Saturated Fluorescence Research Articles

Breaking the Diffraction Barrier Using Fluorescence Emission Difference Microscopy

We propose a novel physical mechanism for breaking the diffraction barrier in the far field. Termed fluorescence emission difference microscopy (FED), our approach is based on the intensity difference between two differently acquired images. When fluorescence saturation is applied, the resolving ability of FED can be further enhanced. A detailed theoretical analysis and a series of simulation tests are performed. The validity of FED in practical use is demonstrated by experiments on fluorescent nanoparticles and biological cells in which a spatial resolution of <λ/4 is achieved. Featuring the potential to realize a high imaging speed, this approach may be widely applied in nanoscale investigations.

Combination of spectral and fluorescence imaging microscopy for wide-field in vivo analysis of microvessel blood supply and oxygenation

Hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence imaging of blood transit time were combined to analyze the oxygenation of and blood flow through microvessel networks. The combination imaging technique was demonstrated in a mouse dorsal window chamber model of a growing Caki-2 human renal cell carcinoma over time. Data from Hb saturation and blood supply time maps show the formation of arteriovenous malformations and shunting of blood directly from arteries to the tumor core and into veins in the periphery of the tumor. Images and data analysis show these malformations result in an oxygenated environment ideal for a tumor to proliferate.

Small-molecule inhibition of the uPAR·uPA interaction: Synthesis, biochemical, cellular, in vivo pharmacokinetics and efficacy studies in breast cancer metastasis

The uPAR·uPA protein–protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5μM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.

Polymerization Inhibition by Triplet State Absorption for Nanoscale Lithography

In the field of photopolymerization, multi-photon lithography1 stands out as a mask-free tool for fabricating three-dimensional structures inside a photocurable resin that is transparent at the wavelength used. This approach enables a great deal of versatility compared with single photon absorption lithography where the thickness of the polymerized parts is limited by the penetration depth of the light in the resin2 and therefore a layer-by-layer approach is necessary to fabricate 3D structures.3 The possibility of producing 3D, free-standing, and arbitrarily complex architectures opens up novel applications in various research fields, e.g., photonic crystals, metamaterials, and biological scaffold fabrication.4–10

The wavelength tunable 589 nm laser output based on singly resonant sum-frequency generation and the measurement of saturate fluorescence spectrum of sodium atom

A wavelength-tunable laser output at 589 nm with high conversion efficiency based on sum-frequency generation by using the technique of single-wavelength extra-cavity resonance is achieved. The two fundamental wavelengths are 1583 nm and 938 nm and the nonlinear crystal is the period-poled lithium niobate. After the frequency of 1583 nm laser was locked to the cavity mode and the frequency of 938 nm laser was scanned, a 589 nm laser output with power of 4.96 mW and wavelength tuning range of 7 GHz was obtained and the stability of the output power is improved effectively with the help of servo feedback loop technique of acousto-optic-modulator. Finally, based on this laser, the saturated fluorescence spectrum of sodium D2 line in the temperature range of 348—413 K (75—140 ℃) were measured. The Doppler-free structures of D2a, D2b and crossover lines on Doppler background were observed, which can provide reference signals for the frequency locking of 589nm laser.

Measurements of NO concentration in NH3-doped CH4 + air flames using saturated laser-induced fluorescence and probe sampling

An experimental study of methane+air flames doped with ammonia (4370ppm of the fuel) has been performed. The goal of this work was to analyze formation of NOx from fuel-N under well-controlled conditions. The Heat Flux method was used for stabilization of non-stretched adiabatic flames on a perforated plate burner at atmospheric pressure. Laser-saturated fluorescence (LSF) and probe sampling were adopted to measure NO concentrations in the post-flame zone. LSF experiments include two series of measurements: in methane+air flames doped with NO and then in flames doped with NH3. In the lean flames seeded with NO, LSF measurements clearly deviates from the model predictions at higher concentrations of NO seeded, that strongly corroborates existence of the lean NO reburning. The modeling accurately predicts [NO] in the neat flame and shows no consumption of NO up to 170ppm seeded. In (CH4+NH3)+air mixtures the NO concentrations measured by LSF are in good agreement with the probe sampling results in the whole range of equivalence ratios 0.65<Φ<1.45. No significant impact of the probe sampling on the NOx measurements in the post-flame zone was observed. Present ammonia conversion data are only in agreement with the measurements of Henshaw et al. and disagree with many other earlier experiments. On the other hand they are accurately reproduced by the flame modeling within experimental uncertainties. Therefore the earlier criticism of the models developed by Konnov and by Skreiberg et al. is not substantiated anymore; these mechanisms are capable in predicting ammonia conversion both in lean and in rich flames. Formation of metal complexes in gas cylinders containing ammonia was put forward to explain inconsistencies between present results and earlier experiments.

Effect of an oxygen pressure injection (OPI) device on the oxygen saturation of patients during dermatological methyl aminolevulinate photodynamic therapy

Methyl aminolevulinate photodynamic therapy (MAL-PDT) (a topical treatment used for a number of precancerous skin conditions) utilizes the combined interaction of a photosensitizer (protoporphyrin IX (PpIX)), light of the appropriate wavelength, and molecular oxygen to produce singlet oxygen and other reactive oxygen species which induce cell death. During treatment, localized oxygen depletion occurs and is thought to contribute to decreased efficacy. The aim of this study was to investigate whether an oxygen pressure injection (OPI) device had an effect on localized oxygen saturation levels and/or PpIX fluorescence of skin lesions during MAL-PDT. This study employed an OPI device to apply oxygen under pressure to the skin lesions of patients undergoing standard MAL-PDT. Optical reflectance spectrometry and fluorescence imaging were used to noninvasively monitor the localized oxygen saturation and PpIX fluorescence of the treatment area, respectively. No significant changes in oxygen saturation were observed when these data were combined for the group with OPI and compared to the group that received standard MAL-PDT without OPI. Additionally, no significant difference in PpIX photobleaching or clinical outcome at 3 months between the groups of patients was observed, although the group that received standard MAL-PDT demonstrated a significant increase (p<0.05) in PpIX fluorescence initially and both groups produced a significant decrease (p<0.05) after light irradiation. In conclusion, with this sample size, this OPI device was not found to be an effective method with which to improve tissue oxygenation during MAL-PDT. Further investigation is therefore required to find a more effective method of MAL-PDT enhancement.

Overview of Live‐Cell Imaging: Requirements and Methods Used

Overview of Live‐Cell Imaging: Requirements and Methods Used

Time-gated Cherenkov emission spectroscopy from linear accelerator irradiation of tissue phantoms

Radiation from a linear accelerator induces Cherenkov emission in tissue, which has recently been shown to produce biochemical spectral signatures that can be interpreted to estimate tissue hemoglobin and oxygen saturation or molecular fluorescence from reporters. The Cherenkov optical light levels are in the range of 10(-6) to 10(-9) W/cm2, which limits the practical utility of the signal in routine radiation therapy monitoring. However, due to the fact that the radiation is pulsed, gated-acquisition of the signal allows detection in the presence of ambient lighting, as is demonstrated here. This observation has the potential to significantly increase the value of Cherenkov emission spectroscopy during radiation therapy to monitor tissue molecular events.

A Fatigue‐Resistant Photoswitchable Fluorescent Protein for Optical Nanoscopy

Like the battery bunny: The novel photoswitchable fluorescent protein rsEGFP can be cycled between its fluorescent and nonfluorescent states more than a thousand times and is, therefore, a superb marker for high-resolution RESOLFT imaging (RESOLFT=reversible saturable optical fluorescence transition) and data storage applications.

Revival of Prism-Based TIR Microscopy - Versatile Tracking of Fluorescent and Scattering Probes with NM-Precision

Current single-molecule microscopy experiments mostly rely on the fluorescence signal of fluorescent proteins, organic dyes or quantum dots attached to proteins of interest. However, these probes suffer from certain limitations, namely limited number of photons before photobleaching, photon blinking, and fluorescence saturation. Consequently, the temporal and spatial resolution by which single molecules can be tracked is limited. Promising candidates for replacing fluorescent probes are gold nanoparticles (GNPs). GNPs exhibit a large scattering cross section in the optical spectrum due to plasmon resonance, provide long-term stability and allow for versatile surface chemistry. Furthermore, because generation of photons relies on elastic scattering, their emission rate does not saturate.Here, we present a camera-based wide-field imaging technique for GNP-labeled proteins using a novel parabolic prism-type total-internal reflection (TIR) microscope. We demonstrate the advantages of GNPs over commonly used fluorescent probes and discuss the pros and cons of prism-type versus objective-type TIR microscopy. We demonstrate that prism-based TIR microscopy allows imaging of fluorescent and scattering probes with high signal-to-noise, excellent control over a wide range of incidence angles, and even illumination intensities within a field of view. When tracking surface-immobilized GNPs (40 nm diameter) we obtained two-dimensional localization precisions as good as 5 angstrom within 30 ms exposure time. We demonstrate localization experiments of kinesin-1 motors labeled with GNPs walking on surface-immobilized microtubules. When GNPs where bound to the tail or motor domains of kinesin-1 we found localization precisions of 3.6 nm and 1.9 nm within 30 ms exposure time, respectively. Furthermore GNP-loaded motors showed 8 nm stepping along microtubules at 3 μM MgATP within image acquisition times of 15 ms. Our method allows for precise localization of biomolecules within short acquisition times over long time scales.

Experimental investigation of spectrum of strontium intercombination transition

We observe the fluorescence and the saturated fluorescence spectra of (5s2)1S0(5s5p)3P1 intercombination transition of thermal strontium atomic beam. Experimental investigation of 88Sr(5s2)1S0(5s5p)3P1 intercombination transition is performed on the different experimental conditions. Our study indicates that experimental parameters affect spectra largely, including temperature, intensity of laser and scanning frequency of laser. Intensity of spectrum is reciprocal of scanning frequency of laser due to long lifetime of (5s5p)3P1 state of Sr atom. Owing to Doppler broadening, transit broadening and other experimental factors, the linewidth of (5s2)1S0(5s5p)3P1 fluorescence spectrum is far more than its natural linewidth and proportional scanning frequency of laser.

Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex

Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4′F]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54–65)(SO3-), characterized thrombin exosite I and II interactions with HCII and heparin in the complex. HCII binding to exosite I of heparin-bound [4′F]FPR-T caused a saturable fluorescence increase, absent with antithrombin. Heparin binding to exosite II and a second weaker site caused fluorescence quenching of [6F]-FFR-T, attenuated by simultaneous Hir-(54–65)(SO3-) binding. Stopped-flow analysis demonstrated ordered assembly of HCII and the [6F]FFR-T·heparin complex, in agreement with tighter heparin binding to thrombin than to HCII. Saturating HCII dependences and bell-shaped heparin dependences of the fluorescence change reported ternary complex formation, consistent with a template mechanism in which the thrombin·heparin complex binds HCII and allowing for interaction of thrombin·(heparin)2 complexes with HCII. Hir-(54–65)(SO3-) displacement in reactions with FPR-blocked and active thrombin indicated a concerted action of the active site and exosite I during ternary complex formation. These studies demonstrate that binding of HCII to the thrombin·heparin complex is dramatically enhanced compared with heparin binding alone and that exosite I is still available for ligand or HCII binding when both heparin binding sites on thrombin are saturated.

Nonlinearity of two-photon Ca2+imaging yields distorted measurements of tuning for V1 neuronal populations

We studied the relative accuracy of drifting gratings and noise stimuli for functionally characterizing neural populations using two-photon calcium imaging. Calcium imaging has the potential to distort measurements due to nonlinearity in the conversion from spikes to observed fluorescence. We demonstrate a dramatic impact of fluorescence saturation on functional measurements in ferret V1 by showing that responses to drifting gratings strongly violate contrast invariance of orientation tuning, a fundamental property of the spike rates. The observed relationship is consistent with saturation that clips the high-contrast tuning curve peaks by ∼40%. The nonlinearity was also apparent in mouse V1 responses to drifting gratings, but not as strong as in the ferret. Contrast invariance holds, however, for tuning curves measured with a randomized grating stimulus. This finding is consistent with prior work showing that the linear portion of a linear-nonlinear system can be recovered with reverse correlation. Furthermore, we demonstrate that a noise stimulus is more effective at keeping spike rates in the linear operating regime of a saturating nonlinearity, which both maximizes signal-to-noise ratios and simplifies the recovery of fast spike dynamics from slow calcium transients. Finally, we uncover spatiotemporal receptive fields by removing the nonlinearity and slow calcium transient from a model of fluorescence generation, which allowed us to observe dynamic sharpening of orientation tuning. We conclude that for two-photon recordings it is imperative that one considers the nonlinear distortion when designing stimuli and interpreting results, especially in sensory areas, species, or cell types with high firing rates.

Oxygen saturation and perfusion changes during dermatological methylaminolaevulinate photodynamic therapy

Methylaminolaevulinate (MAL)-photodynamic therapy (PDT) is a successful topical treatment for a number of (pre)cancerous dermatological conditions. In combination, light of the appropriate wavelength, the photosensitizer protoporphyrin IX (PpIX) and tissue oxygen result in the production of singlet oxygen and reactive oxygen species inducing cell death. This study investigates real-time changes in localized tissue blood oxygen saturation and perfusion in conjunction with PpIX fluorescence monitoring for the first time during dermatological MAL-PDT. Oxygen saturation, perfusion and PpIX fluorescence were monitored noninvasively utilizing optical reflectance spectroscopy, laser Doppler perfusion imaging and a fluorescence imaging system, respectively. Patients attending for standard dermatological MAL-PDT were recruited to this ethically approved study and monitored prior to, during and after light irradiation. Significant reductions in mean blood oxygen saturation (P < 0·005) and PpIX fluorescence (P < 0·001) were observed within the first minute of irradiation (4·75 J cm(-2) ) while, in contrast, perfusion was observed to increase significantly (P < 0·01) during treatment. The changes in oxygen saturation and PpIX fluorescence were positively correlated during the initial phase of treatment (r(2) = 0·766). Rapid reductions in the localized blood oxygen saturation have been observed for the first time to occur clinically within the initial minutes of light irradiation and positively correlate with the concurrent PpIX photobleaching. Furthermore, perfusion increases, suggesting that the microvasculature compensates for the PDT-induced oxygen depletion.

Influence of Carbohydrates on the Interaction of Procyanidin B3 with Trypsin

The biological properties of procyanidins, in particular their inhibition of digestive enzymes, have received much attention in the past few years. Dietary carbohydrates are an environmental factor that is known to affect the interaction of procyanidins with proteins. This work aimed at understanding the effect of ionic food carbohydrates (polygalacturonic acid, arabic gum, pectin, and xanthan gum) on the interaction between procyanidins and trypsin. Physical-chemical techniques such as saturation transfer difference-NMR (STD-NMR) spectroscopy, fluorescence quenching, and nephelometry were used to evaluate the interaction process. Using STD-NMR, it was possible to identify the binding of procyanidin B3 to trypsin. The tested carbohydrates prevented the association of procyanidin B3 and trypsin by a competition mechanism in which the ionic character of carbohydrates and their ability to encapsulate procyanidins seem crucial leading to a reduction in STD signal and light scattering and to a recovery of the proteins intrinsic fluorescence. On the basis of these results, it was possible to grade the carbohydrates in their aggregation inhibition ability: XG > PA > AG ≫ PC. These effects may be relevant since the coingestion of procyanidins and ionic carbohydrates are frequent and furthermore since these might negatively affect the antinutritional properties ascribed to procyanidins in the past.

Determination of photophysical parameters of chlorophyll α in photosynthetic organisms using the method of nonlinear laser fluorimetry

We study the possibility of solving the multiparameter inverse problem of nonlinear laser fluorimetry of molecular systems with high local concentration of fluorophores (by the example of chlorophyll α molecules in photosynthetic organisms). The algorithms are proposed that allow determination of up to four photophysical parameters of chlorophyll α from the experimental fluorescence saturation curves. The uniqueness and stability of the inverse problem solution obtained using the proposed algorithms were assessed numerically. The laser spectrometer, designed in the course of carrying out the work and aimed at nonlinear laser fluorimetry in the quasi-stationary and nonstationary excitation regimes is described. The algorithms, proposed in this paper, are tested on pure cultures of microalgae Chlorella pyrenoidosa and Chlamydomonas reinhardtii under different functional conditions.

Far-field optical nanoscopy with reduced number of state transition cycles

We report on a method to reduce the number of state transition cycles that a molecule undergoes in far-field optical nanoscopy of the RESOLFT type, i.e. concepts relying on saturable (fluorescence) state transitions induced by a spatially modulated light pattern. The method is exemplified for stimulated emission depletion (STED) microscopy which uses stimulated emission to transiently switch off the capability of fluorophores to fluoresce. By switching fluorophores off only if there is an adjacent fluorescent feature to be recorded, the method reduces the number of state transitions as well as the average time a dye is forced to reside in an off-state. Thus, the photobleaching of the sample is reduced, while resolution and recording speed are preserved. The power of the method is exemplified by imaging immunolabeled glial cells with up to 8-fold reduced photobleaching.

Estimating Cell Count and Distribution in Labeled Histological Samples Using Incremental Cell Search

Cell proliferation is critical to the outgrowth of biological structures including the face and limbs. This cellular process has traditionally been studied via sequential histological sampling of these tissues. The length and tedium of traditional sampling is a major impediment to analyzing the large datasets required to accurately model cellular processes. Computerized cell localization and quantification is critical for high-throughput morphometric analysis of developing embryonic tissues. We have developed the Incremental Cell Search (ICS), a novel software tool that expedites the analysis of relationships between morphological outgrowth and cell proliferation in embryonic tissues. Based on an estimated average cell size and stain color, ICS rapidly indicates the approximate location and amount of cells in histological images of labeled embryonic tissue and provides estimates of cell counts in regions with saturated fluorescence and blurred cell boundaries. This capacity opens the door to high-throughput 3D and 4D quantitative analyses of developmental patterns.

Flame structure measurements of NO in premixed hydrogen–nitrous oxide flames

Experimental NO flame profiles were measured for N2-diluted, H2–N2O premixed flames stabilized on a flat flame apparatus at equivalence ratios ranging from 0.95 to 1.55. NO measurements were obtained using saturated laser-induced fluorescence and calibrated by microprobe post-flame sampling utilizing Fourier transform infrared (FTIR) spectroscopy. These experimental data were compared with a burner-stabilized flame model using three different published chemical mechanisms incorporating H2/O2/NxOy chemistry. For the stoichiometric flame condition, peak NO model predictions using the three chemical mechanisms differed by 38%, whereas these model predictions varied by over 47% for the fuel-rich conditions examined. NO predictions in the post-flame region using the Konnov chemical mechanism were generally within the estimated experimental uncertainty, whereas the model predictions using the other two mechanisms fell below the lower uncertainty bounds. The normalized spatial NO profile predictions for all three chemical mechanisms showed reasonable agreement with the normalized experimental NO profiles for near-stoichiometric H2–N2O flames; however, there was appreciable disparity between the experimental and model predictions for fuel-rich, H2–N2O flames. Sensitivity and rate-of-production analyses show that NHx/NO chemical interactions, as well as NHx/NxHy recombination reactions under fuel-rich conditions, are important to accurately modeling NO profiles in hydrogen-nitrous oxide flames. The experimental data provide constraints for the rate constants of important NHx/NxHy chemical interactions, and some modifications were considered for improving comparisons between model predictions and experiments.