Fluorescence Excitation Spectroscopy Research Articles

Room-Temperature Solution Fluorescence Excitation Correlation Spectroscopy.

Single-molecule fluorescence spectroscopy is a powerful tool for investigating the physical properties of individual molecules, yet elucidating the fast fluctuation dynamics of freely diffusing single molecules in solution at room temperature, where a variety of chemical and biological processes occur, remains challenging. In this study, we report on fluorescence excitation correlation spectroscopy of room-temperature solutions, which enables the study of spontaneous fluctuation of the excitation spectrum with microsecond time resolution. By employing Fourier transform spectroscopy with broadband femtosecond pulses and time-correlated single-photon counting, we achieved fluorescence excitation spectroscopy of a room-temperature solution at the single-molecule level. Building upon this single-molecule measurement, we obtained an excitation wavelength-resolved fluorescence autocorrelation function in the microsecond to millisecond range, demonstrating the potential of this method to elucidate fast, spontaneous, time-dependent changes of excitation spectra in statistically equilibrated systems. With further development, this method will allow the study of spectral exchange associated with transitions between sub-ensembles of solution-phase molecules with unprecedented time resolution.

On-the-Fly Simulation of Two-Dimensional Fluorescence-Excitation Spectra.

Two-dimensional (2D) fluorescence-excitation (2D-FLEX) spectroscopy is a recently proposed nonlinear femtosecond technique for the detection of photoinduced dynamics. The method records a time-resolved fluorescence signal in its excitation- and detection-frequency dependence and hence combines the exclusive detection of excited state dynamics (fluorescence) with signals resolved in both excitation and emission frequencies (2D electronic spectroscopy). In this work, we develop an on-the-fly protocol for the simulation of 2D-FLEX spectra of molecular systems, which is based on interfacing the classical doorway-window representation of spectroscopic responses with trajectory surface hopping simulations. Applying this methodology to gas-phase pyrazine, we show that femtosecond 2D-FLEX spectra can deliver detailed information that is otherwise obtainable via attosecond spectroscopy.

High-Resolution Cryogenic Spectroscopy of Single Molecules in Nanoprinted Crystals.

We perform laser spectroscopy at liquid helium temperatures (T = 2 K) to investigate single dibenzoterrylene (DBT) molecules doped in anthracene crystals of nanoscopic height fabricated by electrohydrodynamic dripping. Using high-resolution fluorescence excitation spectroscopy, we show that zero-phonon lines of single molecules in printed nanocrystals are nearly as narrow as the Fourier-limited transitions observed for the same guest-host system in the bulk. Moreover, the spectral instabilities are comparable to or less than one line width. By recording super-resolution images of DBT molecules and varying the polarization of the excitation beam, we determine the dimensions of the printed crystals and the orientation of the crystals' axes. Electrohydrodynamic printing of organic nano- and microcrystals is of interest for a series of applications, where controlled positioning of quantum emitters with narrow optical transitions is desirable.

Intensive processing reduces quality of grains: a triangulation of three assessment methods

ABSTRACT The presented study evaluated different manufacturing procedures of breakfast cereals in view of their ability to preserve the natural quality of the raw material, as this is recommended by the EU Regulation 2018/848 for organic farming and the Demeter guidelines. From four crops (spelt, buckwheat, rice and quinoa), different products were manufactured: flakes, roasted flakes from cooked grains, extrudates from dough, and puffed grains. The samples (in total n = 18) were analysed for positive (thiamine, folate, lysine) and negative (acrylamide) health-related constituents, delayed fluorescence by Fluorescence Excitation Spectroscopy (FES) and food-induced emotions by the EmpathicFoodTest (EFT). The results showed that the four crops reacted similarly to the manufacturing processes, but the sensitivity of the crop was different for the health-related constituents, with spelt and rice showing greater sensitivity with respect to acrylamide formation and thiamine loss, than buckwheat and quinoa (though differences at the individual crop level were not statistically verified). The different assessment methods led to largely consistent evaluations of the processing methods: after extrusion, puffing and roasting flakes, significant quality losses were observed. Depending on the parameter of the test method, the classification of the degradation due to processing differed slightly: lysine degradation was significantly higher in puffed products (especially in rice and spelt) and acrylamide concentration was high (> 200 µg kg-1) in roasted and puffed spelt. The methods EFT and FES rated extruded samples particularly unfavourably. The least harmful process in all parameters was the production of flakes.

Tip-enhanced two-photon excited fluorescence spectroscopy of monolayer black phosphorus

The optical properties of monolayer black phosphorus (BP) in two-photon excited fluorescence (TPEF) are pivotal for photoluminescence applications at the single molecular level. In this study, we theoretically examine the TPEF properties of monolayer BP using a tip-enhanced spectroscopy system. We detail the influence of parameters such as the tip radius, the distance between the tip and the BP film, the incidence angle and the tip half-cone angle on the fluorescence excitation and emission enhancement of monolayer BP. This examination employs the finite element method. Our findings reveal that the signal enhancement in TPEF is substantially improved, approximately two orders of magnitude greater than that in single-photon excited fluorescence (SPEF), attributed to the high-order nonlinear coupling effect. By optimising the system configuration, we determined that the TPEF enhancement factor with monolayer BP can reach as high as eight orders of magnitude under an excitation wavelength of 800 nm. The highest spatial resolutions achievable for TPEF and SPEF spectroscopy are also 4.4 nm and 5.3 nm, respectively. These results provide an in-depth understanding of the TPEF enhancement mechanism and are invaluable for designing and applying BP-based optoelectronic devices.

(Invited) Diameter-Dependent Stacking of Squaraine Dye Molecules inside Chirality-Sorted Single-Wall Carbon Nanotubes

Filling of single-wall carbon nanotubes (SWCNTs) with organic dye molecules is a promising strategy to add new functionalities, for instance by aligning dipolar dyes in a one-dimensional (1D) head-to-tail coupled chain to obtain a giant enhancement of their nonlinear optical response,[1] or by photo-sensitizing the tubes in new wavelength ranges by excitation energy transfer (EET) from the dye to the SWCNTs, useful for photo-conversion applications.[2] The 1D stacking of molecules inside SWCNTs modulates both the properties of the molecules and of the SWCNTs. We found that the EET of squaraine-dye-filled SWCNTs reveals a pronounced SWCNT-diameter-dependence of the dye absorption, indicating a diameter-dependent stacking. By performing IR fluorescence excitation spectroscopy on a range of chirality-enriched samples of dye-filled SWCNTs with different chirality distributions, each shifted dye absorption wavelength observed through the EET can be unambiguously assigned to a specific encapsulating SWCNT chirality.[3] This reveals a systematic trend with diameter that can be associated with the J-aggregate- and H-aggregate-like interactions expected in close-packed single- and double-file stackings, as corroborated by a simple geometric model, and enables tuning of the optical properties of the 1D molecular arrays through the selection of proper SWCNT diameters.[1] S. Cambré, J. Campo, C. Beirnaert, C. Verlackt, P. Cool, W. Wenseleers, Nature Nanotechnol. 10(3) 248-252 (2015). [2] S. van Bezouw, D.H. Arias, R. Ihly, S. Cambré, A.J. Ferguson, J. Campo, J.C. Johnson, J. Defillet, W. Wenseleers, J.L. Blackburn, ACS Nano 12(7) 6881-6894 (2018). [3] S. Forel, H. Li, S. van Bezouw, J. Campo, L. Wieland, W. Wenseleers, B.S. Flavel, S. Cambré, Nanoscale 14, 8385-8397 (2022).

Two-dimensional fluorescence excitation spectroscopy: A novel technique for monitoring excited-state photophysics of molecular species with high time and frequency resolution.

We propose a novel UV/Vis femtosecond spectroscopic technique, two-dimensional fluorescence-excitation (2D-FLEX) spectroscopy, which combines spectral resolution during the excitation process with exclusive monitoring of the excited-state system dynamics at high time and frequency resolution. We discuss the experimental feasibility and realizability of 2D-FLEX, develop the necessary theoretical framework, and demonstrate the high information content of this technique by simulating the 2D-FLEX spectra of a model four-level system and the Fenna-Matthews-Olson antenna complex. We show that the evolution of 2D-FLEX spectra with population time directly monitors energy transfer dynamics and can thus yield direct qualitative insight into the investigated system. This makes 2D-FLEX a highly efficient instrument for real-time monitoring of photophysical processes in polyatomic molecules and molecular aggregates.

Transient Stimulated Raman Excited Fluorescence Spectroscopy

The pursuit of better sensitivity has always been one of the central themes in Raman spectroscopy. Recently, all-far-field single-molecule Raman spectroscopy has been demonstrated by a novel hybrid spectroscopy that couples Raman scattering with fluorescence emission. However, such frequency-domain spectroscopy lacks efficient hyperspectral excitation methods and encounters intrinsic strong fluorescence backgrounds from electronic transitions, hindering its applications in advanced Raman spectroscopy and microscopy. Here we report the ultrafast time-domain spectroscopy counterpart named transient stimulated Raman excited fluorescence (T-SREF): excited by two successive broadband femtosecond pulse pairs (i.e., the pump and Stokes pulses) with time-delay scanning, strong vibrational wave packet interference is revealed on the time-domain fluorescence trace, resulting in background-free spectra of the corresponding Raman modes after the Fourier transform. T-SREF achieves background-free Raman spectra of electronic-coupled vibrational modes with sensitivity up to the level of a few molecules, which paves the way for supermultiplexed fluorescence detection and molecular dynamics sensing.

Obtaining mutant Chlorella vulgaris strains with excellent self-flocculation properties and high wastewater treatment efficiency via atmospheric and room temperature plasma technique

In this study, atmospheric and room temperature plasma technique (ARTP) was applied to generate mutants of Chlorella vulgaris to improve its self-flocculation. Four high self-flocculation mutant strains named SDJZM-1, SDJZM-2, SDJZM-3 and SDJZM-4 were obtained. Among them, SDJZM-1showed the highest self-flocculation efficiency of 92.34 %, and the lowest self-flocculation efficiency of SDJZM-2 was still increased by 78.85 % compared to the original type. In addition, four mutants showed high capability to uptake nutrients from wastewater. Especially, mutant SDJZM-4 increased the removal of total nitrogen, phosphorus and ammonia nitrogen by 29.39 %, 22.72 % and 14.82 %, respectively, in comparison with wild-type strain. Three-dimensional excitation and emission matrix fluorescence spectroscopy analysis showed that more protein was found in mutant strains, and a significant correlation between the protein content in bound extracellular polymeric substances (B-EPS) and flocculation efficiency was observed. The main self-flocculation mechanism of mutant strains was the bridging and sweeping made by B-EPS.

Relaxation dynamics of 3He and 4He clusters and droplets studied using near infrared and visible fluorescence excitation spectroscopy.

The relaxation dynamics of electronically excited 3He and 4He clusters and droplets is investigated using time-correlated near-infrared and visible (NIR/VIS) fluorescence excitation spectroscopy. A rich data set spanning a wide range of cluster and droplet sizes is produced. The spectral features broadly follow the vacuum ultraviolet excitation (VUV) spectra. However, when the NIR/VIS spectra are normalised to the VUV fluorescence, regions with distinctly different cluster size and isotope dependence are identified, enabling deeper insight into the relaxation mechanism. Particle density, location of atomic-like states and their principal quantum number, n, are found to play an important role in the relaxation. For states with n = 3 and higher, only energy within the surface region is transferred to excited atoms which are subsequently ejected from the surface and fluoresce in vacuum. For states with n = 2, energy from the entire region within clusters and droplets is transferred to the surface, leading to the ejection of excited atoms and excimers. Here, the energy is transferred by excitation hopping, which competes with radiative and non-radiative decay, making ejection and NIR/VIS fluorescence inefficient in increasingly larger droplets.

Rotational and deperturbation analysis of the interacting A2Π1/2, v = 11 ∼ B2Σ+, v = 6 states of yttrium monoxide (YO) molecule

The YO molecules in gas-phase were formed by the reaction of the laser produced yttrium plasma with molecular oxygen and subsequently cooled by free-jet expansion. The YO molecules were studied by laser-induced excitation and dispersed fluorescence (DF) spectroscopy at 0.1 cm-1 spectral resolution. The B2Σ+ state was observed in vibrations v=5 and 6 from the X2Σ+ ground state. The anomalously low spin-rotation constant of the B2Σ+, v=6 level compared to the v=0-5 vibrational levels of the B state indicated perturbation. Investigation of the wavelength filtered excitation spectra led to the observation of the A2Π1/2, v=11 spin-orbit component lying 28.5 cm-1 below the B2Σ+, v=6 state. The deperturbation analysis of the interacting pair of B2Σ+, v=6 ∼ A2Π1/2,v=11 states was carried out by incorporating the spin-orbit, spin-electronic and L-uncouple interaction parameters as off-diagonal matrix elements in the 2Σ+-2Π Hamiltonian. The molecular constants of v=11 of the A and v=6 of the B states followed the vibrational trend after deperturbation. The e-parity level of the B2Σ+, v=6 and A2Π1/2, v=11 crossed after J=25.5 and deperturbation analysis estimated J-dependent configuration mixing up to ∼50% for the e-parity and 16% for the f-parity levels. The value of the configuration mixing coefficient of the interacting A and the B states, obtained from the intensity analysis of the observed vibronic bands in the DF spectra, is in agreement with the depertubation analysis. In addition, the Ω=½ spin-orbit components of the A2Π state was observed in v=12 and 13 vibrations and were found to be unperturbed.

PVDF membranes modified with diblock copolymer PEO-b-PMMA as additive: Effects of copolymer and barrier pore size on filtration performance and fouling in a membrane bioreactor

In the present work, filtration performance and fouling behavior of four poly(vinylidene difluoride) (PVDF) membranes with different composition and molecular weight cut-off (MWCO) were investigated in a lab-scale submerged membrane bioreactor (MBR) system, treating real pharmaceutical wastewater. Poly(ethylene oxide)-block-poly(methyl methacrylate) (PEO-b-PMMA) diblock copolymer or FeCl2 or the combination of PEO-b-PMMA and FeCl2 were used as special additives during membrane formation. A 30-day filtration experiment was performed using four membranes in the same aeration tank simultaneously, and filtration performance of the membranes was investigated over the entire filtration period. Fouling parameters were calculated for all membranes and the TMP-step method was used to determine the critical flux of the membranes. Formed cake layers onto the surface of the membranes at the end of each filtration run were collected and extracellular polymeric substances (EPSs), excitation and emission matrix (EEM) fluorescence spectroscopy, and Fourier transform infrared (FTIR) spectroscopy analyses were performed for the cake layers. Collected cake layers after EPSs extraction were dried and their masses were measured to estimate anti-biofilm formation potential of the membranes. Obtained results revealed that incorporation of the PEO-b-PMMA diblock copolymer into the PVDF membrane accompany with FeCl2 salt which tailors the MWCO and prevents the increment of the pore size improves membrane performance and reduces its fouling propensity, in a way that membrane with smaller MWCO containing copolymer revealed lower flux decline, higher critical flux, higher flux recovery ratio (FRR) and lower biofilm mass per area. Moreover, EPS and EEM analyses revealed that membrane surface chemistry has considerable effect on the composition of the cake layers. Finally, chemical oxygen demand (COD) removal efficiency proved that MWCO does not have obvious effect on effluents’ quality. The present study confirms the importance of the surface chemistry and MWCO on fouling behavior of the membranes in the MBR system and reveals that the addition of the PEO-b-PMMA diblock copolymer to the PVDF membrane improves antifouling property of the membrane considerably.

Spectral Dependence of the Energy Transfer from Photosynthetic Complexes to Monolayer Graphene.

Fluorescence excitation spectroscopy at cryogenic temperatures carried out on hybrid assemblies composed of photosynthetic complexes deposited on a monolayer graphene revealed that the efficiency of energy transfer to graphene strongly depended on the excitation wavelength. The efficiency of this energy transfer was greatly enhanced in the blue-green spectral region. We observed clear resonance-like behavior for both a simple light-harvesting antenna containing only two chlorophyll molecules (PCP) and a large photochemically active reaction center associated with the light-harvesting antenna (PSI–LHCI), which pointed towards the general character of this effect.

Differentiation of eggs from organic and conventional production systems using fluorescence excitation spectroscopy (FES)

ABSTRACT The decay curves of the delayed luminescence of egg yolks after excitation with white, yellow and blue light were investigated, using fluorescence excitation spectroscopy (FES), and the relationships to the origin of the eggs were examined. FES measurements were done by exciting the sample with light of defined wavelengths, representing the colours white (w), blue (bl) and yellow (ye), and the subsequent photon emission was detected. A total of 147 samples (each sample consisted of 10 eggs) were analysed, which originated from samples collected directly from the farm as well as from retail markets. Classification by partitioning and Tukey HSD tests were used to show differences between the origin of the eggs. It was shown that the induced emission of the conventional eggs was different compared with that of the organic eggs, which was reflected especially in the long-time emission parameters R40-w and R80-ye/bl, with lower values for conventional samples. Classification was possible with the parameters R40-w and R80-ye/bl, allowing correct classifications of 91.8% and 95.9% of the samples, respectively.

Measurement of absolute light yield and quantum efficiency of LaBr3:Ce scintillator detector using SiPM

This brief communication presents our work to determine the absolute light yield and quantum efficiency of LaBr3:Ce scintillator by comparison and also by direct pulse measurement method using SiPM. The first part presents use of the simpler comparison method to determine the light yields of different scintillators using the known yield of NaI(Tl) as reference. In the second part we have determined the absolute light yield and quantum efficiency of LaBr3:Ce crystal by using a SiPM photo detector. Our measured value is in good agreement with the light yield reported by previous measurements using PMT and excitation fluorescence spectroscopy. Quantum efficiencies for scintillation detectors have been determined by using both PMTs and photo detectors, namely APDs by previous authors. This communication is possibly the first report on the determination of quantum efficiency of LaBr3:Ce using SiPM photo detector. The simple and effective method presented here would allow to determine the light yield of any scintillation detector.

Spectroscopic Study of the B 1Π State of NaH.

In this study, the B1Π excited state of NaH has been experimentally studied for the first time. Pulsed laser-induced fluorescence excitation spectroscopy was used to investigate the B1Π electronic state of NaH. A total of 48 ro-vibronic transitions were observed, including within the B–X (0–0) and B–X (0–1) transition bands. Only one B-state vibrational level was identified, and a series of PQR lines, with eight e-parity and eight f-parity sublevels (v′ = 0, J′ = 1–8), were assigned. The level assignment was supported by a comparison of the experimental line positions with the ab initio calculations, the dispersed laser-induced fluorescence spectrum of the NaH B1Π → X1∑+ emission, and the V-type optical–optical double resonance spectra. The Dunham-type coefficients, the mean internuclear distance, the harmonic vibrational frequency ω, and the dissociation energies D0 and De of the B1Π state were determined.

Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts.

Tolyporphins are distinctive tetrapyrrole natural products found singularly in a filamentous cyanobacterial-microbial holobiont (termed HT-58-2) from Micronesia. The absorption and fluorescence features of tolyporphins resemble those of chlorophyll a, complicating direct analysis of culture samples. Treatment of the crude (unfractionated) organic extract (CH2 Cl2 /2-propanol, 1:1) of HT-58-2 cultures with NaBH4 in methanol causes reduction of the peripheral ketone auxochromes, whereupon tolyporphins (predominantly 7,17-dioxobacteriochlorins) exhibit a bathochromic shift (λabs ˜ 676 → ˜ 700 nm) and chlorophyll a (a 131 -oxochlorin) exhibits a hypsochromic shift (λabs 665 → 634 nm). Fluorescence excitation spectroscopy (at 368 and 491 nm with λem 710 nm) enabled detection of reduced tolyporphins amidst abundant reduced chlorophyll a (1:19 ratio), a detection sensitivity >5 times that without reduction. The resulting assay combines simple sample preparation from non-axenic cultures at microscale quantities (2 mL, 2 μm), absence of any fractionation procedures, and fluorescence detection. Tolyporphins were readily detected in cultures of HT-58-2 at reasonable growth periods in the absence of environmental stressors, which was not possible previously.

(Invited) Combining Absorption, Wavelength-Dependent Raman and Fluorescence-Excitation Spectroscopy with High-Resolution Transmission Electron Microscopy for Determining the Chiral Purity of Sorted SWCNT Samples

Single-wall carbon nanotubes (SWCNTs) possess unique electronic and optical properties that depend strongly on their exact chiral structure. Recent progress in the structure sorting of specific SWCNT chiralities, with increasing chiral purity[1], demands an effective characterization methodology to be developed to accurately determine the chiral composition of any SWCNT chirality. Very often, optical spectroscopy is used to assess the chiral composition of a sample, but absorption cross-sections, PL quantum efficiencies and Raman cross-sections are all depending on the exact chiral structure,[2] and can be strongly influenced by other factors such as the specific internal and external environment of the SWCNTs.[3] In this work, we systematically compare the chirality distribution obtained from 3 different optical spectroscopic techniques, i.e. absorption, wavelength-dependent Raman and fluorescence-excitation spectroscopy with the chirality distribution obtained from high-resolution transmission electron microscopy for both chirality-sorted and unsorted SWCNT samples. This combined approach demonstrates the importance of using a multi-technique characterization strategy for a reliable determination of SWCNT chirality distribution.[4][1] J.A. Fagan, Nanoscale Adv. 1, 3307 (2019) and references therein[2] V.N. Popov, Nano Lett. 4, 1795 (2004)[3] S. Cambré et al, Angew. Chem. – Int Ed. 50, 2764 (2011); ACS nano 6, 2649 (2012)[4] A. Castan et al, Carbon 171, 968 (2021)

(Invited) Ultrasonication-Induced Inner Shell Extraction from Double‐Wall Carbon Nanotubes: Characterisation By Ultracentrifugation and in Situ Raman and Fluorescence-Excitation Spectroscopy

We show that ultrasonication extracts inner tubes from double-wall carbon nanotubes (DWCNT), and characterize the process by density gradient ultracentrifugation (DGU) combined with in situ spectroscopic characterization in the density gradient. Our observations also cast new light on the question whether or not the inner tubes of DWCNTs fluoresce, which has been the subject of a long-standing debate, even though single‐DWCNT experiments have already shown drastic PL‐quenching for the inner CNT [1]. Indeed, in previous studies to characterise the PL from DWCNTs, they were typically solubilised using sonication, while here we see that upon sonication, the extraction of the inner tubes from the DWCNTs leads to much more, efficiently fluorescing SWCNTs. First, the DWCNTs were purified from SWCNTs and bundles using density gradient ultracentrifugation (DGU). Then, sonication is applied, after which the extracted inner tubes were separated from the DWCNTs by DGU and characterised by in situ resonant Raman and PL‐spectroscopy as a function of height in the centrifuge tube [2]. These subsequent in situ DGU experiments unambiguously prove the extraction of inner SWCNTs from DWCNTs, even by very brief sonication, and sonication-time-dependent measurements show that the process quickly saturates.[1] D. Levshov, R. Parret, H.-N. Tran, T. Michel, T.T. Cao, V.C. Nguyen, R. Arenal, V.N. Popov, S.B. Rochal, J.-L. Sauvajol, A.-A. Zahab, M. Paillet, Phys. Rev. B 96, 195410 (2017). [2] S. Cambré, P. Muyshondt, R. Federicci, W. Wenseleers, Nanoscale 7, 20015 (2015).

Calcium carbonate labeling for the characterization of self-healing cracks in cement-based materials

Biomineralization has been widely used to develop self-healing cement-based materials for infrastructure maintenance. To characterize the crack repair depth of cement-based materials after microbial self-healing, we used two methods to label calcium carbonate, the main product of microbial mineralization. One method involved the adsorption of Cu2+ by microbial calcium carbonate to form Cu2(OH)2CO3, which surrounded the repair products. The other method involved labeling the mineralized products by doping them with Eu3+ to form CaCO3: Eu3+. X-ray diffraction was used to analyze bicarbonate before and after labeling in solution and in the cement-based materials. Fluorescence excitation and emission spectroscopy was used to analyze CaCO3: Eu3+. X-ray computed tomography was used to characterize the depth of the repair products in cement-based materials labeled using the two methods. Both labeling methods indicated a similar repair depth of 3 mm, demonstrating that these methods can be used to evaluate the efficacy of microbial self-healing in cement-based materials.