavelength, that the polarization modulation approach even mitigates the need of pulsed lasers by using standard cw lasers and that it allows the DIC prism to be placed at an arbitrary angle. As a consequence the presented experimental solutions for 2fFCS can be more easily implemented into commercial laser scanning microscopes.

We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy.

A high-sensitivity bioaerosol monitor based on a single 365 nm LED is developed and a calibration approach is discussed for the first time. The fluorescence detection system, which is the core part of the monitor, contains an optical detection module and a fluorescence signal processor configured with a phase sensitive detector (PSD). B800 fluorescent microspheres and staphylococcus are used for performance evaluation of the monitor. B800 microspheres are appropriate as calibration material. The experimental results demonstrate the PSD plays a significant role in improving sensitivity and signal-to-noise ratio (SNR) of detection. Our monitor can detect staphylococcus concentration above 1800 cfu/L of air stably.

We present a polymer biochip with embedded optics which allows the detection of supercritical angle fluorescence (SAF) without losses due to total internal reflection within the substrate. The chip design comprises structured spherical and aspherical optical elements on the bottom, while the top is chemically functionalized for direct binding of biomolecules. Furthermore, this design facilitates integration in lab-on-a-chip systems with appropriate microfluidics. In the confocal optical setup an ellipsoidal mirror is used for collection of SAF light above the critical angle of the water-polymer interface, which is detected by a photon-counting detector. The work presented here represents a proof of concept for performing sensitive and rapid point-of-care testing, using this low-cost, robust and disposable optical biochip platform. The performance of the platform was validated using direct binding DNA and human IgG assays which yielded low limits of detection 10 pM for DNA and 10 pg/ml for human IgG.

We have developed a novel noncontact setup to implement a cone shell illumination and detection configuration using axicon lenses for depth-sensitive fluorescence measurements. The setup was demonstrated experimentally to be capable of detecting fluorescence from a two-layered turbid agar phantom with a larger sensitivity to the deep layer and a larger range of sensitivity to either layer than a conventional cone configuration implemented by a microscope objective lens. Furthermore, the axicon lens-based setup eliminates the need of moving the objective lens up or down to achieve depth-sensitive measurements, which effectively improves the consistency of optical coupling and thus would be preferred in a clinical setting.

A modified spatial filtering method that improves the sensitivity of single-beam and mode-mismatched thermal lens spectroscopy (TLS) for fluorescence quantum yield measurement is presented. The method is based on the detection of the external part of a laser beam transmitted by the fluorescent sample (eclipsing detection mode). The experimental results show that the signal/noise (S/N) ratio of the absolute quantum yield of Rh6G can be enhanced up to ~1400% using the eclipsing detection mode on the TLS experimental setup. The method was evaluated by measuring the fluorescence quantum yield of varying concentration of ethanolic solutions of Rhodamine 6G.

The broadband parametric fluorescence from a nonlinear crystal can be used as a compact primary source instead of a blackbody for absolute measurements of instrument spectral efficiency. We describe such a setup for measuring the instrument spectral response function in the wavelength range from 450 to 1000 nm. We perform angle-resolved imaging spectroscopy of conical parametric fluorescence in a beta-barium borate crystal pumped by a 405-nm diode laser. The experimental angle-resolved spectra and the generation efficiency of parametric down-conversion agree with a plane-wave theoretical analysis.

We report significant enhancements in Er(3+) luminescence as well as in Raman intensity in silver nanoparticles embedded zinc-tellurite glass. Surface enhanced Raman scattering effect is highlighted for the first time in tellurite glass containing silver NPs resulting in an enhanced Raman signal (~10 times). SAED manifest the growth of Ag(0) nanoparticles along the (111) and (200) crystallographic planes having average diameter in the range 14-36 nm. Surface plasmon resonance bands are observed in the range 484-551 nm. Furthermore, four prominent photoluminescence bands undergo significant enhancements up to 3 times. The enhancement is majorly attributed to the local field effect of silver NPs.

In this work we present a LIDAR sensor devised for the acquisition of time resolved laser induced fluorescence spectra. The gating time for the acquisition of the fluorescence spectra can be sequentially delayed in order to achieve fluorescence data that are resolved both in the spectral and temporal domains. The sensor can provide sub-nanometric spectral resolution and nanosecond time resolution. The sensor has also imaging capabilities by means of a computer-controlled motorized steering mirror featuring a biaxial angular scanning with 200 μradiant angular resolution. The measurement can be repeated for each point of a geometric grid in order to collect a hyper-spectral time-resolved map of an extended target.

We report a spectrally resolved fluorescence lifetime imaging system based on time gated single photon detection with a fixed gate width of 200 ps and 7 spectral channels. Time gated systems can operate at high count rates but usually have large gate widths and sample only part of the fluorescence decay curve. In the system presented in this work, the fluorescence signal is sampled using a high speed transceiver. An error analysis is carried out to characterize the performance of both lifetime and spectral detection. The effect of gate width and spectral channel width on the accuracy of estimated lifetimes and spectral widths is described. The performance of the whole instrument is evaluated at count rates of up to 12 MHz. Accurate fluorescence lifetimes (error < 2%) are recorded at count rates as high as 5 MHz. This is limited by the PMT performance, not by the electronics. Analysis of the large spectral lifetime image sets is challenging and time-consuming. Here, we demonstrate the use of lifetime and spectral phasors for analyzing images of fibroblast cells with 2 different labeled components. The phasor approach provides a fast and intuitive way of analyzing the results of spectrally resolved fluorescence lifetime imaging experiments.

We propose a localized surface plasmon microscope that provides simultaneous imaging of refractive index and fluorescent intensity distributions. We show experimental images of fluorescent and transparent particles under circular pupil illumination to confirm simultaneous high-resolution imaging. Furthermore, we investigate applicability of annular pupil illumination employing two axicons to improve energy efficiency in the fluorescent imaging and find that a brighter image is obtainable by maintaining high spatial resolution for both imaging modes.

A capillary microresonator platform for refractometric sensing is demonstrated by coating the interior of thick-walled silica capillaries with a sub-wavelength layer of high refractive index, dye-doped polymer. No intermediate processing, such as etching or tapering, of the capillary is required. Side illumination and detection of the polymer layer reveals a fluorescence spectrum that is periodically modulated by whispering gallery mode resonances within the layer. Using a Fourier technique to calculate the spectral resonance shifts, the fabricated capillary resonators exhibited refractometric sensitivities up to approximately 30 nm/RIU upon flowing aqueous glucose through them. These sensors could be readily integrated with existing biological and chemical separation platforms such as capillary electrophoresis and gas chromatography where such thick walled capillaries are routinely used with polymer coatings. A review of the modelling required to calculate whispering gallery eigenmodes of such inverted cylindrical resonators is also presented.

We investigate the fundamental transmission characteristics of nanosecond-duration, 10 kHz repetition rate, ultraviolet (UV) laser pulses through state-of-the-art, UV-grade fused-silica fibers being used for hydroxyl radical (OH) planar laser-induced fluorescence (PLIF) imaging. Studied in particular are laser-induced damage thresholds (LIDTs), nonlinear absorption, and optical transmission stability during long-term UV irradiation. Solarization (photodegradation) effects are significantly enhanced when the fiber is exposed to high-repetition-rate, 283 nm UV irradiation. For 10 kHz laser pulses, two-photon absorption is strong and LIDTs are low, as compared to those of laser pulses propagating at 10 Hz. The fiber characterization results are utilized to perform single-laser-shot, OH-PLIF imaging in pulsating turbulent flames with a laser that operates at 10 kHz. The nearly spatially uniform output beam that exits a long multimode fiber becomes ideal for PLIF measurements. The proof-of-concept measurements show significant promise for extending the application of a fiber-coupled, high-speed OH-PLIF system to harsh environments such as combustor test beds, and potential system improvements are suggested.

A significant increase in the use of the herbicide glyphosate has generated many questions about its residual accumulation in the environment and possible damage to crops. In this study, changes in chlorophyll a (chl-a) fluorescence induced by glyphosate in three varieties of glyphosate-resistant soybean plants were determined with an in vivo analysis based on a portable laser-induced fluorescence system. Strong suppression of chl-a fluorescence was observed for all plants treated with the herbicide. Moreover, the ratio of the emission bands in the red and far-red regions (685 nm/735 nm) indicates that the application of glyphosate led to chlorophyll degradation. The results also indicated that the use of glyphosate, even at concentrations recommended by the manufacturer, suppressed chl-a fluorescence. In summary, this study shows that fluorescence spectroscopy can detect, in vivo, very early changes in the photosynthetic status of transgenic soybeans treated with this herbicide.

A new concept of compact biochip for surface plasmon-enhanced fluorescence assays is reported. It takes advantage of the amplification of fluorescence signal through the coupling of fluorophore labels with confined and strongly enhanced field intensity of surface plasmons. In order to efficiently excite and collect the emitted fluorescence light via surface plasmons on a metallic sensor surface, (reverse) Kretschmann configuration is combined with diffractive optical elements embedded on the chip surface. These include a concentric relief grating for the imaging of highly directional surface plasmon-coupled emission to a detector. Additional linear grating is used for the generating of surface plasmons at the excitation wavelength on the sensor surface in order to increase the fluorescence excitation rate. The reported approach offers the increased intensity of fluorescence signal, reduced background, and compatibility with nanoimprint lithography for cost-effective preparation of sensor chip. The presented approach was implemented for biosensing in a model immunoassay experiment in which the limit of detection of 11 pM was achieved.

Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, transport, flow velocities and concentrations in an imaging mode. SPIM-FCS records correlation functions over a whole plane in a sample, which requires array detectors for recording the fluorescence signal. Several types of image sensors are suitable for FCS. They differ in properties such as effective area per pixel, quantum efficiency, noise level and read-out speed. Here we compare the performance of several low light array detectors based on three different technologies: (1) Single-photon avalanche diode (SPAD) arrays, (2) passive-pixel electron multiplying charge coupled device (EMCCD) and (3) active-pixel scientific-grade complementary metal oxide semiconductor cameras (sCMOS). We discuss the influence of the detector characteristics on the effective FCS observation volume, and demonstrate that light sheet based SPIM-FCS provides absolute diffusion coefficients. This is verified by parallel measurements with confocal FCS, single particle tracking (SPT), and the determination of concentration gradients in space and time. While EMCCD cameras have a temporal resolution in the millisecond range, sCMOS cameras and SPAD arrays can extend the time resolution of SPIM-FCS down to 10 μs or lower.

Current optical fiber probes for fluorescence spectroscopy struggle with large luminescence background and low detection sensitivities that challenge the detection of fluorescent molecules at sub-micromolar concentration. Here we report the demonstration of a hollow-core photonic crystal fiber (HC-PCF) probe for remote fluorescence sensing with single molecule sensitivity down to nanomolar concentrations, where both the excitation and fluorescence beams are counter-propagating through the same fiber. A 20 μm polystyrene microsphere is used to efficiently excite and collect the fluorescence from the sample solution thanks to a photonic nanojet effect. Compared to earlier work with silica fibers, the new HC-PCF-microsphere probe achieves a 200x improvement of the signal-to-noise ratio for a single molecule detection event, and a 1000x reduction of the minimum detectable concentration. The device is implemented with fluorescence correlation spectroscopy to distinguish between molecules of similar fluorescence spectra based on the analysis of their translational diffusion properties, and provides similar performance as conventional confocal microscopes.

An optofluidic water-jet waveguide on chip for fluorescence analysis is presented. A high-speed water stream produced by means of a microchannel acts at the same time as the solution to analyze and as the collecting optical waveguide. The absence of solid walls and the very optically smooth surface of the liquid stream permits a strong increase of the signal-to-noise ratio. The device layout allows a self-alignment direct coupling of a water-jet waveguide with a multimode optical fiber connected to the detector. The performances of the integrated system are tested on Cy5 water solutions. For a 4.5 mm long water-jet waveguide, the measured limit of detection (LOD) is 2.56 nM and filter-free detection is possible with an LOD of 6.11 nM.

This work reports on the implementation of different absorption micro-filters based on a dye-doped hybrid organic-inorganic xerogel polymeric material synthesized by the sol-gel process. Microstructures containing eight different filter widths were fabricated in polydimethylsiloxane (PDMS), bonded to glass substrates and filled with the corresponding dye doped polymeric material by a soft lithography approach. The filtering capacity as a function of dye concentration and filter width was studied and revealed a linear dependence with both parameters, as expected according to the Beer-Lambert law. Zero passband transmittance values and relatively sharp stopband regions were achieved with all the filters, also showing rejection levels between -6 dB and -55 dB. Finally, such filters were monolithically integrated into a disposable fluorescence-based photonic lab-on-a-chip (PhLoC) approach. Calibration curves carried out with a model fluorophore target analyte showed an over two-fold increase in sensitivity and a thirty-fold decrease of the limit of detection (LOD) compared with the values recorded using the same PhLoC system but without the polymeric filter structure. The results presented herein clearly indicate the feasibility of these xerogel-based absorbance filtering structures for being applied as low-cost optical components that can be easily incorporated into disposable fluorescence-based photonic lab on a chip systems.

A lidar for measuring fluorescence from atmospheric aerosols was constructed with a third harmonic Nd:YAG laser, a 1-m diameter telescope, and a 32-channel time-resolved photon-counting spectrometer system. Fluorescence spectrum and vertical distribution of fluorescent aerosols in the lower atmosphere were observed during the nighttime with excitation at 355 nm. Relatively strong broad fluorescence was observed from Asian dust and air-pollution aerosols transported from urban and industrial areas. Rough estimates of the fluorescence efficiency were given for these aerosols. The intensity of the total fluorescence over the spectral range from 420 to 510 nm was comparable to that of nitrogen vibrational Raman scattering. That indicates the possibility of making a compact Raman-Mie-fluorescence lidar for aerosol monitoring.

A laser line-scanning instrument was developed to optimize the near-field enhancement capability of a one-dimensional photonic crystal (PC) for excitation of surface-bound fluorophores. The excitation laser beam is shaped into an 8 μm × 1 mm line that is focused along the direction of the PC grating, while remaining collimated perpendicular to the grating. Such a beam configuration offers high excitation power density while simultaneously providing high resonant coupling efficiency from the laser to the PC surface. Using a panel of 21 immunofluorescence assays on the PC surface in a microarray format, the approach achieves an enhancement factor as high as 90-fold between on-resonance and off-resonance illumination. The instrument provides a capability for sensitive and inexpensive analysis of cancer biomarkers in clinical applications.

We characterize a novel fluorescence microscope which combines the high spatial discrimination of a total internal reflection epi-fluorescence (epi-TIRF) microscope with that of stimulated emission depletion (STED) nanoscopy. This combination of high axial confinement and dynamic-active lateral spatial discrimination of the detected fluorescence emission promises imaging and spectroscopy of the structure and function of cell membranes at the macro-molecular scale. Following a full theoretical description of the sampling volume and the recording of images of fluorescent beads, we exemplify the performance and limitations of the TIRF-STED nanoscope with particular attention to the polarization state of the laser excitation light. We demonstrate fluorescence correlation spectroscopy (FCS) with the TIRF-STED nanoscope by observing the diffusion of dye molecules in aqueous solutions and of fluorescent lipid analogs in supported lipid bilayers in the presence of background signal. The nanoscope reduced the out-of-focus background signal. A lateral resolution down to 40-50 nm was attained which was ultimately limited by the low lateral signal-to-background ratio inherent to the confocal epi-TIRF scheme. Together with the estimated axial confinement of about 55 nm, our TIRF-STED nanoscope achieved an almost isotropic and less than 1 attoliter small all-optically induced measurement volume.

We experimentally investigate the fluorescence photon emission characteristics for single q-dots by using optical nanofibers. We demonstrate that single q-dots can be deposited along an optical nanofiber systematically and reproducibly with a precision of 5 μm. For single q-dots on an optical nanofiber, we measure the fluorescence photon numbers coupled into the nanofiber and the normalized photon correlations, by varying the excitation laser intensity. We estimate the fluorescence photon coupling efficiency into the nanofiber guided modes.

Fluorescence Correlation Spectroscopy (FCS) yields measurement parameters (number of molecules, diffusion time) that characterize the concentration and kinetics of fluorescent molecules within a supposedly known observation volume. Absolute derivation of concentrations and diffusion constants therefore requires preliminary calibrations of the confocal Point Spread Function with phantom solutions under perfectly controlled environmental conditions. In this paper, we quantify the influence of optical aberrations on single photon FCS and demonstrate a simple Adaptive Optics system for aberration correction. Optical aberrations are gradually introduced by focussing the excitation laser beam at increasing depths in fluorescent solutions with various refractive indices, which leads to drastic depth-dependent bias in the estimated FCS parameters. Aberration correction with a Deformable Mirror stabilizes these parameters within a range of several tens of μm into the solution. We also demonstrate, both theoretically and experimentally, that the molecular brightness scales as the Strehl ratio squared.

Fluorometers are widely used in ecosystem observing to monitor fluorescence signals from organic compounds, as well as to infer geophysical parameters such as chlorophyll or CDOM concentration, but measurements are susceptible to variation caused by biofouling, instrument design, sensor drift, operating environment, and calibration rigor. To collect high quality data, such sensors need frequent checking and regular calibration. In this study, a wide variety of both liquid and solid fluorescent materials were trialed to assess their suitability as reference standards for performance assessment of in situ fluorometers. Criteria used to evaluate the standards included the spectral excitation/emission responses of the materials relative to fluorescence sensors and to targeted ocean properties, the linearity of the fluorometer's optical response with increasing concentration, stability and consistency, availability and ease of use, as well as cost. Findings are summarized as a series of recommended reference standards for sensors deployed on stationary and mobile platforms, to suit a variety of in situ coastal to ocean sensor configurations. Repeated determinations of chlorophyll scale factor using the recommended liquid standard, Fluorescein, achieved an accuracy of 2.5%. Repeated measurements with the recommended solid standard, Plexiglas Satinice® plum 4H01 DC (polymethylmethacrylate), over an 18 day period varied from the mean value by 1.0% for chlorophyll sensors and 3.3% for CDOM sensors.

We discuss circular scanning Fluorescence Correlation Spectroscopy (sFCS) as a simple extension of standard FCS for accurate, robust and fast diffusion measurements on membranes. The implementation is based on a straightforward conversion of a conventional FCS instrument to a sFCS device by mounting a mirror onto a two-axis piezo scanner. The measurement volume is scanned in a circle with sub-micron radius, allowing the determination of diffusion coefficients and concentrations without any a priori knowledge of the size of the detection volume. This is highly important in measurements on two-dimensional surfaces, where the volume size, and therefore the quantitative outcome of the experiment, is determined by the relative position of the surface and the objective focus, a parameter difficult to control in practice. The technique is applied to diffusion measurements on model membrane systems: supported lipid bilayers and giant unilamellar vesicles. We show that the method is insensitive to membrane positioning and to disturbing processes on faster or slower time scales than diffusion, and yields accurate results even for fluctuating or drifting membranes. Its robustness, short measurement times, and small size of the probed area makes this technique particularly attractive for analyzing the properties of membranes and molecules diffusing and interacting within them.

A multivariate optical computer has been constructed consisting of a spectrograph, digital micromirror device, and photomultiplier tube that is capable of determining absolute concentrations of individual components of a multivariate spectral model. We present experimental results on ternary mixtures, showing accurate quantification of chemical concentrations based on integrated intensities of fluorescence and Raman spectra measured with a single point detector. We additionally show in simulation that point measurements based on principal component spectra retain the ability to classify cancerous from noncancerous T cells.

A single-wavelength spectral-imaging-based Thai jasmine rice breed identification is demonstrated. Our nondestructive identification approach relies on a combination of fluorescent imaging and simple image processing techniques. Especially, we apply simple image thresholding, blob filtering, and image subtracting processes to either a 545 or a 575 nm image in order to identify our desired Thai jasmine rice breed from others. Other key advantages include no waste product and fast identification time. In our demonstration, UVC light is used as our exciting light, a liquid crystal tunable optical filter is used as our wavelength seclector, and a digital camera with 640 active pixels × 480 active pixels is used to capture the desired spectral image. Eight Thai rice breeds having similar size and shape are tested. Our experimental proof of concept shows that by suitably applying image thresholding, blob filtering, and image subtracting processes to the selected fluorescent image, the Thai jasmine rice breed can be identified with measured false acceptance rates of <22.9% and <25.7% for spectral images at 545 and 575 nm wavelengths, respectively. A measured fast identification time is 25 ms, showing high potential for real-time applications., (© 2011 Optical Society of America)

We investigate the influence of circular corrugations surrounding a central nanoaperture to further enhance the fluorescence count rate per emitter and control its emission directionality. Adding a single corrugation already allows to significantly increase the fluorescence signal as compared to a bare nanoaperture. A complete fluorescence characterization quantifies the excitation and emission gains contributing to the fluorescence enhancement process as the number of corrugations is increased.

Laser light excitation of fluorescent markers offers highly sensitive and specific analysis for bio-medical or chemical analysis. To profit from these advantages for applications in the field or at the point-of-care, a plastic lab-on-a-chip with integrated organic semiconductor lasers is presented here. First order distributed feedback lasers based on the organic semiconductor tris(8-hydroxyquinoline) aluminum (Alq3) doped with the laser dye 4-dicyanomethylene-2-methyl-6-(p-dimethylaminostyril)-4H-pyrane (DCM), deep ultraviolet induced waveguides, and a nanostructured microfluidic channel are integrated into a poly(methyl methacrylate) (PMMA) substrate. A simple and parallel fabrication process is used comprising thermal imprint, DUV exposure, evaporation of the laser material, and sealing by thermal bonding. The excitation of two fluorescent marker model systems including labeled antibodies with light emitted by integrated lasers is demonstrated.

A two-wavelength laser-induced fluorescence (LIF) instrument has been developed and used to characterize individual biological aerosol particles, including biological warfare (BW) agent surrogates. Fluorescence in discrete spectral bands from widely different species, and also from similar species under different growth conditions were measured and compared. The two-wavelength excitation approach was found to increase discrimination among several biological materials, and especially with respect to diesel exhaust particles, a common interferent for LIF BW detection systems. The spectral characteristics of a variety of biological materials and ambient air components have been studied as a function of aerosol particle size and incident fluence.

We report high-power operation of a broadband superfluorescent source with an all-fiber configuration. The superfluorescent source yields an amplified spontaneous emission (ASE) output of 43.2 and 25.1 W from both ends, respectively. The maximum combined ASE output is 68.3 W with a slope efficiency of 78.6%. The wavelength bandwidths (FWHM) of the counterpropagating and copropagating ASE output are 31.5 and 18.9 nm, respectively. The fused angle-polished side-pumping coupler is applied to set up the all-fiber configuration. Furthermore, the influence of reflectivity on the laser threshold of the ASE sources is studied theoretically. The experimental results agree with the theoretical analysis.

Simultaneous time- and wavelength-resolved fluorescence spectroscopy (STWRFS) was developed and tested for the dynamic characterization of atherosclerotic tissue ex vivo and arterial vessels in vivo. Autofluorescence, induced by a 337 nm, 700 ps pulsed laser, was split to three wavelength sub-bands using dichroic filters, with each sub-band coupled into a different length of optical fiber for temporal separation. STWRFS allows for fast recording/analysis (few microseconds) of time-resolved fluorescence emission in these sub-bands and rapid scanning. Distinct compositions of excised human atherosclerotic aorta were clearly discriminated over scanning lengths of several centimeters based on fluorescence lifetime and the intensity ratio between 390 and 452 nm. Operation of STWRFS blood flow was further validated in pig femoral arteries in vivo using a single-fiber probe integrated with an ultrasound imaging catheter. Current results demonstrate the potential of STWRFS as a tool for real-time optical characterization of arterial tissue composition and for atherosclerosis research and diagnosis.

Biophysical imaging tools exploit several properties of fluorescence to map cellular biochemistry. However, the engineering of a cost-effective and user-friendly detection system for sensing the diverse properties of fluorescence is a difficult challenge. Here, we present a novel architecture for a spectrograph that permits integrated characterization of excitation, emission and fluorescence anisotropy spectra in a quantitative and efficient manner. This sensing platform achieves excellent versatility of use at comparatively low costs. We demonstrate the novel optical design with example images of plant cells and of mammalian cells expressing fluorescent proteins undergoing energy transfer.

PbS quantum dots are promising active emitters for use with high-quality Si nanophotonic devices in the telecommunications-band. Measurements of low quantum dot densities are limited both because of low fluorescence levels and the challenges of single photon detection at these wavelengths. Here, we report on methods using a fiber taper waveguide to efficiently extract PbS quantum dot photoluminescence. Temperature dependent ensemble measurements reveal an increase in emitted photons concomitant with an increase in excited-state lifetime from 58.9 ns at 293 K to 657 ns at 40 K. Measurements are also performed on quantum dots on high-Q (>10(5)) microdisks using cavity-resonant, pulsed excitation.