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Chemometric analysis for extraction of individual fluorescence spectrum and lifetimes from a target mixture

The present invention is a system for chemometric analysis for the extraction of the individual component fluorescence spectra and fluorescence lifetimes from a target mixture. The present invention combines a processor with an apparatus for generating an excitation signal to transmit at a target mixture and an apparatus for detecting the emitted signal from the target mixture. The present invention extracts the individual fluorescence spectrum and fluorescence lifetime measurements from the frequency and wavelength data acquired from the emitted signal. The present invention uses an iterative solution that first requires the initialization of several decision variables and the initial approximation determinations of intermediate matrices. The iterative solution compares the decision variables for convergence to see if further approximation determinations are necessary. If the solution converges, the present invention then determines the reduced best fit error for the analysis of the individual fluorescence lifetime and the fluorescence spectrum before extracting the individual fluorescence lifetime and fluorescence spectrum from the emitted signal of the target mixture.

Hallidy, William H.

Canopy Level Solar Induced Fluorescence for Vegetation in Controlled Experiments

Solar induced chlorophyll fluorescence (SIF) was retrieved from high resolution reflectance spectra acquired one meter above saplings of three deciduous tree species during springtime (three weeks after leaf flush) and in late summer when foliage was mature. SIF was determined by application of the Fraunhofer Line Depth (FLD) Principal to above-canopy spectra acquired with an Analytical Spectral Devices (ASD) Fieldspec spectroradiometer (3.2 nm resolution with 1.2 nm sampling interval). SIF retrievals were made at the two atmospheric oxygen (O2) absorption features that occur in the chlorophyll fluorescence (ChlF) region (660 -780 nm). These telluric features are 02V, the broader and deeper feature centered at 760 nm, but located on the shoulder of the far-red ChlF peak at 740 nm; and 023, a narrow feature centered at 688 nm that is positioned near the red ChlF peak at 685 nm. Supporting, coincident leaf level fluorescence, reflectance, photochemical and other measurements were also made. At the leaf level, these measurements included in situ photosynthetic capacity (Pmax) and light adapted total chlorophyll fluorescence (Fs') collected at steady state under high light and controlled chamber conditions (e.g., temperature, PAR, humidity, and COz); optical properties (reflectance, transmittance, absorptance); chlorophyll and carotenoid content; specific leaf mass; carbon (C) and nitrogen (N) content; fluorescence emission spectra at multiple excitation wavelengths; the ChlF contribution to red (R) and far-red (FR) reflectance; fluorescence imagery; and fluorescence excitation-emission matrices (EEMs). The tree species examined were tulip poplar (Liriodendron tulipifera L.), red maple (Acer rubrum L.), and sweetgum (Liquidambar styraczflua L.), and each had been provided four levels of N augmentation (0, 19, 37, and 75 kg Nhectare seasonally) to simulate atmospheric deposition from air pollution. Whole-plant SIF measurements of these species were compared with SIF estimates derived using FluorMOD, a radiative transfer model that includes fluorescence properties of foliage, when provided with our supporting measurements. Simulated values for SIF were also compared with similar estimates made over three years for corn (Zea mays L) crops under N treatments (20, 50,70, and 140 kg Nhectare) and with corn and pepper (Capsicum annuum L.) plants provided dimethyl urea (DCMU, 0 and 5 x 10" M) which were grown in pots and grouped in artificial canopies for spectral measurements. For near-surface measurements of healthy and stressed vegetation, red SIF retrieved at 688 nm (023) varied between 2-7 mW/m(exp 2)/nm/sr while far-red SIF retrieved at 760 nm (O2A) varied between 0.5 and 4.0 mW/m(exp 2)/nm/sr. Typical values for the SIF red/far-red ratio ranged between 1.75 and 4.0. Relationships of SIF to spectral reflectance indices and foliar photochemical indices were examined, such as photosynthetic light use efficiency. Initial investigations of the variability in these measurements at the same leaf temperature for sunlit and shaded foliage showed us that shaded foliage produced higher ChlF and light use efficiency. Other factors that affect SIF determinations are discussed. These results will assist in determine the expected intensity of the SIF signal from vegetation near the surface, as well as to identify enhancements needed for FluorMOD (or other such models), and will assist in determining relationships of SIF to reflectance indices and carbon dynamics.

Middleton, E. M.

Filling-in of Near-infrared Solar Lines by Terrestrial Fluorescence and Other Geophysical Effects: Simulations and Space-based Observations from SCIAMACHY and GOSAT

Global mapping of terrestrial vegetation fluorescence from space has recently been accomplished with high spectral resolution (nu/nu greater than 35 000) measurements from the Japanese Greenhouse gases Observing SAellite (GOSAT). These data are of interest because they can potentially provide global information on the functional status of vegetation including light-use efficiency and global primary productivity that can be used for global carbon cycle modeling. Quantifying the impact of fluorescence on the O2-A band is important as this band is used for photon pathlength characterization in cloud- and aerosol-contaminated pixels for trace-gas retrievals including CO2. Here, we examine whether fluorescence information can be derived from space using potentially lower-cost hyperspectral instrumentation, i.e., more than an order of magnitude less spectral resolution (nu/nu approximately 1600) than GOSAT, with a relatively simple algorithm. We discuss laboratory measurements of fluorescence near one of the few wide and deep solar Fraunhofer lines in the long-wave tail of the fluorescence emission region, the calcium (Ca) II line at 866 nm that is observable with a spectral resolution of approximately 0.5 nm. The filling-in of the Ca II line due to additive signals from various atmospheric and terrestrial effects, including fluorescence, is simulated. We then examine filling-in of this line using the SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY) satellite instrument. In order to interpret the satellite measurements, we developed a general approach to correct for various instrumental artifacts that produce false filling-in of solar lines in satellite measurements. The approach is applied to SCIAMACHY at the 866 nm Ca II line and to GOSAT at 758 and 770 nm on the shoulders of the O2-A feature where there are several strong solar Fraunhofer lines that are filled in primarily by vegetation fluorescence. Finally, we compare temporal and spatial variations of SCIAMACHY additive signals with those of GOSAT and the Enhanced Vegetation Index (EVI) from the MODerate-resolution Imaging Spectroradiometer (MODIS). Although the derived additive signals from SCIAMACHY are extremely weak at 866 nm, their spatial and temporal variations are consistent with chlorophyll a fluorescence or another vegetation-related source. We also show that fillingin occurs at 866 nm over some barren areas, possibly originating from luminescent minerals in rock and soil.

vegetation

Leaf-level chlorophyll fluorescence and reflectance spectra of high latitude plants

Little is known about the chlorophyll fluorescence spectra for high latitude plants. A FluoWat leaf clip was used to measure leaf-level reflectance and chlorophyll fluorescence spectra of leaves of common high latitude plants to examine general spectral characteristics of these species. Fluorescence yield (Fyield) was calculated as the ratio of the emitted fluorescence divided by the absorbed radiation for the wavelengths from 400 nm up to the wavelength of the cut-off for the FluoWat low pass filter (either 650 or 700 nm). The Fyield spectra grouped into distinctly different patterns among different plant functional types. Black spruce (Picea mariana) Fyield spectra had little red fluorescence, which was reabsorbed in the shoot, but displayed a distinct far-red peak. Quaking aspen (Populus tremuloides) had both high red and far-red Fyield peaks, as did sweet coltsfoot (Petasites frigidus). Cotton grass (Eriophorum spp.) had both red and far-red Fyield peaks, but these peaks were much lower than for aspen or coltsfoot. Sphagnum moss (Sphagnum spp.) had a distinct Fyield red peak but low far-red fluorescence. Reindeer moss lichen (Cladonia rangiferina) had very low fluorescence levels, although when damp displayed a small red Fyield peak. These high latitude vegetation samples showed wide variations in Fyield spectral shapes. The Fyield values for the individual red or far-red peaks were poorly correlated to chlorophyll content, however the ratio of far-red to red Fyield showed a strong correlation with chlorophyll content. The spectral variability of these plants may provide information for remote sensing of vegetation type but may also confound attempts to measure high latitude vegetation biophysical characteristics and function using solar induced fluorescence (SIF).

K Fred Huemmrich

KrF laser-induced OH fluorescence imaging in a supersonic combustion tunnel

Planar fluorescence images of OH in a continuous-flow, electrical-resistively heated, high enthalpy, hydrogen-air combustion tunnel, induced by a tunable KrF laser, were recorded. These images were compared to previously recorded fluorescence images induced by a doubled-dye laser under similar conditions. Images induced by the doubled-dye laser system demonstrated a severe distortion caused by absorption and fluorescence trapping. By contrast, images of the fluorescence induced by the tunable KrF laser retained the symmetry properties of the flow. Based on signal-to-noise ratio measurements the yield of the fluorescence induced by the doubled-dye laser is larger than the fluorescence yield induced by the KrF laser. The measurements in the present facility of OH fluorescence induced by the KrF laser were limited by the photon-statistical noise. Based 2 on this result, doubled-dye laser systems are recommended for OH imaging in small and OH lean (less than 10 exp 15/cu cm) facilities. KrF lasers should be selected otherwise.

Quagliaroli, T. M.

Laser-saturated fluorescence measurements in laminar sooting diffusion flames

The hydroxyl radical is known to be one of the most important intermediate species in the combustion processes. The hydroxyl radical has also been considered a dominant oxidizer of soot particles in flames. In this investigation the hydroxyl concentration profiles in sooting diffusion flames were measured by the laser-saturated fluorescence (LSF) method. The temperature distributions in the flames were measured by the two-line LSF technique and by thermocouple. In the sooting region the OH fluorescence was too weak to make accurate temperature measurements. The hydroxyl fluorescence profiles for all four flames presented herein show that the OH fluorescence intensities peaked near the flame front. The OH fluorescence intensity dropped sharply toward the dark region of the flame and continued declining to the sooting region. The OH fluorescence profiles also indicate that the OH fluorescence decreased with increasing height in the flames for all flames investigated. Varying the oxidizer composition resulted in a corresponding variation in the maximum OH concentration and the flame temperature. Furthermore, it appears that the maximum OH concentration for each flame increased with increasing flame temperature.

Wey, Changlie

Measurement of the fluorescence of crop residues: A tool for controlling soil erosion

Management of crop residues, the portion of a crop left in the field after harvest, is an important conservation practice for minimizing soil erosion and for improving water quality. Quantification of crop residue cover is required to evaluate the effectiveness of conservation tillage practices. Methods are needed to quantify residue cover that are rapid, accurate, and objective. The fluorescence of crop residue was found to be a broadband phenomenon with emission maxima at 420 to 495 nm for excitations of 350 to 420 nm. Soils had low intensity broadband emissions over the 400 to 690 nm region for excitations of 300 to 600 nm. The range of relative fluorescence intensities for the crop residues was much greater than the fluorescence observed of the soils. As the crop residues decompose their blue fluorescence values approach the fluorescence of the soil. Fluorescence techniques are concluded to be less ambiguous and better suited for discriminating crop residues and soils than reflectance methods. If properly implemented, fluorescence techniques can be used to quantify, not only crop residue cover, but also photosynthetic efficiency in the field.

Daughtry, C. S. T.

Preparation and Characterization of Fluorescent Derivatives of Lysozyme

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. However, its use in macromolecular crystal growth studies is hampered by the necessity of preparing fluorescent derivatives where the probe does not markedly affect the crystal packing. Alternatively, one can prepare derivatives of limited utility if it is known that they will not affect the specific goals of a given study. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme, covalently attaching fluorescent probes to two different sites on the protein molecule. The first site is the side chain carboxyl group of ASP 101. Amine containing probes such as lucifer yellow, cascade blue, and 5- (2-aminoethyl) aminonapthalene-l-sulfonic acid (EDANS) have been attached using a carbodiimide coupling procedure. ASP 101 lies within the active site cleft, and it is believed that the probes are "buried" within that cleft. This is supported by the fact that all such derivatives have been found to crystallize, with the crystals being fluorescent. Tetragonal crystals of the lucifer yellow derivative have been found to diffract to at least 1.9 A resolution. X-ray diffraction data has been acquired and we are now working on the structure of this derivative. The second group of derivatives is to the N-terminal amine group. The derivatization reaction is performed by using a succinimidyl ester of the probe to be attached. Fluorescent probes such as pyrene acetic acid, 5-carboxyfluorescein, and Oregon green have been attached to this site. We have had little success in crystallizing these derivatives, probably because this site is part of the contact region between the 43 helix chains. However, these sites do not interfere with formation of the 43 helices and the derivatives are suitable for study of their formation in solution. The derivatives are being characterized by steady state and lifetime fluorescence methods, and the presentation will discuss these results.

Smith, Lori

Development of Highly Fluorescent Materials Based on Thiophenylimidazole Dyes

Organic fluorescent materials are expected to find many potential applications in optical devices and photo-functionalized materials. Although many investigations have been focused on heterocyclic compounds such as coumarins, bipyridines, rhodamines, and pyrrole derivatives, little is known for fluorescent imidazole materials. We discovered that one particular class of imidazole derivatives is highly fluorescent. A series of monomeric and polymeric based fluorescent dyes were prepared containing a thiophene unit at the second position of the imidazole ring. Dependence of fluorescence efficiency on parameters such as solvent polarity and substituent groups has been investigated. It was found that a formyl group at the 2-position of the thiophene ring dramatically enhance fluorescence properties. Ion recognition probes indicated their potential as sensor materials. These fluorophores have flexibility for introduction of versatile substituent groups that could improve the fluorescence efficiency and sensor properties.

Santos, Javier

Monitoring the Photosynthetic Apparatus During Space Flight: Interspecific Variation in Chlorophyll Fluorescence Signatures Induced by Different Root Zone Stresses

Chlorophyll fluorescence has been used extensively as a tool to indicate stress to the photosynthetic apparatus in green plants. A rise in fluorescence has been attributed to the blockage of photosystem II photochemistry, and patterns of fluorescence decay (quenching) from dark adapted leaves can be related to specific photochemical and non-photochemical deexcitation pathways of light trapped by the photosynthetic apparatus and thus result in characteristically different fluorescence signatures. Four distantly related plant species, Hypocharis radicata (Asteraceae), Brassica rapa (Brassicaceae), Spinacea oleracea (Chenopodiaceae) and Triticum aestivum (Poaceae), were grown hydroponically for three weeks before the initiation of three different root zone stresses (10 mM Cu, 100 mM NaCl and nitrogen deficient nutrition). After 10 days, characteristic fluorescence signatures for each stress could be noted although the degree varied between species. Fast kinetics analysis showed a reduction in plastoquinone pool size for copper and nitrogen stress for all species but a more species specific result with NaCl stress. Photochemical quenching kinetics varied between species and stress treatments from no quenching in S. oleracea in copper treatments to increased photochemical quenching in NaCl treatments. Non-photochemical quenching kinetics demonstrated a distinct pattern between stresses for all species. Copper treatments characteristically exhibited a shallow, flat non-photochemical quenching profile suggesting a general blockage of electron transport whereas NaCl treatments exhibited a slow rising profile that suggested damage to thylakoid acidification kinetics and nitrogen deficiency exhibited a fast rising and declining profile that suggested an altered state 1-state 2 transition regulated by the phosphorylation of LHCII. These results demonstrate characteristic fluorescence signatures for specific plant stresses that may be applied to different, unrelated plant species. In addition, the potential use of chlorophyll fluorescence for both basic research and diagnostic physiology in space biology is presented.

Bubenheim, David L.

Validation of fluorescent-labeled microspheres for measurement of relative blood flow in severely injured lungs

The aim of the study was to validate a nonradioactive method for relative blood flow measurements in severely injured lungs that avoids labor-intensive tissue processing. The use of fluorescent-labeled microspheres was compared with the standard radiolabeled-microsphere method. In seven sheep, lung injury was established by using oleic acid. Five pairs of radio- and fluorescent-labeled microspheres were injected before and after established lung injury. Across all animals, 175 pieces were selected randomly. The radioactivity of each piece was determined by using a scintillation counter. The fluorescent dye was extracted from each piece with a solvent without digestion or filtering. The fluorescence was determined with an automated fluorescent spectrophotometer. Perfusion was calculated for each piece from both the radioactivity and fluorescence and volume normalized. Correlations between flow determined by the two methods were in the range from 0.987 +/- 0.007 (SD) to 0.991 +/- 0.002 (SD) after 9 days of soaking. Thus the fluorescent microsphere technique is a valuable tool for investigating regional perfusion in severely injured lungs and can replace radioactivity.

Non-NASA Center

Filling-In of Broad Far-Red Solar Lines by Terrestrial Fluorescence and Atmospheric Raman Scattering as Detected by SCIAMACHY Satellite Measurements

Global mapping of terrestrial vegetation fluorescence from space has recently been accomplished with high spectral resolution measurements from the Japanese Greenhouse gases Observing SATellite (GOSAT). These data are of interest because they can potentially provide global information on the functional status of vegetation including light use efficiency and global primary productivity that can be used for global carbon cycle modeling. Quantifying the impact of fluorescence on the O2-A band is important as this band is used for cloud- and aerosol-characterization for other trace-gas retrievals including CO2. Here, we demonstrate that fluorescence information can be derived from space using potentially lower-cost hyperspectral instrumentation, i.e., more than an order of magnitude less spectral resolution than GOSAT, with a relatively simple algorithm. As a demonstration, we use the filling-in of one of the few wide and deep solar Fraunhofer lines in the red and far-red chlorophylla fluorescence bands, the calcium II line near 866 nm, to retrieve fluorescence with the SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY) satellite instrument. Although the signal from vegetation fluorescence is extremely weak at 866 nm, our results suggest that useful information may be obtained after adjustments are made to the observed spectra to correct for instrumental artifacts. We compare fluorescence from SCIAMACHY with that retrieved at 758 and 770 nm from similarly-corrected GOSAT data as well with the Enhanced Vegetation Index (EVI) from the MODerate-resolution Imaging Spectroradiometer (MODIS). We also show that filling-in occurs at 866 nm over barren areas, possibly originating from luminescent minerals in rock and soil.

Joiner, J.

Fluorescence Signatures of Rare Earth Metals during Precipitation in Various Conditions

Fluorescence spectroscopy is a widely used sensor methodology that analyzes light emitted from a compound or element as it decays from an excited state. This technique is very sensitive and selective, which is ideal to characterize analytes at lower limits of detection. Key example targets of significant industry and research interest include rare earth elements (REEs) such as dysprosium (Dy) and europium (Eu). These are widely used in advanced technologies including semiconductors, electric vehicle motors, lasers, and permanent magnets. Identifying new sources and responsible reutilization of REEs is essential, and new approaches to extract and recycle REEs could be notably enhanced through the integration of on-line sensors. The sensors can support faster process design, informed scale-up, and cost-effective deployment. This study covers the initial exploration of applying fluorescence-based on-line monitoring to REEs within a precipitation process. This study demonstrates the successful scale-up of a fluorescence -based sensing approach, from stationary cuvettes and small-volume microfluidic devices to continuous flow systems operating at the bench scale (10-25mL). This work also provides initial insight into the challenges of signal’s effects and utility within a turbid environment. Using a modular design for monitoring flowing solutions in a flow tube, fluorescence can be characterized for a variety of analytical targets. In this study, detection performance parameters between the cuvette and flow tube system were compared. Additionally, the response of Dy during precipitation by sodium bicarbonate in the two measurement designs was explored. This letter represents a starting point to bridge the gap between traditional fluorescence sensor measurements in a cuvette to future developments that explore the ability to integrate fluorescence sensors into extraction and separation processes at industrially relevant scales.

fluorescence

A new relative referencing method for crop monitoring using chlorophyll fluorescence

The measurement of plant chlorophyll fluorescence has been used for many years as a method to monitor a plant's health status. These types of methods have been mostly relegated to the laboratory. The newly developed Relative Referencing Method allows for the measurement of chlorophyll fluorescence under artificial lighting conditions. The fluorescence signal can be determined by first taking a reference signal measurement, then a second measurement with an additional fluorescence excitation source. The first signal can then be subtracted from the second and the plant's chlorophyll fluorescence due to the second lighting source can be determined. With this simple approach, a photosynthesizing plant can be monitored to detect signs of water stress. Using this approach experiments on tomato plants have shown that it was possible to detect water stress, while the plants were continuously illuminated by fluorescent lamps. This method is a promising tool for the remote monitoring of crops grown in a CELSS-type application. Published by Elsevier Science Ltd on behalf of COSPAR.

Plant Physiology

Characterization of Fluorescence Signals in Synthetic Anorthite

In-situ resource utilization (ISRU) is a key capability to enable a long term presence on the Moon and other planetary bodies. For example, efforts have been underway to develop processes to utilize the lunar regolith to produce building materials and extract useful resources. In processes that utilize the regolith directly to produce construction materials, such as in various sintering methods, characterizing the composition of the regolith is important as the composition can affect the optimal process parame-ters. In processes that involve extracting resources such as iron, oxygen, hydrogen, etc., identifying areas with higher abundance of a particular resource will be critical since different regions of the Moon exhibit differing regolith composition. To this end, Ra-man is one technique that can rapidly characterize the mineralogy of rock samples. This technique has al-ready been deployed on Mars to detect various miner-als including olivine, carbonates, phosphates, etc. Besides characterizing the mineralogy, Raman spectra often contain unwanted fluorescence signals that can mask Raman peaks. In some cases, the fluorescence signals are narrow enough to aid in the identification of specific elements in soils. In this work, we explore fluorescence peaks in a synthetic anorthite sample, which are at the same position as Ruby fluorescence peaks attributed to chromium. These fluorescence peaks, as well as similar fluorescence peaks attributed to other elements, could have important implications for ISRU and the mining of critical resources as has been previously described.

Raman Spectroscopy

Raman spectroscopy of Ryugu particles and their extracted residues: Fluorescence background characteristics and similarities to CI chondrites

We present here an investigation of Ryugu particles recovered by the Hayabusa2 space mission and their extracted carbonaceous acid residues using Raman spectroscopy. Raman parameters of Ryugu intact grains and their acid residues are characterized by broad D (defect induced) and G (graphite) band widths, indicating the presence of polyaromatic carbonaceous matter with low thermal maturity. Raman spectra of Ryugu particles and CI (type 1) chondrites exhibit stronger laser-induced fluorescence backgrounds compared to Type 2 and Type 3 carbonaceous chondrites. The high fluorescence signatures and wide bandwidths of the D and G bands of Ryugu intact grains are similar to the Raman spectra observed in CI chondrites, reflecting the low structural order of their aromatic carbonaceous matter, and strengthening the link between Ryugu particles and CI chondrites. The high fluorescence background intensity of the Ryugu particles is due to multiple causes, but it is likely that the relative abundance of geometry-bearing macromolecular organic matter in total organic carbon contents makes a large contribution to the fluorescence intensities. Locally observed high fluorescence in the acid-extracted residues of Ryugu is due to nitrogen-bearing outlier phase. The high fluorescence signature is one consequence of the low degree of thermal maturity of the organic matter and supports evidence that the Ryugu particles have escaped significant parent body thermal metamorphism.

Ryugu particles

A fluorescent-protein spin qubit

Quantum bits (qubits) are two-level quantum systems that support initialization, readout and coherent control1. Optically addressable spin qubits form the foundation of an emerging generation of nanoscale sensors. The engineering of these qubits has mainly focused on solid-state systems. However, fluorescent proteins, rather than exogenous fluorescent probes, have become the gold standard for in vivo microscopy because of their genetic encodability. Although fluorescent proteins possess a metastable triplet state, they have not been investigated as qubits. Here we realize an optically addressable spin qubit in enhanced yellow fluorescent protein. A near-infrared laser pulse enables triggered readout of the triplet state with up to 20% spin contrast. Using coherent microwave control of the enhanced-yellow-fluorescent-protein spin at liquid-nitrogen temperatures, we measure a (16 ± 2) μs coherence time under Carr–Purcell–Meiboom–Gill decoupling. We express the qubit in mammalian cells, maintaining contrast and coherent control despite the complex intracellular environment. Finally, we demonstrate optically detected magnetic resonance in bacterial cells at room temperature with contrast up to 8%. Our results introduce fluorescent proteins as a powerful qubit platform that paves the way for applications in the life sciences, such as nanoscale field sensing and spin-based imaging modalities.

Feder, Jacob S. [Univ. of Chicago, IL (United Stat

Waveform-dependent air fluorescence from neutral and ionic nitrogen molecules

Laser-induced air fluorescence in the ultraviolet regime is primarily attributed to transitions between the C and B states in excited neutral N 2 molecules and between the B and X states in N$^+_2$ ions. However, the mechanism underlying the former remains contentious, as direct population to the C state by light fields is forbidden by electron spin constraints. In this work, we investigate the mechanism of air fluorescence from excited neutral N 2 molecules by carrier-envelope phase–stabilized sub–4 femtosecond pulses. Our results show that fluorescence from N$^+_2$ ions reaches a maximum with cosine-like pulses, while fluorescence from excited neutral N 2 molecules peaks with sine-like pulses. In addition, by scanning the chirp of the driving pulse, we find that ionic fluorescence is maximized with chirp-free pulses, whereas neutral fluorescence favors negatively chirped pulses. These observations, supported by classical trajectory Monte Carlo simulations, support the mechanism of intersystem crossing from excited spin-singlet states, which are populated via recollision-induced strong-field excitation.

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