Two-photon nitric oxide laser-induced fluorescence measurements in a diesel engine

A two-photon nitric oxide (NO) laser-induced fluorescence (LIF) technique was developed and applied to study in-cylinder diesel combustion. The technique prevents many problems associated with in-cylinder, single-photon NO planar-laser-induced fluorescence measurements, including fluorescence interference from the Schumann-Runge bands of hot O2, absorption of a UV excitation beam by in-cylinder gases, and difficulty in rejecting scattered laser light while simultaneously attempting to maximize fluorescence signal collection. Verification that the signal resulted from NO was provided by tuning of the laser to a vibrational off-resonance wavelength that showed near-zero signal levels, which resulted from either fluorescence or interference at in-cylinder pressures of as much as 20 bar. The two-photon NO LIF signal showed good qualitative agreement with NO exhaust-gas measurements obtained over a wide range of engine loads.     

Monitoring photosensitizer concentration by use of a fiber-optic probe with a small source-detector separation

We present a noninvasive method to track the concentration of photodynamic therapy drugs in real time. The method is based on measurements of backscattered and fluorescent light with a steady-state fluorescence spectrometer. The ratio of the fluorescent light to the scattered light is found to be linearly proportional to the absorption coefficient of the photosensitizer. The fiber-optic probe used for the measurements has a small source-detector separation; therefore the measurements could be performed through the working channel of an endoscope.     

Modeling of fiber-optic fluorescence probes for strongly absorbing samples

The dynamic range of fiber-optic fluorescent probes such as single fibers and fiber bundles is calculated for strongly absorbing samples, such as process liquids, foodstuffs, and lubricants. The model assumes an excitation beam profile based on a Lambertian light source and uses analytical forms of the collection efficiency, followed by an Abel transformation and numerical integration. It is found that the effect of primary absorption of the excitation light and secondary absorption of the fluorescence is profound. For fiber bundles and bifurcated fiber probes, the upper accessible concentration limit is roughly given by the absorption length of the primary and secondary absorption. Fluorescence detectors that are placed at right angles to the excitation beam axis or collinear to the beam axis are equally strongly affected by secondary absorption. A probe in which the same fiber is used for excitation and for collection of the fluorescence emerges as the fiber probe with the largest accessible concentration range.     

Metal nanoparticle plasmon-enhanced light-harvesting in a photosystem I thin film

Silver metal nanoparticle (NP) enhanced fluorescence is investigated in thin films of cyanobacterial Photosystem I trimer complexes (PSI) by correlating confocal laser scanning microscopy, dark-field imaging, and fluorescence lifetime measurements. PSI represents an interesting light-harvesting complex with a 20 nm diameter that is not uniformly contained within the surface-localized plasmon field of the NPs. With weak far-field illumination, 5- to 20-fold fluorescence enhancement is observed for PSI complexes adjacent to NPs, arising from efficient nanoparticle light collection and subsequent localized, surface plasmon excitation of PSI. Enhanced PSI fluorescence is detected most prominently near "rafts" of aggregated NPs that more completely fill the confocal field of view. These results demonstrate opportunities to probe energy transfer within photosynthetic complexes using plasmonic excitation and to design nanostructures for optimizing artificial light-harvesting systems.     

Spatially correlated fluorescence/AFM of individual nanosized particles and biomolecules

Individual fluorescent polystyrene nanospheres (<10-100-nm diameter) and individual fluorescently labeled DNA molecules were dispersed on mica and analyzed using time-resolved fluorescence spectroscopy and atomic force microscopy (AFM). Spatial correlation of the fluorescence and AFM measurements was accomplished by (1) positioning a single fluorescent particle into the near diffraction-limited confocal excitation region of the optical microscope, (2) recording the time-resolved fluorescence emission, and (3) measuring the intensity of the excitation laser light scattered from the apex of an AFM probe tip and the AFM topography as a function of the lateral position of the tip relative to the sample substrate. The latter measurements resulted in concurrent high-resolution (approximately 10-20 nm laterally) images of the laser excitation profile of the confocal microscope and the topography of the sample. Superposition of these optical and topographical images enabled unambiguous identification of the sample topography residing within the excitation region of the optical microscope, facilitating the identification and structural characterization of the nanoparticle(s) or biomolecule(s) responsible for the fluorescence signal observed in step 2. These measurements also provided the lateral position of the particles relative to the laser excitation profile and the surrounding topography with nanometer-scale precision and the relationship between the spectroscopic and structural properties of the particles. Extension of these methods to the study of other types of nanostructured materials is discussed.     

Multiple-fiber probe design for fluorescence spectroscopy in tissue

The fiber-optic probe is an essential component of many quantitative fluorescence spectroscopy systems, enabling delivery of excitation light and collection of remitted fluorescence in a wide variety of clinical and laboratory situations. However, there is little information available on the role of illumination--collection geometry to guide the design of these components. Therefore we used a Monte Carlo model to investigate the effect of multifiber probe design parameters--numerical aperture, fiber diameter, source--collection fiber separation distance, and fiber-tissue spacer thickness--on light propagation and the origin of detected fluorescence. An excitation wavelength of 400 nm and an emission wavelength of 630 nm were simulated. Noteworthy effects included an increase in axial selectivity with decreasing fiber size and a transition with increasing fiber-tissue spacer size from a subsurface peak in fluorophore sensitivity to a nearly monotonic decrease typical of single-fiber probes. We provide theoretical evidence that probe design strongly affects tissue interrogation. Therefore application-specific customization of probe design may lead to improvements in the efficacy of fluorescence-based diagnostic devices.     

Liquid core waveguide for full imaging of electrophoretic separations

We employed a liquid core waveguide to image both DNA electrophoresis separations and isoelectric focusing of proteins. The utility of the system is demonstrated for DNA fragment sizing and protein separations. The system utilizes the liquid-core waveguide as an efficient window for both the excitation of separated samples and the collection of light through total internal reflectance, with an ability to detect target molecules in the zeptomolar range. Scanning the excitation laser along the length of the electrophoresis capillary excites individually separated analyte bands, while the fluorescence is collected end-on by an optical fiber coupled to a photomultiplier, thus, creating an image of the separation along the length of the capillary.     

Laser-induced fluorescence detection system for microfluidic chips based on an orthogonal optical arrangement

In this work, a simple LIF detection system based on an orthogonal optical arrangement for microfluidic chips was developed. Highly sensitive detection was achieved by detecting the fluorescence light emitted in the microchannel through the sidewall of the chip to reduce scattered light interference from the laser source. A special crossed-channel configuration, with a 1.5-mm distance from the separation channel to the sidewall of the glass chip, was designed in order to facilitate collection of emitted fluorescence light through the sidewall. The significant difference in intensity distribution of scattered laser light on the chip plane observed in this study was fully exploited to optimize S/N ratio of detected signals by rejection of scattered light, both through systematic measurements and employing ray-tracing simulation. A fluorescence collection angle of 45 degrees in the chip plane gave the best result, with a scattered light intensity 1/38 of that obtained at an angle of 90 degrees. Sodium fluorescein and fluorescein isothiocyanate-labeled amino acids were used as model samples to demonstrate the performance of the LIF system. A detection limit (S/N = 3) of 1.1 pM fluorescein was obtained, which is comparable to that of optimized confocal LIF systems for chip-based capillary electrophoresis. Apart from the high detection power, the system also has the advantages of simple optical structure, compactness, and ease in building.     

Quantitative transmission Raman spectroscopy of pharmaceutical tablets and capsules

Quantitative analysis of pharmaceutical formulations using the new approach of transmission Raman spectroscopy has been investigated. For comparison, measurements were also made in conventional backscatter mode. The experimental setup consisted of a Raman probe-based spectrometer with 785 nm excitation for measurements in backscatter mode. In transmission mode the same system was used to detect the Raman scattered light, while an external diode laser of the same type was used as excitation source. Quantitative partial least squares models were developed for both measurement modes. The results for tablets show that the prediction error for an independent test set was lower for the transmission measurements with a relative root mean square error of about 2.2% as compared with 2.9% for the backscatter mode. Furthermore, the models were simpler in the transmission case, for which only a single partial least squares (PLS) component was required to explain the variation. The main reason for the improvement using the transmission mode is a more representative sampling of the tablets compared with the backscatter mode. Capsules containing mixtures of pharmaceutical powders were also assessed by transmission only. The quantitative results for the capsules' contents were good, with a prediction error of 3.6% w/w for an independent test set. The advantage of transmission Raman over backscatter Raman spectroscopy has been demonstrated for quantitative analysis of pharmaceutical formulations, and the prospects for reliable, lean calibrations for pharmaceutical analysis is discussed.     

A new hybrid photomultiplier tube as detector for scintillating crystals

In this work, we have attentively studied the performance of a new hybrid photomultiplier tube (HPMT) as detector for photons from scintillating crystals. The HPMT is equipped with a YAP window in order to improve light collection and increase measured light response from scintillating crystals. Several measurements have been performed on BGO, LSO, CsI(Tl) and NaI(Tl) planar crystals having three different surface treatments as well as on YAP : Ce and CsI(Tl) matrices. Such crystals have been coupled to two HPMTs, one equipped with a YAP window (Y-HPMT) and the other with a conventional quartz window (Q-HPMT). Measurements on crystals coupled to the Y-HPMT have shown a consistent improvement of the light response, thanks to the presence of the YAP window. Indeed, the light response measured with the Y-HPMT was on average equal to 1.5, 2.1 and 2.6 times that obtained with the Q-HPMT for planar crystals with white painted (diffusive), fine ground and polished rear surfaces, respectively. With regards to crystal matrices, we measured a light response increase of about 1.2 times.     

M13 phage-functionalized single-walled carbon nanotubes as nanoprobes for second near-infrared window fluorescence imaging of targeted tumors

Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas.     

Three- and four-photon absorption of a multiphoton absorbing fluorescent probe

High-order multiphoton excitation processes are becoming a reality for fluorescence imaging and phototherapy treatment because they afford minimization of scattered light losses and a reduction of unwanted linear absorption in the living organism transparency window, making them less susceptible to photodamage, while improving the irradiation penetration depth and spatial resolution. We report the four-photon-excited fluorescence emission of (7-benzothiazol-2-yl-9,-didecylfluoren-2-yl)diphenylamine in hexane and its four-photon absorption cross section sigma4' = 8.1 x 10(-109) cm8 s3 photon(-3) for the transition S0 --> S1 when excited at 1600 nm with a tunable optical parametric generator (OPG) pumped by picosecond laser pulses. When pumped at 1200 nm, three-photon absorption was observed, corresponding to the same transition.     

Half-ball lens couples a beveled fiber probe for depth-resolved spectroscopy: Monte Carlo simulations

In this study, we propose a beveled fiber-optic probe coupled with a half-ball lens for improving the depth-resolved fluorescence measurements of epithelial tissue. The Monte Carlo (MC) simulation results show that for a given excitation-collection fiber separation, the probe design with a bevel-angled collection fiber is more sensitive to detect fluorescence photons emitted from the shallow layer of tissue, whereas the flat-tip collection fiber is in favor of probing fluorescence photons originating from deeper tissue areas. This compact half-ball lens-beveled fiber probe design has the potential to facilitate the depth-resolved fluorescence detection of epithelial tissue.     

Highly efficient optical detection of surface-generated fluorescence

We present a theoretical study of a new highly efficient system for optical light collection, designed for ultrasensitive fluorescence detection of surface-bound molecules. The main core of the system is a paraboloid glass segment acting as a mirror for collecting the fluorescence. A special feature of the system is its ability to sample not only fluorescence that is emitted below the angle of total internal reflection (the critical angle) but also particularly the light above the critical angle. As shown, this is especially advantageous for collecting the fluorescence of surface-bound molecules. A comparison is made with conventional high-aperture microscope objectives. Furthermore, it is shown that the system allows not only for highly efficient light collection but also for confocal imaging of the detection region, which is of great importance for rejecting scattered light in potential applications such as the detection of only a few molecules.     

Temperature and pressure imaging using infrared planar laser-induced fluorescence

A new diagnostic technique for measurements of temperature and pressure distribution in gaseous flows has been developed. The technique, based on infrared planar laser-induced fluorescence (IR-PLIF), is applicable to all IR-active species. A simple two-line excitation approach is used for measurements of temperature, while pressure measurements utilize online excitation on one rotational line and offline excitation on another. A demonstration of the technique in a supersonic underexpanded jet of 30% CO2 and 70% N2 was performed, and the results are, to the best of our knowledge, the first demonstration of temperature and pressure imaging using IR-PLIF. The developed diagnostic shows potential for single-shot two-dimensional measurements of temperature and pressure in gaseous flows.     

Method for tracing the position of an alien object embedded in a random particulate medium

We have developed a new technique based on light scattering experiments for tracing an alien particle with deterministic potential in a random collection of particles. We have shown that, via a sequence of measurements of light scattered to a far field of a scattering collection, it is possible to locate the center of the alien particle. The analysis of the stability of reconstruction is provided, and it is demonstrated via simulations that the results are stable for sufficiently large wavelength of the incident light and in cases when the size of the alien particle is comparable with the size of the typical particle in the collection.     

Two-photon excitation fluorescence microscopy with a high depth of field using an axicon

In conventional two-photon excitation fluorescence microscopy, the numerical aperture of the objective determines the lateral resolution and the depth of field. In some situations, as with functional imaging of dynamic events distributed in live biological tissue, an improved temporal resolution is needed; as a consequence, it is imperative to use optics with a high depth of field to simultaneously image objects at different axial positions. With a conventional microscope objective, increasing the depth of field is achieved at the expense of lateral resolution. To overcome this limitation, we have incorporated an axicon in a two-photon excitation fluorescence microscopy system; measurements have shown that an axicon provides a depth of field in excess of a millimeter, while the lateral resolution is maintained at the micrometer scale. Thus axicon-based two-photon microscopy has been shown to yield a high-resolution projection image of a sample with a single 2D scan of the laser beam while maintaining the improved tissue penetration typical of two-photon microscopy.     

Elimination of fluorescence and scattering backgrounds in luminescence lifetime measurements using gated-phase fluorometry

A new gated form of phase fluorometry for measuring lifetimes is presented. The technique uses a square-wave excitation and gates the detector on only during the off period of the excitation. Using a long-lived sample, this eliminates or reduces errors from scattered light and short-lived fluorescences. Using a square-wave modulated excitation source with a 50% duty cycle, traditional data treatment can be used after, at most, a simple pi/2 phase adjustment. A combination of theory and experimental results demonstrates the validity of this new gated method and its utility for eliminating or reducing background. The results are precise, accurate, eliminate scattering errors, and greatly reduce errors due to short-lived fluorescence impurities. Errors from fluorescence bleed-through into the detection period or a slow excitation source turn off can be mitigated by using an offset time prior to gating the detector on.     

Improvement in the measurement of index modulation along an optical fiber grating by movement of the probe spot perpendicularly to the fiber axis

We demonstrate a large improvement in the efficiency of the method proposed by Krug et al. [Opt. Lett. 20, 1767 (1995)] to measure the amplitude of the refractive-index modulation along a fiber Bragg grating. The basic idea consists of using what to our knowledge is a new modulation scheme for the probe beam that not only allows the user to get a better discrimination of the probe light incoherently scattered by the fiber from that scattered by the grating but also facilitates alignment of the setup.     

Method to reduce errors of droplet sizing based on the ratio of fluorescent and scattered light intensities (laser-induced fluorescence/Mie technique)

The droplet sizing accuracy of the laser technique, based on the ratio of laser-induced fluorescence (LIF) and scattered light (Mie) intensities from droplets, is examined. We develop an analytical model of the ratio of fluorescent to scattered light intensities of droplets, which shows that the LIF/Mie technique is susceptible to sizing errors that depend on the mean droplet size and the spread of the droplet size distribution. The sizing uncertainty due to the oscillations of the scattered light intensity as a function of droplet size is first quantified. Then, a new data processing method is proposed that can improve the sizing uncertainty of the technique for the sprays that were examined in this study by more than 5% by accounting for the size spread of the measured droplets, while improvements of 25% are possible when accounting for the mean droplet size. The sizing accuracy of the technique is evaluated in terms of the refractive index of liquid, scattering angle, and dye concentration in the liquid. It is found that the proposed approach leads to sizing uncertainty of less than 14% when combined with light collection at forward scattering angles close to 60° and the lowest fluorescent dye concentration in the liquid for all refractive indices.