Lamp-based native fluorescence detection of proteins in capillary electrophoresis

The potential of a recently developed lamp-based fluorescence detector for the analysis of underivatised proteins by capillary electrophoresis (CE) was investigated. Fluorescence detection (Flu) was achieved using optical light guides to deliver excitation light from a Xenon–Mercury lamp to the capillary detection window and to collect fluorescence emission and lead it to a photomultiplier. The performance of the detector was evaluated by monitoring the native fluorescence of the amino acid tryptophan and the proteins α-chymotrypsinogen A, carbonic anhydrase II, lysozyme and trypsinogen upon excitation at 280 nm. The test compounds were analysed using background electrolytes (BGEs) of sodium phosphate at pH 3.0 and 11.3. The results were compared to experiments of CE with UV absorbance detection. For tryptophan, a linear fluorescence response was obtained with a dynamic range of over 4 orders of magnitude, and a limit of detection (LOD) of 6.7 nM. This LOD was a factor of 200 more favourable than UV detection at 280 nm, and a factor of 20 better than detection at low-UV wavelengths. All tested proteins showed linear fluorescence responses up to 250 μg/mL. LODs were typically in the 10–20 nM range. These LODs were a factor of 25 lower than for UV detection at 280 nm, and comparable to UV detection at low-UV wavelengths. Overall, Flu yields much more stable baselines, especially with a BGE of high pH. The applicability of CE–Flu is demonstrated by the analysis of a degraded protein mixture, and of an expired formulation of the protein drug human growth hormone, indicating that protein degradation products can be selectively detected.     

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.     

Enantioselective micellar electrokinetic chromatography of dl‐amino acids using (+)‐1‐(9‐fluorenyl)‐ethyl chloroformate derivatization and UV‐induced fluorescence detection

Chiral analysis of dl ‐amino acids was achieved by micellar electrokinetic chromatography coupled with UV‐excited fluorescence detection. The fluorescent reagent (+)‐1‐(9‐fluorenyl)ethyl chloroformate was employed as chiral amino acid derivatizing agent and sodium dodecyl sulfate served as pseudo‐stationary phase for separating the formed amino acid diastereomers. Sensitive analysis of (+)‐1‐(9‐fluorenyl)ethyl chloroformate‐amino acids was achieved applying a xenon‐mercury lamp for ultraviolet excitation, and a spectrograph and charge‐coupled device for wavelength‐resolved emission detection. Applying signal integration over a 30 nm emission wavelength interval, signal‐to‐noise ratios for derivatized amino acids were up to 23 times higher as obtained using a standard photomultiplier for detection. The background electrolyte composition (electrolyte, pH, sodium dodecyl sulfate concentration, and organic solvent) was studied in order to attain optimal chemo‐ and enantioseparation. Enantioseparation of 12 proteinogenic dl ‐amino acids was achieved with chiral resolutions between 1.2 and 7.9, and detection limits for most derivatized amino acids in the 13–60 nM range (injected concentration). Linearity (coefficients of determination > 0.985) and peak‐area and migration‐time repeatabilities (relative standard deviations lower than 2.6 and 1.9%, respectively) were satisfactory. The employed fluorescence detection system provided up to 100‐times better signal‐to‐noise ratios for (+)‐1‐(9‐fluorenyl)ethyl chloroformate‐amino acids than ultraviolet absorbance detection, showing good potential for d ‐amino acid analysis.     

A lamp light-emitting diode-induced fluorescence detector for capillary electrophoresis

A light-emitting diode-induced fluorescence detector (LED-FD) for capillary electrophoresis was constructed and evaluated. A lamp LED with an enhanced emission spectrum and a band pass filter was used as the excitation light source. Refractive index matching fluid (RIMF) was used in the detection cell to reduce scattering light and the noise level. The limit of detection (LOD) for fluorescein was 1.5 nM (SNR=3). The system exhibited linear responses in the range of 1 x 10(-8) to 5 x 10(-6)M (R=0.999). Application of the lamp LED-FD for the analysis of FITC-labeled ephedra herb extract by capillary electrophoresis was demonstrated.     

Optical fiber light-emitting diode-induced fluorescence detection for capillary electrophoresis

A highly sensitive optical fiber light-emitting diode (LED)-induced fluorescence detector for CE has been constructed and evaluated. In this detector, a violet or blue LED was used as the excitation source and an optical fiber with 40 microm OD was used to transmit the excitation light. The upper end of the fiber was inserted into the separation capillary and was situated right at the detection window. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a cutoff filter before reaching the photomultiplier tube. Output signals were recorded and processed with a computer using in-house written software. The present CE/fluorescence detector deploys a simple and inexpensive optical system that requires only an LED as the light source. Its utility was successfully demonstrated by the separation and determination of amino acids (AAs) labeled with naphthalene-2,3-dicarboxaldehyde (NDA) and FITC. Low detection limits were obtained ranging from 17 to 23 nM for NDA-tagged AAs and 8 to 12 nM for FITC-labeled AAs (S/N=3). By virtue of such valuable features as low cost, convenience, and miniaturization, the presented detection scheme was proven to be attractive for sensitive fluorescence detection in CE.     

Design and evaluation of capillary coupled with optical fiber light‐emitting diode induced fluorescence detection for capillary electrophoresis

A new detector, capillary coupled with optical fiber LED‐induced fluorescence detector (CCOF‐LED‐IFD, using CCOF for short), is introduced for CE. The strategy of the present work was that the optical fiber and separation capillary were, in the parallel direction, fastened in a fixation capillary with larger inner diameter. By employing larger inner diameter, the fixation capillary allowed the large diameter of the optical fiber to be inserted into it. By transmitting an enhanced excitation light through the optical fiber, the detection sensitivity was improved. The advantages of the CCOF‐CE system were validated by the detection of riboflavin, and the results were compared to those obtained by the in‐capillary common optical fiber LED‐induced fluorescence detector (IC‐COF‐LED‐IFD, using COF for short). The LODs of CCOF‐CE and COF‐CE were 0.29 nM and 11.0 nM (S/N = 3), respectively. The intraday (n = 6) repeatability and interday (n = 6) reproducibility of migration time and corresponding peak area for both types of CE were all less than 1.10 and 3.30%, respectively. The accuracy of the proposed method was judged by employing standard addition method, and recoveries obtained were in the range of 98.0–102.4%. The results indicated that the sensitivity of the proposed system was largely improved, and that its reproducibility and accuracy were satisfactory. The proposed system was successfully applied to separate and determine riboflavin in real sample.     

In-column fiber-optic laser-induced fluorescence detection for CE

A highly sensitive in-column fiber-optic LIF detector for CE has been constructed and evaluated. In this detection system, a 457-nm diode-pumped solid-state blue laser was used as the excitation light source and an optical fiber (40 mum od) was used to transmit the excitation light. One end of the optical fiber was inserted into the separation capillary and was in situ positioned at the detection window. The other end of the fiber was protruded from the capillary to capture the excitation light beam from the blue laser. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a yellow color filter before reaching the photomultiplier tube. The present CE-fluorescence detection is a simple and compact optical system. It reduces the laser scattering effect from the capillary and fiber as compared to the conventional LIF detection for CE. Its utility was successfully demonstrated by the separation and determination of D-penicillamine labeled with naphthalene-2,3-dicarboxaldehyde. The detection limit was 0.8 nM (S/N = 3). The present detection scheme has been proven to be attractive for sensitive fluorescence detection for CE.     

Dual wavelength excitation fluorescence detector for capillary electrophoresis using a pulsed bi-colour light emitting diode

A simple and novel two-colour fluorescence detector for capillary electrophoresis was created using a single bi-colour light emitting diode (LED), multi-band pass excitation and emission filters and a single detector. Excitation light from a blue/red (470/635 nm) bi-colour LED was filtered through a 390/482/563/640 nm multi-band bandpass filter, with emitted light filtered through a 446/523/600/677 nm multi-band bandpass filter before being detected using a photon counting detector. Sequential pulsing of the blue/red LED and deconvolution of the collected fluorescence data allowed extracted electropherograms to be obtained corresponding to excitation with the blue and red LEDs. Optimisation of the pulsed LED conditions revealed an optimum LED on-time of 50 ms, off-time of 30 ms with a pulsed current of 40 mA, giving an effective data acquisition rate of 6.25 Hz. The characteristics of this system were validated by the simultaneous separation and determination of six fluorescent dyes: fluorescein, FITC, coumarin 334, dibromo(R)fluorescein (Ex/Em 470/525 nm), and Cy 5 and the Agilent Bioanalyser DNA dye (Ex/Em 635/670 nm). Under optimum conditions, the detection limits for FITC, fluorescein and Cy 5 were 69 nM, 42 nM and 289 nM (S/N = 3), respectively. These were lower than those obtained with continuous operation of the individual wavelengths at a constant current of 20 mA, but were slightly higher than those obtained using dedicated single wavelength filter combinations designed specifically for use with these fluorophores. The intraday repeatability (n = 6) of migration times was less than 1.0% and less than 3.4% for peak areas, while interday (n = 3) migration time and peak area reproducibility were less than 0.9% and 3.6%, respectively. This simple detector is capable of performing quantitative two-wavelength excitation without the need for complex optics and light source configurations.     

An ellipsoidal mirror for detection of laser-induced fluorescence in capillary electrophoresis system: applications for labelled antibody analysis

An LIF detector was integrated into a CE system which uses a ball lens to focus the laser beam on the CE capillary. The detector employs an ellipsoid that is glued on the capillary window, to permit the collection of the fluorescence in the capillary. This 'trapped' fluorescence stays in the capillary because the angle of the silica/air interface is greater than the critical angle. The performance of this new detector setup is found to be identical to the collinear setup using the same ball lens. An application to the analysis of FITC-labeled IgG was optimized using a 14 cm effective length capillary. The LOD of an FITC-labeled IgG2 at an excitation wavelength of 488 nm was 150 pg/mL, which was 10 times better than the LOD recorded with slab gel silver staining. Using a tetramethylrhodamine (TAMRA)-labeled IgG2 and a 532 nm excitation wavelength the LOD is 50 pg/mL. The electropherograms of four different commercial FITC conjugates of IgG were studied. The presence of aggregates was observed in two samples while close kinetics of reduction was observed between free aggregates and high aggregates concentration samples. The integrated LIF detector provides an extremely powerful and convenient tool for antibody analysis and should be useful for therapeutic MAb control in pharmaceutical facilities.     

FLUORESCENCE OF MELANIN-DEPENDENCE UPON EXCITATION WAVELENGTH AND CONCENTRATION

Abstract— An introduction to the fundamental characteristics of synthetic melanin fluorescence is presented. The particular difficulties associated with the detection and reduction of the relatively weak signal are discussed and a technique is described for correcting the fluorescence spectra for attenuation of the excitation and emission beams. Spectra are reported for the excitation wavelength range 340–400 nm and an emission range of 360–560 nm. The concentration dependence of the corrected fluorescence signal is examined and is shown to be linear. The variation of the fluorescence spectra with excitation wavelength suggests a two-component fluorescence, for the wavelength range studied. The presence of an isosbestic point in the spectra is used to identify the fluorophores as components of a reaction equilibrium. The possible relationship of this equilibrium to that associated with the melanin photo ESR is discussed     

Fluorescence detection in capillary electrophoresis using a variable wavelength epi-fluorescence microscope

A capillary electrophoresis fluorescence detector is described. A high-pressure mercury lamp with a filter block allowed the selection of a particular excitation waveband. Detection was performed on-column, the fluorescence emission was monitored and measured with a silicon photodiode detector with a built-in amplifier. The concentration limit of detection (CLOD) of 0.4 ng/mL was obtained for rhodamine B, a fluorescent indicator. Based on an estimated injection volume of 2.5 nL, the mass limit of detection (MLOD) was 2.1×10^–18 mol. The separation of three fluorescent indicators: thionine, eosin yellowish and rhodamine B, was achieved in less than 6 min. The separation of nine porphyrin-free acids using the system developed was also demonstrated. The advantages and potential of using an epi-illumination microscope as a versatile and sensitive fluorescence detection system for capillary electrophoresis are described.     

Characterisation of interferon α‐2b by liquid chromatography and mass spectrometry techniques

Interferon α‐2b produced by Escherichia coli consists of 165 amino acids and contains two disulphide bonds; its purity was confirmed by LC‐UV (DAD)‐FLD and LC‐MS techniques. A C₄ column was used with UV detection at 214 nm; diode array detector (DAD) spectra were recorded from 200–400 nm and fluorescence detection was performed at specific wavelengths of trypthophan emission and excitation. Peptide mapping was performed with trypsin. Peptides produced by trypsin digestion were analysed by LC‐UV (DAD)‐FLD, LC‐MS, and LC‐MS/MS using a C₁₈ column. Amino acid sequence coverage was about 95%. UV spectra in the range from 200 nm to 400 nm, emission (Em) and excitation (Ex) spectra of each separated peptide were additionally compared with spectra of the same peptide produced by digestion of European Pharmacopaeia interferon α‐2b standard (spectral matching). The chromatogram of any interferon α‐2b (drug substance or certificated standard) sample produced in the same manner with the same amino acid composition should be similar to the chromatogram obtained by the method described in this paper. Molecular masses of peptides were obtained from MS experiments and MS/MS experiments gave additional structural information. The molecular mass of interferon α‐2b was obtained by MALDI‐TOF MS analysis in linear mode, with an accuracy comparable to the theoretical average mass ± 5 atomic mass units. The molecular mass was obtained from the deconvoluted ESI mass spectrum.     

A simple microfluidic system for efficient capillary electrophoretic separation and sensitive fluorimetric detection of DNA fragments using light-emitting diode and liquid-core waveguide techniques

A miniaturized CE system has been developed for fast DNA separations with sensitive fluorimetric detection using a rectangle type light-emitting diode (LED). High sensitivity was achieved by combining liquid-core waveguide (LCW) and lock-in amplification techniques. A Teflon AF-coated silica capillary on a compact 6x3 cm baseplate served as both the separation channel for CE separation and as an LCW for light transmission of fluorescence emission to the detector. An electronically modulated LED illuminated transversely through a 0.2 mm aperture, the detection point on the LCW capillary without focusing, and fluorescence light was transmitted to the capillary outlet. To simplify the optics and enhance collection of light from the capillary outlet, an outlet reservoir was designed, with a light transmission window, positioned directly in front of a photomultiplier tube (PMT), separated only by a high pass filter. Automated sample introduction was achieved using a sequential injection system through a split-flow interface that allowed effective release of gas bubbles. In the separation of a phiX174 HaeIII DNA digest sample, using ethidium bromide as labeling dye, all 11 fragments of the sample were effectively resolved in 400 s, with an S/N ratio comparable to that of a CE system with more sophisticated LIF.     

Sensitive determination of sulfonamides in environmental water by capillary electrophoresis coupled with both silvering detection window and in‐capillary optical fiber light‐emitting diode‐induced fluorescence detector

A new detector, silvering detection window and in‐capillary optical fiber light‐emitting diode‐induced fluorescence detector (SDW‐ICOF‐LED‐IFD), is introduced for capillary electrophoresis (CE). The strategy of the work was that half surface of the detection window was coated with silver mirror, which could reflect the undetected fluorescence to the photomultiplier tube to be detected, consequently enhancing the detection sensitivity. Sulfonamides (SAs) are important antibiotics that achieved great applications in many fields. However, they pose a serious threat on the environment and human health when they enter into the environment. The SDW‐ICOF‐LED‐IFD‐CE system was used to determine fluorescein isothiocyanate (FITC)‐labeled sulfadoxine (SDM), sulfaguanidine (SGD) and sulfamonomethoxine sodium (SMM‐Na) in environmental water. The detection results obtained by the SDW‐ICOF‐LED‐IFD‐CE system were compared to those acquired by the CE with in‐capillary optical fiber light‐emitting diode‐induced fluorescence detection (ICOF‐LED‐IFD‐CE). The limits of detection (LODs) of SDW‐ICOF‐LED‐IFD‐CE and ICOF‐LED‐IFD‐CE were 1.0–2.0 nM and 2.5–7.7 nM (S/N = 3), respectively. The intraday (n = 6) and interday (n = 6) precision of migration time and corresponding peak area for both types of CE were all less than 0.86% and 3.68%, respectively. The accuracy of the proposed method was judged by employing standard addition method, and recoveries obtained were in the range of 92.5–102.9%. The results indicated that the sensitivity of the SDW‐ICOF‐LED‐IFD‐CE system was improved, and that its reproducibility and accuracy were satisfactory. It was successfully applied to analyze SAs in environmental water.     

Dual‐Luminophore‐Labeled Gold Nanoparticles with Completely Resolved Emission for the Simultaneous Imaging of MMP‐2 and MMP‐7 in Living Cells under Single Wavelength Excitation

Although considerable effort has been devoted to the design of various nanoprobes for the fluorescent detection of multiple biomarkers in a single assay, they often suffer from emission‐overlapping, owing to small Stokes shifts and wide emission spectra, which results in cross‐talk and inaccurate quantification. Herein, we report the design and synthesis of a new nanoprobe for multienzyme detection with completely resolved emission peaks under single‐wavelength excitation. The probe was assembled by attaching a cleavable peptide spacer, which was comprised from a matrix metalloproteinase‐2 (MMP‐2) substrate and a MMP‐7 substrate, onto the surface of gold nanoparticles (AuNPs) through cysteine residues. A lanthanide complex, BCTOT‐Eu^III (BCTOT=1,10‐bis(5′‐chlorosulfo‐thiophene‐2′‐yl)‐4,4,5,5,6,6,7,7‐octafluorodecane‐1,3,8,10‐tetraone), and 7‐amino‐4‐methylcoumarin (AMC) were attached to the N terminus and the C terminus of the peptide, respectively. In the presence of one or both targeting enzymes, the substrate was cleaved and fluorescence resonance energy transfer (FRET) between the dyes and AuNPs was prohibited, thereby resulting in the dramatic fluorescence emission of dyes. Importantly, there was no emission cross‐talk between the two dyes, thereby ensuring accurate detection of each enzyme. Based on this, the simultaneous fluorescence image of MMP‐2 and MMP‐7 was accomplished in living cells under single wavelength excitation. The apparent differences in the fluorescence imaging indicated distinct differences between the expression levels of MMPs between the human normal liver cells and the human hepatoma cells.     

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

An LIF detector was integrated into a CE system based on silver mirror coating detection window and small‐angle optical deflection from collinear configuration. For this detection scheme, the incident light beam was focused on capillary through the edge of a lens, resulting in a small deflection angle that deviated 18° from the collinear configuration. Meanwhile, the excitation light and emitted fluorescence were effectively reflected by silver mirror coating at the detection window. The fluorescence was collected through the center of the same lens and delivered to a PMT in the vertical direction. In contrast to conventional collinear LIF detection systems, the fluorescence intensity was greatly enhanced and the background level was significantly eliminated. FITC and FITC‐labeled amino acids were used as model analytes to evaluate the performance with respect to design factors of this system. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems. All the results indicate that the proposed detection scheme will be promising for development of sensitive and low‐cost CE system.     

DIRECT RECORDING OF THE INITIALLY EXCITED AND THE SOLVENT RELAXED FLUORESCENCE EMISSION SPECTRA OF TRYPTOPHAN BY PHASE SENSITIVE DETECTION OF FLUORESCENCE

Abstract Phase sensitive detection of fluorescence was used to directly record the initially excited and the solvent-relaxed emission spectra of N-acetyl-L-tryptophanamide in propylene glycol. Emission from the initially excited state was suppressed by adjusting the phase sensitive detector to be out of phase with the emission on the short wavelength side of the fluorescence spectrum. Then, the phase sensitive intensities revealed the emission spectrum of the solvent relaxed state. Similarly, the emission from the solvent relaxed state was suppressed by adjusting the detector to be out of phase with the emission on the long wavelength side of the spectrum, allowing the spectrum of the initially excited state to be directly recorded. Distinct emission spectra could be recorded when the solvent relaxation time was comparable to the fluorescence lifetime. At higher or lower temperatures, emission occurs predominantly from a single state, and suppression of the fluorescence signal at any arbitrary wavelength resulted in suppression of the entire emission. A simple theory is described which allows the spectral relaxation times to be estimated from the phase sensitive intensities. From this analysis we obtained an activation energy for spectral relaxation of 3 kcal/mol. This activation energy is smaller than that found for the temperature dependence of fluorescence depolarization, 7.8 kcal/mol. We attribute this difference to the smaller molecular motions required for spectral relaxation.
The method of phase sensitive detection of fluorescence shows excellent resolving power and sensitivity, and this method should facilitate measurement of spectral relaxation around tryptophan residues in proteins.     

Two‐Photon STED Spectral Determination for a New V‐Shaped Organic Fluorescent Probe with Efficient Two‐Photon Absorption

Two‐photon stimulated emission depletion (STED) cross sections were determined over a broad spectral range for a novel two‐photon absorbing organic molecule, representing the first such report. The synthesis, comprehensive linear photophysical, two‐photon absorption (2PA), and stimulated emission properties of a new fluorene‐based compound, (E)‐2‐{3‐[2‐(7‐(diphenylamino)‐9,9‐diethyl‐9H‐fluoren‐2‐yl)vinyl]‐5‐methyl‐4‐oxocyclohexa‐2,5‐dienylidene} malononitrile ( 1 ), are presented. Linear spectral parameters, including excitation anisotropy and fluorescence lifetimes, were obtained over a broad range of organic solvents at room temperature. The degenerate two‐photon absorption (2PA) spectrum of 1 was determined with a combination of the direct open‐aperture Z‐scan and relative two‐photon‐induced fluorescence methods using 1 kHz femtosecond excitation. The maximum value of the 2PA cross section ～1700 GM was observed in the main, long wavelength, one‐photon absorption band. One‐ and two‐photon stimulated emission spectra of 1 were obtained over a broad spectral range using a femtosecond pump–probe technique, resulting in relatively high two‐photon stimulated emission depletion cross sections (～1200 GM). A potential application of 1 in bioimaging was demonstrated through one‐ and two‐photon fluorescence microscopy images of HCT 116 cells incubated with micelle‐encapsulated dye.     

Fluorescence Lifetime Spectroscopy of Glioblastoma Multiforme¶

Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time‐resolved, laser‐induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time‐resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time‐resolved spectra were detected in a 360–550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral‐ (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high‐grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectraland the time domain can enhance the ability of fluorescencebased techniques to diagnose and detect brain tumor margins intraoperatively.     

Compact fiber-optic fluorosensor employing light-emitting ultraviolet diodes as excitation sources

A compact fluorosensor using three different ultraviolet light-emission diodes as excitation sources for fiber-optic recording of fluorescence spectra from samples is described. A compact integrated spectrometer with linear array wavelength recording is used, yielding a spectral resolution of about 8 nm. In two system implementations ultraviolet light-emitting diodes at 300, 340 and 395 nm, or at 360, 385 and 410 nm were used as excitation sources with typical emission halfwidths of 12 nm, each combined with a matching long-path colored-glass filter automatically brought into the fluorescence light flow for suppression of reflected light. Spectra from measurements on vegetation, human skin tumors and a rare-earth ion-based thermographic phosphor were recorded to illustrate the system performance.