Nonlinear Fluorescence Research Articles

An investigation of porous structure in molecularly imprinted polymer for sensor development: Non-linear fluorescence quenching of 17β-estradiol bound inside MIP submicron particles by sodium nitrite and methacrylamide

17β-Estradiol (E2) molecularly imprinted and non-imprinted poly(methacrylic acid-co-ethylene glycol dimethacrylate) submicron particles (MIP and NIP) have been synthesized in the presence and absence of template E2 molecules. The sizes of MIP and NIP particles were determined via a nanoDLS particle size analyzer to be 477 ± 11 and 373 ± 21 nm. After these naturally non-fluorescent MIP and NIP particles bound with E2 molecules as a fluorophore, strong fluorescence at λ em = 310 ± 1 nm was detectable when excited by light of λ ex = 280 ± 1 nm. Sodium nitrite and methacrylamide were selected as two fluorescence quenchers with different ionic/molecular sizes. They both exhibited non-linear quenching behavior in the Stern–Volmer plots. The smaller quencher (sodium nitrite) ionized in water to produce nitrite anions that easily penetrated into the porous MIP particles and quenched the fluorescence emission from the E2 molecules bound inside. It produced a dynamic quenching constant ( K sv) of 23.0 ± 1.3 M −1 when the concentration of E2–MIP particles was 2.5 mg/mL in water. The larger quencher (methacrylamide) was sterically hindered in penetrating the pores of MIP particles. Its dynamic quenching constant ( K sv) hence became small, to be less than 1.0 ± 0.5 M −1. This approach enabled an investigation of the porous structure in MIP particles for development of a novel E2 sensing scheme.

Quantitative analysis for nonlinear fluorescent spectra based on edges matching

A novel spectra-edge-matching approach is proposed for the quantitative analysis of the nonlinear fluorescence spectra of the air impurities excited by a femtosecond laser. The fluorescence spectra are first denoised and compressed, both by wavelet transform, and several peak groups are then picked from each spectrum according to a threshold of intensity and are used to extract the spectral features through principal component analysis. It is indicated that the first two principle components actually cover up to 98% of the total information and are sufficient for the final concentration analysis. The analysis reveals a monotone relationship between the spectra intensity and the concentration of the air impurities, suggesting that the femtosecond laser induced fluorescence spectroscopy along with the proposed spectra analysis method can become a powerful tool for monitoring environmental pollutants.

Effect of anthracene doping on potassium hydrogen phthalate crystals

The influence of the highly fluorescent dopant, anthracene (over a concentration range from 5 × 10−4 to 1.2 × 10−2 mol dm−3) on the nonlinear optical properties and fluorescence intensity of potassium hydrogen phthalate (KHP) single crystals grown at 30 °C by a slow evaporation solution growth technique (SEST) has been investigated. Powder XRD and FTIR spectral analyses confirm the slight distortion of the structure of crystal because of doping. UV–Visible study shows that the transparency is not affected much by the dopant. The SEM investigation reveals that KHP suffers from crack development. Thermal analysis indicates that there is no decomposition of the crystal up to the melting point. It is interesting to observe that additions of small quantity anthracene to KHP results in the enhancement of fluorescence intensity. The fluorescence intensity dependence on dopant concentration is observed. Interestingly, second harmonic generation (SHG) efficiency of KHP is dramatically improved by doping with small quantities of anthracene.

Dipole spectrum structure of nonresonant nonpertubative driven two-level atoms

We analize the dipole spectrum of a two-level atom excited by a non-resonant intense monochromatic field, under the electric dipole approximation and beyond the rotating wave approximation. We show that the apparently complex spectral structure can be completely described by two families: harmonic frequencies of the driving field and field-induced nonlinear fluorescence. Our formulation of the problem provides quantitative laws for the most relevant spectral features: harmonic ratios and phases, non-perturbative Stark shift, and frequency limits of the harmonic plateau. In particular, we demonstrate the locking of the harmonic phases at the wings of the plateau opening the possibility of ultra-short pulse generation through harmonic filtering.

Multiphoton excitation fluorescence microscopy in planar membrane systems

The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26+/-2 mN/m and 28+/-3 mN/m for DOPC and DPPC, respectively.

Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections

Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.

Instantaneous Temperature Imaging of Diffusion Flames Using Two-Line Atomic Fluorescence

This work investigates the first demonstration of nonlinear regime two-line atomic fluorescence (NTLAF) thermometry in laminar non-premixed flames. The results show the expediency of the technique in the study of the reaction zone and reveals interesting findings about the indium atomization process. Indium fluorescence is observed to be strongest at the flame-front, where the temperature exceeds 1000 K. The uncertainty in the deduced temperature measurement is approximately 6%. The temperature profile across the reaction zone shows good agreement with laminar flame calculations. The advantages and inherent limitations of the technique are discussed.

Intrinsically Resolution Enhancing Probes for Confocal Microscopy

In recent years different implementations of superresolution microscopy based on targeted switching (STED, GSD, and SSIM) have been demonstrated. The key elements to break the diffraction barrier are two distinct molecular states that generate a saturable nonlinear fluorescence response with respect to the excitation intensity. In this paper, we demonstrate that a nonlinearity can even be encoded in fluorescent probes, which then increase the resolution of a standard confocal microscope. This nonlinearity is achieved by an intensity dependent blocking of the resonance energy transfer between a donor and one or more acceptor fluorophores, utilizing radical anion states of the acceptor. In proof-of-principle experiments, we demonstrate a significant resolution increase using probes with different numbers of acceptor fluorophores. Quantitative description by a theoretical model paves the way for the development of fluorescent probes that can more than double the resolution of essentially any confocal microscope in all three dimensions.

Advances in polarization sensitive multiphoton nano-bio-imaging

In this talk, we shall shortly review four main directions of ongoing research in our laboratories, directed at the conception and demonstration of a variety of innovative configurations in nanoscale multiphoton imaging. A common feature to all of these directions appears to be the central role played by the involvement of polarization features, both in- and outgoing, moreover so in view of the tensorial aspects inherent to nonlinear schemes such second-harmonic generation, electro-optic modulation or two-photon fluorescence which will ne emphasized. These advances relate to the new domain of nonlinear ellipsometry in multiphoton imaging (1), of high relevance to fundamental aspects of nanophotonics and nanomaterial engineering as well as towards basic life science issues. The four domains to be shortly reported are: a) polarization resolved second-harmonic generation in semiconductor QD's with record small sizes in the 10-12 nm range (2) b) original use of two-photon confocal polarization resolved microscopy in DNA stained by two photon fluorescent dyes in different LC phases arrangements so as to characterize these as well as ascertain the respective DNA-dye orientation (intercalant or groves) (3) c) elaboration and demonstration of an electrooptic confocal microscope in a highly sensitive interferometric and homodyne detection configuration allowing to map weak electric potentials such as in artificial functionalized membranes, the dynamical investigation of firing and propagation aspects of action potentials in neurones being currently the next step (4) d) original plasmon based enhanced nanoscale confocal imaging involving a dual detection scheme (fluorescence imaging and ATR plasmon coupling in reflection) whereby adequate preparation and switching of the incoming polarization state between radial, linear and azimuthal configurations, entail different images and plasmon enhancement levels (5).

A classification method for nonlinear fluorescent spectra based on edges matching

A pre-process of feature extraction and classification approach based on spectrum edges matching is proposed to analyze the complicated nonlinear fluorescence spectra emitted by the interaction between femto-second (fs) laser and the impurities in air. The spectra data is denoised and compressed from 3979 points to 664 points using wavelet (WT) transform. By similarity analysis we create the characteristic spectra of 3 kinds of gases and the weights for classification. A new method of classification is proposed based on the edges matching and the comparison with characteristic spectra. Compared with existing methods，our method can not only get 100% classification accuracy，but also gives the characteristic position and the matching degree. The analysis of the matching degree shows that our method works well at low concentrations and has a potential application of identifying gases of lower concentration.

Multiplexed Digital Quantification of mRNA Abundance: a Highly Reproducible, PCR–independent Assessment of BAALC, ERG, and MN1 mRNA Levels in Acute Myeloid Leukemia (AML) Bone Marrow (BM) and Peripheral Blood (PB) Samples.

Abstract 1599Poster Board I-625The greatest obstacle to routine clinical testing of gene expression levels has been the lack of reproducibility of currently used methodologies, such as quantitative reverse transcriptase PCR (qRT-PCR) and microarray expression profiling. While these assays are useful for retrospective analyses of batched samples, they cannot be used for upfront evaluation of individual patients (pts) for molecular risk and treatment assignment. To overcome this barrier, we tested a recently developed, high throughput, PCR-independent, digital quantification technology, the nCounter system (Nanostring® Technologies). This system counts individual mRNA molecules, rather than measuring non-linear fluorescence generated by PCR-amplified targets (eg qRT-PCR). Using 72 AML samples and spike-in controls, we and collaborators demonstrated that the nCounter system is highly reproducible, sensitive, and accurate to femtomolar concentrations (Payton, J, et al. JCI 119:1714-26; Geiss, G, et al. Nat Biotech 26:317-25). Here we validated this technology using an independent set of 101 pts with a diagnosis of de novo cytogenetically normal AML. At diagnosis, pts presented with FAB subtypes M0, M1, M2, M4, M5(A, B), had a median age of 43 years (range 19-59), median white blood count of 28.5× 103/μL (range 1.4-273.0), median of 69% BM blasts (range 22-95) and median of 65% PB blasts (range 0-97). Paired BM and PB specimens were available for 27 pts; blast percentages were ≥ 20% for all paired specimens. We used the nCounter system to measure mRNA abundance (‘counts‘) of 27 genes whose expression correlates with clinical and/or pathological criteria, including 3 genes associated with prognosis (BAALC, ERG, MN1), and control/housekeeping genes (GAPDH, ABL, Actin). Briefly, mononuclear cells from pretreatment BM or PB were enriched on Ficoll-Hypaque gradients and RNA was isolated using Trizol reagent; 100ng of total RNA was assayed in triplicate by nCounter according to the manufacturer's protocols. The nCounter results demonstrated substantial reproducibility, with a median CV [coefficient of variation, (standard deviation/mean *100)] <6% across replicates. In addition, the nCounter counts for BAALC, ERG, and MN1 normalized to ABL were highly correlated with the ABL-normalized qRT-PCR results. Significant correlation was observed for all 3 genes, with the following Spearman correlation coefficients: BAALC r = 0.9, ERG r = 0.7, and MN1 r = 0.8 (all p<0.001). Correlation of BAALC, ERG, and MN1 nCounter counts with the expression levels measured by Affymetrix® HG-U133 plus 2.0 microarrays were also tested. Summary measures of microarray gene expression levels were computed using the Robust Multichip Average method, which incorporates quantile normalization of arrays. Significant correlation of nCounter and microarray results was observed, with Spearman correlation coefficients as follows: BAALC r = 0.96, ERG r = 0.8, and MN1 r = 0.8 (all p<0.001). For the 27 sets of paired samples, nCounter results for BM and PB were also significantly correlated, with Spearman correlation coefficients of BAALC r = 0.9, ERG r = 0.7, and MN1 r = 0.6 (all p<0.001). Because RNA quickly degrades if not promptly isolated from PB or BM, and degraded RNA often fails qRT-PCR assays, we determined whether RNA quality affected nCounter performance by assessment of standard quality parameters, including ratio of absorbance at 260 and 280 nm (260:280, a measure of RNA purity, acceptable 1.8-2.0) and RNA Quality Index (RQI, which assesses 18S:28S rRNA ratio and RNA degradation, 7-10 acceptable). Quality ranged from very high, with 260:280 ratios >1.9 and RQI scores >9, to relatively low, with 260:280 ratios <1.8, RQI scores <4, and degraded RNA visible on the Experion® RNA chip. Such a range of RNA quality is consistent with our experience with clinical specimens, which may be delayed in transit to the laboratory. Nevertheless, fewer than 3% of nCounter assays failed to generate acceptable results (11/393 assays), likely because no PCR step is required. Our results show that the nCounter system is a rapid, relatively inexpensive ($0.72/assay), and highly reproducible methodology that will be very useful for routine diagnostic testing of prognostic gene expression and upfront molecular-risk assessment for treatment guidance in AML pts. DisclosuresNo relevant conflicts of interest to declare.

Maximization of nonlinear fluorescence from ultrasmall (≤2 nm) semiconductor quantum dots to be used for deep tissue imaging

We propose the use of ultrasmall semiconductor quantum dots (USQDs) for specialized bio-imaging applications, and discuss methods for enhancing fluorescent signals from USQDs to be used for two-photon-absorption based deep tissue imaging. In particular, we report optimizing the excitation wavelength for two-photon absorption-induced fluorescence (TPAF) in CdSe/ZnS SQDs and demonstrate a 68-fold enhancement in the fluorescence signal when the TPAF excitation wavelength is changed from 900 nm to 780 nm.

Nonlinear fluorescence quenching of newly synthesized coumarin derivative by aniline in binary mixtures

The fluorescence quenching of newly synthesized coumarin (chromen-2-one) derivative, 4-(5-methyl-3-phenyl-benzofuran-2-yl)-6-chloro-chromen-2-one (MPBClC) by aniline in different binary solvent mixtures of benzene and acetonitrile have been reported by steady state fluorescence measurements. All the measurements were carried out at room temperature (296 K). A positive curvature from linearity was observed in the Stern–Volmer (S–V) plot in all the solvent mixtures. Various rate parameters for the fluorescence quenching have been determined by sphere of action static quenching model and finite sink approximation model. The results show that the positive curvature in the S–V plot is due to both static and dynamic quenching processes. Further, with the use of finite sink approximation model, it is concluded that these bimolecular reactions are diffusion-limited.

Filamentation “remote” sensing of chemical and biological agents/pollutants using only one femtosecond laser source

All matters in the path of filaments induced by an intense femtosecond laser pulse propagating in air could be fragmented and result in the emission of characteristic fluorescence spectra from the excited fragments. The fluorescence spectra exhibit specific signatures (fingerprints) that can be used for the identification of various substances including chemical and biological species. In this paper, we present an overview of the recent progress in our laboratory concerning the “remote” sensing of chemical and biological agents/pollutants in air using filamentation-induced nonlinear fluorescence techniques.

Development of temperature imaging using two-line atomic fluorescence

This work aims to advance understanding of the coupling between temperature and soot. The ability to image temperature using the two-line atomic fluorescence (TLAF) technique is demonstrated. Previous TLAF theory is extended from linear excitation into the nonlinear fluence regime. Nonlinear regime two-line atomic fluorescence (NTLAF) provides superior signal and reduces single-shot uncertainty from 250 K for conventional TLAF down to 100 K. NTLAF is shown to resolve the temperature profile across the stoichiometric envelope for hydrogen, ethylene, and natural gas flames, with deviation from thermocouple measurements not exceeding 100 K, and typically ≲30 K. Measurements in flames containing soot demonstrate good capacity of NTLAF to exclude interferences that hamper most two-dimensional thermometry techniques.

Enhancing Signal to Noise Ratio in linear and non-linear excitation microscopy

The resolution capability of an optical system can be completely characterized by the vectorial diffraction theory[1], which defines the intensity distribution of a point like source imaged by a lens assuming ideal imaging conditions. Unfortunately, these conditions can not be completely reached as noise affects a recorded microscope image. A detailed characterization of the imaging process in linear and non-linear fluorescence microscopy allows to evaluate the noise deterioration effect on the resolution capability.

MEH‐PPV photobleaching control by femtosecond pulse shaping

AbstractIn the work reported here we were able to control the photobleaching of poly[2‐methoxy‐5‐(2′‐ethyl‐hexyloxy)‐1,4‐phenylene vinylene] (MEH‐PPV), excited by two‐photon absorption, using femtosecond pulse shaping. By applying a cosine‐like spectral phase mask, we observe a reduction of three times in the photobleaching rate, while the fluorescence intensity decreases by 20%, in comparison to the values obtained with a Fourier‐transform‐limited pulse. These results demonstrate an interesting trade‐off between photobleaching rate and nonlinear fluorescence intensity. The possible mechanism behind this process is discussed in terms of the pulse spectral profile and the absorbance band of MEH‐PPV. (© 2009 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Beyond the diffraction-limit biological imaging by saturated excitation microscopy

We demonstrate high-resolution fluorescence imaging in biological samples by saturated excitation (SAX) microscopy. In this technique, we saturate the population of fluorescence molecules at the excited state with high excitation intensity to induce strong nonlinear fluorescence responses in the center of laser focus, which contributes the improvement of the spatial resolution in three dimensions. Using SAX microscopy, we observed stained microtubules in HeLa cells with improved spatial resolution. We also measured the relation of the fluorescence and excitation intensity with several kinds of fluorescence dyes and, in the results, confirmed that SAX microscopy has the potential to observe any kind of fluorescence samples in current usage.

Fluorescence-detected two-dimensional electronic coherence spectroscopy by acousto-optic phase modulation

Two-dimensional electronic coherence spectroscopy (ECS) is an important method to study the coupling between distinct optical modes of a material system. Such studies often involve excitation using a sequence of phased ultrashort laser pulses. In conventional approaches, the delays between pulse temporal envelopes must be precisely monitored or maintained. Here, we introduce a new experimental scheme for phase-selective nonlinear ECS, which combines acousto-optic phase modulation with ultrashort laser excitation to produce intensity modulated nonlinear fluorescence signals. We isolate specific nonlinear signal contributions by synchronous detection, with respect to appropriately constructed references. Our method effectively decouples the relative temporal phases from the pulse envelopes of a collinear train of four sequential pulses. We thus achieve a robust and high signal-to-noise scheme for phase-selective ECS to investigate the resonant nonlinear optical response of photoluminescent systems. We demonstrate the validity of our method using a model quantum three-level system-atomic Rb vapor. Moreover, we show how our measurements determine the resonant complex-valued third-order susceptibility.

Nonlinear imaging study of extracellular matrix in chemical‐induced, developmental dissecting aortic aneurysm: Evidence for defective collagen type III

Using a recent model of dissecting aortic aneurysm (DAA) caused by in utero exposure to semicarbazide, we examined the elastin and collagen using standard methods and two nonlinear imaging techniques, multiphoton fluorescence (MPF) and second harmonic generation (SHG) microscopy. Sprague-Dawley rat dams were given semicarbazide (6.13 mg/kg/day i.p.) from gestational days 14 to 20 (GD14-20). Fetuses were harvested on GD20 and pups on postnatal day 1 (PND1), PND7, and PND28; matched controls were from dams treated with saline. Aortic immunohistopathology and collagen/elastin signal intensity via MPF and SHG microscopy at an excitation wavelength of 800 nm were studied. Massive DAA of the aortic arch occurred in nearly 100% of pups at birth (i.e., no GD20 fetuses showed lesions). MPF and SHG demonstrated that collagen was significantly degraded at GD20 and in newborns, but normalized by PND28. GD20 fetuses and newborn pups showed a decreased content of medial and adventitial collagen type III in pooled aortas by Western blot and immunohistochemistry. In 7- and 28-day-old pups resolution of DAA blood in vascular media and a recovery of stainable collagen type III was found. Elastin in healed DAA (PND28 pups) was focally disorganized. MPF and SHG microscopy provide sensitive and high-resolution information on aortic elastin and collagen. In this model of DAA, collagen displays aberrant imaging quality likely linked to a marked decrease in collagen type III in the developing extracellular matrix. Birth Defects Research (Part A) 2008.