Fluorescence Rejection Research Articles

Methanol-to-Olefins Studied by UV Raman Spectroscopy as Compared to Visible Wavelength: Capitalization on Resonance Enhancement.

Resonance Raman spectroscopy can provide insights into complex reaction mechanisms by selectively enhancing the signals of specific molecular species. In this work, we demonstrate that, by changing the excitation wavelength, Raman bands of different intermediates in the methanol-to-hydrocarbons reactions can be identified. We show in particular how UV excitation enhances signals from short-chain olefins and cyclopentadienyl cations during the induction period, while visible excitation better detects later-stage aromatics. However, visible excitation is prone to fluorescence that can obscure Raman signals, and hence, we show how fast fluorescence rejection techniques like Kerr gating are necessary for extracting useful information from visible excitation measurements.

Superior performance of catalytic ozonation on molecular-level transformation of effluent organic matter and self-cleaning property in catalytic ozonation membrane reactor

Catalytic ozonation membrane reactor (COMR) is one of the most promising technologies for municipal wastewater reclamation. Herein, we evaluated superior performance of catalytic ozonation in COMR through filtration of effluent organic matter (EfOM) using the origin and three catalytic membranes. We found that filtration primarily contributed to rejection of protein-like fluorescence enriched in relatively high-oxygen compounds (O/C >0.4) with high molecular weight and catalytic ozonation further removed humic-like materials and highly unsaturated, aromatic and reduced compounds (O/C<0.4; H/C<1.3), demonstrating complementary function of catalytic ozonation and filtration. The superior performance of catalytic ozonation than ozonation is reflected in higher removal of medium molecular weighted humic-like materials and S-containing compounds, enhancing the mineralization and reaction extent of EfOM. Additionally, catalytic ozonation exhibited strong self-cleaning property and eliminated irreversible membrane fouling. Our findings provide molecular-level insights into the reaction mechanism governing the COMR towards municipal wastewater reclamation.

Application of deep raman spectroscopy to detect ammonium nitrate concealed in color fabrics

Detection of concealed Improvised Explosive Devices (IEDs) is a prerequisite for forensic and homeland security investigations. In this case, fabrics are common barriers for hidden explosives to be seen or detected. Deep Raman spectroscopy is a well-known detection method that provides detailed vibrational information about the depth profiling of Raman-active materials. In this research, a combination of space-resolved and time-resolved Raman techniques was successfully used to acquire high-quality ammonium nitrate Raman spectra concealed in white and brown synthetic fabric as a highly fluorescing barrier. The signal-to-background noise ratio was calculated in all the experiments. Concerning the result for white synthetic fabric, the optimum value was at 180 ns gate delay and 2 mm offset. These values were at 100 ns gate delay and 3 mm offset for brown synthetic fabrics. The role of retarded Raman photons in offset and gate delay variations was discussed too. As the results showed, implementing TR-SORS to identify the IEDs concealed in color fabrics caused the accelerated rejection of high fluorescence from the container surface and led to improved Raman signal.

Time-Resolved Raman Spectrometer With High Fluorescence Rejection Based on a CMOS SPAD Line Sensor and a 573-nm Pulsed Laser

A time-resolved Raman spectrometer is demonstrated based on a 256 × 8 single-photon avalanche diodes fabricated in CMOS technology (CMOS SPAD) line sensor and a 573-nm fiber-coupled diamond Raman laser delivering pulses with duration below 100-ps full-width at half-maximum (FWHM). The collected backscattered light from the sample is dispersed on the line sensor using a custom volume holographic grating having 1800 lines/mm. Efficient fluorescence rejection in the Raman measurements is achieved due to a combination of time gating on sub-100-ps time scale and a 573-nm excitation wavelength. To demonstrate the performance of the spectrometer, fluorescent oil samples were measured. For organic sesame seed oil having a continuous wave (CW) mode fluorescence-to-Raman ratio of 10.5 and a fluorescence lifetime of 2.7 ns, a signal-to-distortion value of 76.2 was achieved. For roasted sesame seed oil having a CW mode fluorescence-to-Raman ratio of 82 and a fluorescence lifetime of 2.2 ns, a signal-to-distortion value of 28.2 was achieved. In both cases, the fluorescence-to-Raman ratio was reduced by a factor of 24-25 owing to time gating. For organic oil, spectral distortion was dominated by dark counts, while for the more fluorescent roasted oil, the main source of spectral distortion was timing skew of the sensor. With the presented postprocessing techniques, the level of distortion could be reduced by 88%-89% for both samples. Compared with common 532-nm excitation, approximately 73% lower fluorescence-to-Raman ratio was observed for 573-nm excitation when analyzing the organic sesame seed oil.

Bent crystal Laue analyser combined with total reflection fluorescence X-ray absorption fine structure (BCLA + TRF-XAFS) and its application to surface studies.

A bent crystal Laue analyser (BCLA) is an X-ray energy analyser used for fluorescence X-ray absorption fine-structure (XAFS) spectroscopy to separate the fluorescence X-ray emission line of a target atom from the elastic scattering X-rays and other fluorescence emission lines. Here, the feasibility of the BCLA for total reflection fluorescence XAFS (TRF-XAFS), which has a long X-ray footprint on the substrate surface owing to grazing incidence, was tested. The focal line of the BCLA was adjusted on the X-ray footprint and the XAFS signal for one monolayer of Pt deposited on a 60 nm Au film with high sensitivity was obtained. Although range-extended XAFS was expected by the rejection of Au fluorescence arising from the Au substrate, a small glitch was found in the Au L3 edge because of the sudden change of the complex refraction index of the Au substrate at the Au edge. This abnormal spectrum feature can be removed by reflectivity correction using Au foil absorption data. BCLA combined with TRF-XAFS spectroscopy (BCLA + TRF-XAFS) is a new technique for the in situ surface analysis of highly dispersed systems even in the presence of a liquid overlayer.

Parallelized shifted-excitation Raman difference spectroscopy for fluorescence rejection in a temporary varying system.

A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted‐excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted‐excitation spectra, has been used to separate Raman scattering from excitation‐shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.

Fast Raman spectral mapping of highly fluorescing samples by time-gated spectral multiplexed detection.

We present a time-gated Raman micro-spectroscopy technique suitable for fast Raman mapping of samples eliciting large laser-induced fluorescence backgrounds. To achieve the required time resolution for effective fluorescence rejection, a picosecond pulsed laser and a single-photon avalanche diode were used. A module consisting of a spectrometer, digital micromirror device, and two prisms was used for high-resolution spectral filtering and multiplexing, which is required for a high chemical specificity and short integration times. With this instrument, we demonstrated time-gated Raman imaging of highly fluorescent samples, achieving acquisition times as short as 3min for 40×40 pixel resolution images.

Raman Imaging Microscopy for Quantitative Analysis of Biological Samples.

Raman imaging microscopy is a powerful tool for label-free imaging of biological samples. It has the advantage of measuring the spatial distribution of endogenous proteins and lipids in cells, as well as obtaining chemical information on these endogenous molecules, such as hydrogen bonding and electrostatic interactions. However, because Raman intensity is very weak compared with fluorescence intensity, obtaining a reliable Raman image requires fast acquisition of a Raman image and rejection of background fluorescence. In this chapter, we describe the procedure for obtaining images of the Raman band of interest using a multipoint technique, which is the fast acquisition method for obtaining an image.

Improved Savitzky-Golay-method-based fluorescence subtraction algorithm for rapid recovery of Raman spectra.

In this paper, we propose an improved subtraction algorithm for rapid recovery of Raman spectra that can substantially reduce the computation time. This algorithm is based on an improved Savitzky-Golay (SG) iterative smoothing method, which involves two key novel approaches: (a)the use of the Gauss-Seidel method and (b)the introduction of a relaxation factor into the iterative procedure. By applying a novel successive relaxation (SG-SR) iterative method to the relaxation factor, additional improvement in the convergence speed over the standard Savitzky-Golay procedure is realized. The proposed improved algorithm (the RIA-SG-SR algorithm), which uses SG-SR-based iteration instead of Savitzky-Golay iteration, has been optimized and validated with a mathematically simulated Raman spectrum, as well as experimentally measured Raman spectra from non-biological and biological samples. The method results in a significant reduction in computing cost while yielding consistent rejection of fluorescence and noise for spectra with low signal-to-fluorescence ratios and varied baselines. In the simulation, RIA-SG-SR achieved 1 order of magnitude improvement in iteration number and 2 orders of magnitude improvement in computation time compared with the range-independent background-subtraction algorithm (RIA). Furthermore the computation time of the experimentally measured raw Raman spectrum processing from skin tissue decreased from 6.72 to 0.094s. In general, the processing of the SG-SR method can be conducted within dozens of milliseconds, which can provide a real-time procedure in practical situations.

Spatially compressed dual-wavelength excitation Raman spectrometer.

The design and operation of a novel dual-laser excitation Raman instrument is described. The use of two lasers of differing wavelengths allows for a Raman spectrum covering all fundamental modes of vibration to be collected while minimizing fluorescence and allowing for spatial compression of the spectrum on an imaging detector. The use of diode lasers with integrated distributed Bragg reflector gratings facilitates the use of an integrated thermoelectric cooler to allow collection of shifted excitation spectra for both of the lasers, further enhancing the rejection of fluorescence. An example is given, which uses seven excitation wavelengths for each laser to reconstruct the Raman spectrum of a solvent in the presence of a highly fluorescent dye by using a sequentially shifted excitation Raman reconstruction algorithm.

Single Photon Avalanche Diode for a Time-Gated Raman Spectrometer

Chemicals and bacteria in drinking water pose significant health risks for human being. To improve the safety of drinking water, sensitive, low-cost, rapid detection methods are required for water monitoring. Both electrical1 and optical2techniques have been used in water quality monitoring. However, optical techniques are sometimes preferred for chemical and biological analysis due to their non-contacting and non-destructive properties. Since water is a weak Raman scatterer, Raman spectroscopy is an especially promising technique for water monitoring. Considering the low concentration of contaminants in water and the small Raman cross section, an ultra-sensitive detector for extremely low light level detection is necessary for the detection system.An avalanche diode biased above the breakdown voltage (Geiger mode)3,4 was designed for the detection system. When biased above breakdown, single photon generated carriers in the depletion region can trigger a self-sustaining avalanche multiplication process, diode in Geiger mode is also named as the single photon avalanche diode (SPAD). To reduce the cost and increase its integration with other electronic systems, this diode was fabricated with the standard CMOS process. Determined by the specific process, the peak detection efficiency is in the visible region, and fluorescence emission becomes the most significant background noise in this situation. Raman signal is usually emitted simultaneously with the excitation, while there is delay between excitation and fluorescence emission5. According to the temporal distribution shown in Fig 1, if synchronizing the detection window with laser excitation and shutting down the detector after the excitation, the fluorescence background can be successfully removed6. Figure 1 Temporal distribution of Raman and fluorescenceTo suppress fluorescence background, the SPAD in this work is operated in the time gated mode. Unlike the free running mode which is always biased above breakdown, SPAD in time gated mode is only biased above its breakdown in a very narrow time window, only photons coincident with this window can be detected. This operation can not only remove fluorescence signal, but also reduce the dark count detection probability. Fig 2 shows the schematic and timing diagram of the proposed pixel circuit. Three on chip pulse generators were designed. Two of them are used to gate on and off the diode, and the third one is for time gated read out. Depending on the arrival time of photons, the out signal will have variable width, which can be used to extract the temporal distribution of photons. The chip has been measured in terms of dark noise, temperature dependence, and detection efficiency. In this presentation, both simulation and measurement results will be discussed.Figure 2: Proposed pixel circuit (a): Schematic; (b) Timing diagram References M. W. Shinwari, M. J. Deen and D. Landheer, “Study of the Electrolyte-Insulator-Semiconductor Field-Effect Transistor (EISFET) with Applications in Biosensor Design,” Microelectronics Reliability, 47(12), 2025-2057 (December 2007). Z. Li, M.J. Deen, Q. Fang, P.R. Selvaganapathy, "Design of a flat field concave-grating-based micro-Raman spectrometer for environmental applications" Appl Optics, 51, 6855-6863 (2012).D Palubiak, MM El-Desouki, O Marinov, MJ Deen, Q Fang, "High-speed, single-photon avalanche-photodiode imager for biomedical applications", IEEE Sensors Journal, 11, 2401-2412, (2011).MM El-Desouki, D Palubiak, MJ Deen, Q Fang, O Marinov, "A Novel, High-Dynamic-Range, High-Speed, and High-Sensitivity CMOS Imager Using Time-Domain Single-Photon Counting and Avalanche Photodiodes", IEEE Sensors Journal, 11, 1078-1083 (2011).J. V. Sinfield, O. Colic, D. Fagerman and C. Monwuba. "A Low Cost Time-Resolved Raman Spectroscopic Sensing System Enabling Fluorescence Rejection," Appl Spectrosc , 64, 201-210 (2010) 6. J. Blacksberg, Y. Maruyama, E. Charbon and G. R. Rossman. "Fast single-photon avalanche diode arrays for laser Raman spectroscopy," Opt Lett , 36, 3672-3674 (2011)

Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination

We demonstrate super-resolution imaging with background fluorescence rejection by interferometric temporal focusing microscopy, in which temporal focusing is combined with structured illumination. The lateral resolution and the optical sectioning capability are simultaneously improved by factors of 1.6 and 1.4, respectively, compared to conventional temporal focusing microscopy. Fluorescent beads (200 nm diameter) that are difficult to distinguish from the background fluorescence in conventional temporal focusing microscopy, are clearly visualized by interferometric temporal focusing microscopy.

非法食品添加剂拉曼检测中的荧光抑制

非法食品添加剂拉曼检测中的荧光抑制

Fluorescence Rejection by Shifted Excitation Raman Difference Spectroscopy at Multiple Wavelengths for the Investigation of Biological Samples

Shifted excitation Raman difference spectroscopy (SERDS) was applied for an effective fluorescence removal in the Raman spectra of meat, fat, connective tissue, and bone from pork and beef. As excitation light sources, microsystem diode lasers emitting at 783 nm, 671 nm, and 488 nm each incorporating two slightly shifted excitation wavelengths with a spectral difference of about 10 cm−1 necessary for SERDS operation were used. The moderate fluorescence interference for 783 nm excitation as well as the increased background level at 671 nm was efficiently rejected using SERDS resulting in a straight horizontal baseline. This allows for identification of all characteristic Raman signals including weak bands which are clearly visible and overlapping signals that are resolved in the SERDS spectra. At 488 nm excitation, the spectra contain an overwhelming fluorescence interference masking nearly all Raman signals of the probed tissue samples. However, the essentially background-free SERDS spectra enable determining the majority of characteristic Raman bands of the samples under investigation. Furthermore, 488 nm excitation reveals prominent carotenoid signals enhanced due to resonance Raman scattering which are present in the beef samples but absent in pork tissue enabling a rapid meat species differentiation.

Deep Raman spectroscopy for the non-invasive standoff detection of concealed chemical threat agents

Deep Raman spectroscopy has been utilized for the standoff detection of concealed chemical threat agents from a distance of 15m under real life background illumination conditions. By using combined time and space resolved measurements, various explosive precursors hidden in opaque plastic containers were identified non-invasively. Our results confirm that combined time and space resolved Raman spectroscopy leads to higher selectivity towards the sub-layer over the surface layer as well as enhanced rejection of fluorescence from the container surface when compared to standoff spatially offset Raman spectroscopy. Raman spectra that have minimal interference from the packaging material and good signal-to-noise ratio were acquired within 5s of measurement time. A new combined time and space resolved Raman spectrometer has been designed with nanosecond laser excitation and gated detection, making it of lower cost and complexity than picosecond-based laboratory systems.

Fast single-photon avalanche diode arrays for laser Raman spectroscopy

We incorporate newly developed solid-state detector technology into time-resolved laser Raman spectroscopy, demonstrating the ability to distinguish spectra from Raman and fluorescence processes. As a proof of concept, we show fluorescence rejection on highly fluorescent mineral samples willemite and spodumene using a 128×128 single-photon avalanche diode (SPAD) array with a measured photon detection efficiency of 5%. The sensitivity achieved in this new instrument architecture is comparable to the sensitivity of a technically more complicated system using a traditional photocathode-based imager. By increasing the SPAD active area and improving coupling efficiency, we expect further improvements in sensitivity by over an order of magnitude. We discuss the relevance of these results to in situ planetary instruments, where size, weight, power, and radiation hardness are of prime concern. The potential large-scale manufacturability of silicon SPAD arrays makes them prime candidates for future portable and in situ Raman instruments spanning numerous applications where fluorescence interference is problematic.

Convex Lens-Induced Confinement for Imaging Single Molecules

Fluorescence imaging is used to study the dynamics of a wide range of single molecules in solution or attached to a surface. Two key challenges in this pursuit are to image immobilized single molecules in the presence of a high level of fluorescent background, and to image freely diffusing single molecules for long times. We present a simple modification to a wide-field fluorescence microscope that addresses both challenges and dramatically improves single-molecule imaging. The Convex Lens Induced Confinement (CLIC) system restricts molecules to a wedge-shaped gap of nanoscale depth. The shallow depth of the imaging volume leads to up to 20-fold greater rejection of background fluorescence than is achieved with total internal reflection fluorescence (TIRF) imaging. The elimination of out-of-plane diffusion leads to approximately 10,000-fold longer diffusion-limited observation time per molecule than is achieved with confocal detection in free solution. The restriction of freely diffusing molecules to gaps whose depth is greater than the molecular diameter provides a simple measure of molecular size. The CLIC system may be implemented using a minor modification to a standard flow cell, and does not require any nanofabrication, nor any custom optics, electronics, or computer control. We demonstrate the advantageous properties of CLIC measurements by imaging singly labeled surface-immobilized DNA molecules in the presence of a high concentration of free dye (up to 2 micromolar); by counting the transmembrane proteins in freely diffusing lipid vesicles; and by measuring the sizes of freely diffusing proteins and DNA with diameters ranging from 3nm to 200 nm. Further, we apply CLIC to probe weak intermolecular interactions in a range of systems.

Convex Lens-Induced Confinement for Imaging Single Molecules

Fluorescence imaging is used to study the dynamics of a wide variety of single molecules in solution or attached to a surface. Two key challenges in this pursuit are (1) to image immobilized single molecules in the presence of a high level of fluorescent background and (2) to image freely diffusing single molecules for long times. Strategies that perform well by one measure often perform poorly by the other. Here, we present a simple modification to a wide-field fluorescence microscope that addresses both challenges and dramatically improves single-molecule imaging. The technique of convex lens-induced confinement (CLIC) restricts molecules to a wedge-shaped gap of nanoscale depth, formed between a plano-convex lens and a planar coverslip. The shallow depth of the imaging volume leads to 20-fold greater rejection of background fluorescence than is achieved with total internal reflection fluorescence (TIRF) imaging. Elimination of out-of-plane diffusion leads to an approximately 10,000-fold longer diffusion-limited observation time per molecule than is achieved with confocal fluorescence correlation spectroscopy. The CLIC system also provides a new means to determine molecular size. The CLIC system does not require any nanofabrication, nor any custom optics, electronics, or computer control.

A Low Cost Time-Resolved Raman Spectroscopic Sensing System Enabling Fluorescence Rejection

This paper describes a novel, compact, fiber-coupled, time-resolved Raman spectroscopy system that takes advantage of recent developments in diode laser and data acquisition technology to exploit the natural temporal separation between Raman and fluorescence phenomena and thereby limits the influence of fluorescence on Raman observations. The unit has been designed to be particularly low cost and is intended to provide the foundation for a wide range of in-line or fieldable sensing devices that can enhance the potential and affordability of in situ chemical analyses. The system operating principles, design, and performance are discussed along with its advantages and tradeoffs relative to traditional continuous wave (CW) Raman techniques. The system relies on a 6.4 kHz repetition rate 900 ps pulsed diode laser operating in the visible wavelength range (532 nm) to enhance the quality of Raman observations relative to CW and infrared systems, particularly for analytes examined in the presence of fluorophores. Time-resolved photon counting, achieved through a combination of off-the-shelf and custom hardware and software, limits the influence of fluorescence on Raman observations under pulsed excitation. The paper presents examples of the quality of Raman signatures that can be obtained with the system for a variety of compounds such as trichloroethylene, benzene, an aqueous nitrate solution, and olive oil. Further, the paper demonstrates an approximately 15-fold improvement in signal-to-noise ratio when comparing long- and short-gated time-resolved photon counting acquisition scenarios for a neat benzene sample doped with rhodamine 6G at a concentration of 1 x 10(-4) M. The system's versatility and effectiveness in the assessment of complex mixtures representative of industrial or field settings is demonstrated through analysis of a gasoline sample. Additional discussion outlines how efficient signal averaging over extended observation periods can enable low concentration chemical analyses, particularly relevant in field settings.

Ultrafast Raman loss spectroscopy

AbstractThis paper deals with a new form of nonlinear Raman spectroscopy called ‘ultrafast Raman loss spectroscopy (URLS)’. URLS is analogous to stimulated Raman spectroscopy (SRS) but is much more sensitive than SRS. The signals are background (noise) free unlike in coherent anti‐Stokes Raman spectroscopy (CARS) and it provides natural fluorescence rejection, which is a major problem in Raman spectroscopy. In addition, being a self‐phase matching process, the URLS experiment is much easier than CARS, which requires specific phase matching of the laser pulses. URLS is expected to be alternative if not competitive to CARS microscopy, which has become a popular technique in applications to materials, biology and medicine. Copyright © 2009 John Wiley &amp; Sons, Ltd.