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[Display omitted] • Oxygen atoms were detected by vacuum UV emission after two-photon excitation. • Time-resolved vacuum UV emission was recorded at varying pressures of He and N 2. • The kinetic analysis of the time profiles was made by the integrated profiles method. • The quenching rate coefficients of O(3p 3P) by He and N 2 have been determined. • State-to-state (3p 3P → 3 s 3S) quenching rate coefficients have been determined. Electronically excited atomic oxygen O (2p 3 3 p 3 P J) was prepared by ultraviolet (225.6 nm) two-photon excitation from the ground state 2 p 4 3 P 2. The 2p 3 3 p 3 P J state transfers to the 2p 3 3 s 3 S state via the infrared transition at 845 nm and/or by collisions with ambient gases. The vacuum ultraviolet (VUV) emission at 130 nm via the transition 2p 3 3 s 3 S → 2 p 4 3 P J was detected, and the time-resolved VUV emission at varying pressures of He and N 2 was recorded. The kinetic analysis made by the integrated profiles method has given the rate coefficients not only for the overall quenching of the 2p 3 3 p 3 P J state but also for the state-to-state quenching 2p 3 3 p 3 P J → 2p 3 3 s 3 S by He and N 2. [ABSTRACT FROM AUTHOR]

Hydroxyl-terminated fourth generation poly(amido amine) dendrimer and folic acid were chemically bound on graphene oxide. The resultant hybrids exhibited one-photon and two-photon fluorescence emission, since the excitation irradiation at 390 and 780 nm on the hybrids brought a fluorescence emission in the visible region around 450 nm. In addition, the photocytotoxicity study revealed that under the two-photon excitation at 780 nm, the hybrids can absorb near-infrared light and generate reactive oxygen species which can oxidize the HeLa cells and cause their death, suggesting the phototherapeutic behavior. Cytotoxicity measurement revealed the high biocompatibility of the hybrids toward HeLa cells. Thus, the present biocompatible hybrids consisting of only dendrimer, folic acid and graphene oxide have potentials as photodynamic therapeutic agents for medical treatment. [ABSTRACT FROM AUTHOR]

In this paper, the two-photon-excited emission spectra of polymer optical fibers doped with the conjugated polymer Poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) have been measured pumping the fibers transversely to their symmetry axis. Measurements include evolutions of the emission spectra with excitation wavelength and with propagation distance, together with an analysis of emission photostability. Comparisons with results for one-photon-excited emission are also presented and discussed. [ABSTRACT FROM PUBLISHER]

ABSTRACT Mapping the activity of neuronal circuits with high resolution in the intact brain is a fundamental step toward understanding brain function. In the last several years, nonlinear microscopy combined with fluorescent activity reporters has become a crucial tool for achieving this goal. In this review article, we will highlight the principles underlying nonlinear microscopy and discuss its application to neuroscience, focusing on recent functional studies in the rodent neocortex in combination with genetically encoded calcium indicators. Microsc. Res. Tech. 77:492-501, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

This paper presents the design and characterization of a three-axis thermomechanical actuator-based endoscopic scanner for obtaining ex vivo two-photon images. The scanner consisted of two sub-systems: 1) an optical system (prism, gradient index lens, and optical fiber) that was used to deliver and collect light during imaging and 2) a small-scale silicon electromechanical scanner that could raster scan the focal point of the optics through a specimen. The scanner can be housed within a 7 mm Ø endoscope port and can scan at the speed of 3 kHz x 100 Hz × 30 Hz along three axes throughout a 125 × 125 × 100 μm3 volume. The high-speed thermomechanical actuation was achieved through the use of geometric contouring, pulsing technique, and mechanical frequency multiplication (MFM), where MFM is a new method for increasing the device cycling speed by pairing actuators of unequal forward and returning stroke speeds. Sample cross-sectional images of 15-μm fluorescent beads are presented to demonstrate the resolution and optical cross-sectioning capability of the two-photon imaging system. [ABSTRACT FROM AUTHOR]

ABSTRACT Fluorescence microscopy is an essential technique for the basic sciences, especially biomedical research. Since the invention of laser scanning confocal microscopy in the 1980s, which enabled imaging both fixed and living biological tissue with 3D precision, high-resolution fluorescence imaging has revolutionized biological research. Confocal microscopy, by its very nature, has one fundamental limitation. Due to the confocal pinhole, deep tissue fluorescence imaging is not practical. In contrast (no pun intended), two-photon fluorescence microscopy allows, in principle, the collection of all emitted photons from fluorophores in the imaged voxel, dramatically extending our ability to see deep into living tissue. Since the development of transgenic mice with genetically encoded fluorescent protein in neocortical cells in 2000, two-photon imaging has enabled the dynamics of individual synapses to be followed for up to 2 years. Since the initial landmark contributions to this field in 2002, the technique has been used to understand how neuronal structure are changed by experience, learning, and memory and various diseases. Here we provide a basic summary of the crucial elements that are required for such studies, and discuss many applications of longitudinal two-photon fluorescence microscopy that have appeared since 2002. J. Comp. Neurol. 522:1708-1727, 2014. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The ability to resolve the spatio-temporal complexity of intracellular 2 distribution is the "Holy Grail" of cellular physiology. In an effort to obtain a minimally invasive approach to the mapping of intracellular 2 tensions, two methods of phosphorescent lifetime imaging microscopy were compared in the current study and gave similar results. These were two-photon confocal laser scanning microscopy with pinhole shifting, and picosecond time-resolved epi-phosphorescence microscopy using a single 0.5 μm focused spot. Both methods utilized coordination complex embedded nanoparticles (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of a titanium-sapphire Tsunami lasers (710-1050 nm). [ABSTRACT FROM AUTHOR]

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects. [ABSTRACT FROM AUTHOR]

A therapy of cancer cells: Two‐photon‐triggered camptothecin delivery (see picture) with nanoimpellers was studied in MCF‐7 breast cancer cells. A fluorophore with a high two‐photon absorption cross‐section was first incorporated in the nanoimpellers. Fluorescence resonance energy transfer (FRET) from the fluorophore to the azobenzene moiety was demonstrated. [ABSTRACT FROM AUTHOR]

Structural dynamics of dendritic spines is one of the key correlative measures of synaptic plasticity for encoding short-term and long-term memory. Optical studies of structural changes in brain tissue using confocal microscopy face difficulties of scattering. This results in low signal-to-noise ratio and thus limiting the imaging depth to few tens of microns. Multiphoton microscopy (MpM) overcomes this limitation by using low-energy photons to cause localized excitation and achieve high resolution in all three dimensions. Multiple low-energy photons with longer wavelengths minimize scattering and allow access to deeper brain regions at several hundred microns. In this article, we provide a basic understanding of the physical phenomena that give MpM an edge over conventional microscopy. Further, we highlight a few of the key studies in the field of learning and memory which would not have been possible without the advent of MpM. [ABSTRACT FROM AUTHOR]

Highlights: [•] We have synthesized a new biphenyl ethylene derivative (BEEASP). [•] The single- and two-photon fluorescent properties of the dye were investigated. [•] The results showed that the dye is sensitive to DNA. [•] The possible interaction mechanism of dye–DNA was groove binding. [ABSTRACT FROM AUTHOR]

A new dinuclear RuII polypyridyl complex, [(bpy)2Ru(H2bpip)Ru(bpy)2]4+ ( RuH2bpip, bpy=2,2-bipyridine, H2bpip=2,6-pyridyl(imidazo[4,5-f][1,10]phenanthroline), was developed to act as a one- and two-photon luminescent probe for biological Cu2+ detection. This RuII complex shows a significant two-photon absorption cross section (400 GM) and displays a remarkable one- and two-photon luminescence switch in the presence of Cu2+ ions. Importantly, RuH2bpip can selectively recognise Cu2+ in aqueous media in the presence of other abundant cellular cations (such as Na+, K+, Mg2+, and Ca2+), trace metal ions in organisms (such as Zn2+, Ag+, Fe3+, Fe2+, Ni2+, Mn2+, and Co2+), prevalent toxic metal ions in the environment (such as Cd2+, Hg2+, and Cr3+), and amino acids, with high sensitivity (detection limit≤3.33×10−8 M) and a rapid response time (≤15 s). The biological applications of RuH2bpip were also evaluated and it was found to exhibit low cytotoxicity, good water solubility, and membrane permeability; RuH2bpip was, therefore, employed as a sensing probe for the detection of Cu2+ in living cells and zebrafish. [ABSTRACT FROM AUTHOR]

Abstract: Polystyrene nanoparticles (PSNPs) with an average size of 85nm and loaded with an oxygen-quenchable luminescent ruthenium complex were used to sense and image oxygen inside cells following 2-photon excitation (2-PE). The ruthenium probe possesses a large two-photon absorption cross-section, and 2-PE is achieved by irradiation in the near infrared with commercially available fs-pulsed laser systems. The luminescence of the dye-loaded PSNPs is strongly quenched by oxygen, and Stern–Volmer plots are linear for both conventional single-photon excitation (1-PE) and for 2-PE. The particles do not show any significant cytotoxicity below a threshold concentration of 5μg/mL and are readily taken up by mammalian cells (MCF-7), presumably via membrane mediated pathways. Thus, the PSNPs promise to be well suited to image the oxygen distribution in living cells and tissues. The 2-PE is considered to be advantageous over conventional imaging techniques because it works in the near-infrared where background absorption and luminescence of biomatter is much weaker than at excitation wavelengths below 600nm. [Copyright &y& Elsevier]

We developed a multi-unit microscope for all-optical inter-layers circuits interrogation. The system performs two-photon (2P) functional imaging and 2P multiplexed holographic optogenetics at axially distinct planes. We demonstrated the capability of the system to map, in the mouse retina, the functional connectivity between rod bipolar cells (RBCs) and ganglion cells (GCs) by activating single or defined groups of RBCs while recording the evoked response in the GC layer with cell-type specificity and single-cell resolution. We then used a logistic model to probe the functional connectivity between cell types by deriving the "cellular receptive field" describing how RBCs impact each GC type. With the capability to simultaneously image and control neuronal activity at axially distinct planes, the system enables a precise interrogation of multi-layered circuits. Understanding this information transfer is a promising avenue to dissect complex neural circuits and understand the neural basis of computations., Competing Interests: Anurag Agrawal is an employee of Double Helix Optics, Inc., (© 2022 The Author(s).)

Flow cytometry is an essential technique in biomedical discovery for cell counting, cell sorting, and biomarker detection. In vivo flow cytometers based on one-photon or two-photon excited fluorescence have been developed for over a decade. One drawback of the laser beam scanning two-photon flow cytometer is that the two-photon excitation volume is limited to the focal spot due to the short Rayleigh range of focused Gaussian beams. Hence, the sampling volume is much smaller than in one-photon flow cytometers, making it challenging to count or detect rare circulating cells in vivo. Non-diffracting light waves like Bessel beams and Airy beams have narrow intensity profiles with an effective spot size as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making them an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Airy beams rather than Gaussian beams is side lobes Airy beams have, which contribute to background noise. Two-photon excitation can reduce this noise, as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using 2D Airy beams to form a light-sheet that intersects the blood vessel a microfluidic channel, which was used to model a blood vessel. The setup can successfully detect and count flowing fluorescent microspheres in a microchannel. [ABSTRACT FROM AUTHOR]

A novel two-photon ratiometric fluorescent pH nanosensor based on fluorescein isothiocyanate (FITC)-doped conjugated polymer nanoparticles is designed for detection of pH in solution and living cells. [Display omitted] • FITC-doped conjugated polymer nanoparticles are designed for two-photon excited fluorescence pH sensing. • The FITC-doped nanosensor possesses outstanding sensitivity, stability, reversibility and two-photon excitability. • The two-photon ratiometric fluorescence signals of the nanosensor show a sensitive response to the pH changes from 4 to 9 in solution (p K a = 6.43). • The biocompatible nanosensor is successfully applied for monitoring and imaging of intracellular pH variation in one- and two-photon excitation mode. Herein, we report a two-photon ratiometric fluorescent pH nanosensor based on conjugated polymer poly(9,9-di-n-octylfluorenyl-2,7-diyl) (PFO) nanoparticles loaded with pH-sensitive fluorescein isothiocyanate (FITC) for intracellular pH monitoring. The obtained nanosensor (FITC-PFO NPs) possesses high sensitivity, excellent stability, good reversibility, favorable two-photon excitability and low cytotoxicity. The ratiometric fluorescence of FITC and PFO (F 517 /F 417) in FITC-PFO NPs solution shows an efficient pH-sensitive response over the pH range from 3 to 10 (p K a = 6.43) under two-photon excitation. Additionally, the FITC-PFO NPs is successfully applied for ratiometric imaging of intracellular pH and its fluctuation in both one-photon and two-photon excitation modes. Overall, the two-photon pH nanosensor based on FITC-PFO NPs exhibits great potential in crucial physiological and biological processes related to intracellular pH fluctuations. [ABSTRACT FROM AUTHOR]

Calcium (Ca) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca concentration ([Ca]) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca] is the use of fluorescent Ca indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca-free and Ca-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca-free and Ca-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca sensitivity is reduced. Nevertheless, quantitative [Ca] recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The response of HeLa cells to subcellular spatially localized two-photon irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. Upon irradiation under these conditions, a localized population of PpIX excited states can be produced with meaningful intracellular spatial resolution; the dimensions of the domain where the incident light flux is high enough for PpIX two-photon absorption are defined by the microscope optics and by the diffraction of light (spot diameter at beam waist of ˜0.5-1.0 μm). In turn, the dimensions of the intracellular domain containing cytotoxic PpIX-sensitized singlet oxygen will likewise be confined. Most importantly, cell response (e.g., morphological signs of cell death) correlates with the light dose delivered and the intracellular domain irradiated. Thus, controlling light delivery can complement other techniques used to impart intracellular spatial localization in mechanistic studies of photoinitiated reactive oxygen species. Such controlled light delivery is also expected to be a particularly useful tool to study the so-called bystander effect in which a selectively-perturbed cell can influence a neighboring cell through intercellular signaling mechanisms. [ABSTRACT FROM AUTHOR]

We report electron energy loss spectroscopy (EELS) and one- and two-photon excited surface-enhanced Raman scattering (SERS) and hyper Raman studies on plasmonic silver nanoaggregates. By comparison with computations, EELS imaging reveals an inverse relationship between local field intensity in an optical experiment and electron energy loss intensity at energies corresponding to excitation wavelengths used for optical probing. This inverse relation exists independent on specific nanoaggregate geometries and is mainly controlled by the gap size between the particles forming the aggregate. The ratio between two- and one-photon excited SERS measured at different excitation wavelengths provides information about local fields in the hottest spots and their dependence on the photon energy. Our data verify experimentally the predicted increase of local optical fields in the hot spots with increasing wave lengths. The reported findings show new experimental ways to characterize local fields of plasmonic nanostructures. This is of particular importance for complex structures which are not easily approachable by computations. [ABSTRACT FROM AUTHOR]

Abstract: We report the synthesis of bioconjugated zinc porphyrin dimers 1a–e designed as photosensitizers for one-photon and two-photon excited photodynamic therapy. These macrocycles are substituted with carbohydrate units (glucose, mannose, lactose) in order to target tumor cells over-expressing lectin membrane receptors. Polarity, singlet oxygen production and in vitro photocytotoxicity are studied to determine their photodynamic therapy potentiality. [Copyright &y& Elsevier]

We demonstrate two-photon-excited single-molecule fluorescence enhancement by single end-to-end self-assembled gold nanorod dimers. We employed biotinylated streptavidin as the molecular linker, which connected two gold nanorods in end-to-end fashion. The typical size of streptavidin of around 5 nm separates the gold nanorods with gaps suitable for the access of fresh dyes in aqueous solution, yet small enough to give very high two-photon fluorescence enhancement. Simulations show that enhancements of more than 7 orders of magnitude can be achieved for two-photon-excited fluorescence in the plasmonic hot spots. With such high enhancements, we successfully detect two-photon-excited fluorescence for a common organic dye (ATTO 610) at the single-molecule, single-nanoparticle level.

Chips of Norway spruce have been impregnated with Na2SO3 and refined at two specific energy consumptions levels at full mill scale. The optical properties of thermomechanical pulps (TMPs) obtained were analyzed in terms of brightness, light scattering, opacity, and autofluorescence by spectral imaging. Even at low sulfite dosage (0.24% sulfite by dry weight) light absorption was reduced, and the brightness was elevated, and a clear dose-response effect was observed. Two-photon spectral imaging (TPSI) showed that sulfonation, impregnation, and refining affect the fluorescence properties differently. Compared to native wood, both processed wood chips and pulp fibers revealed blue-shifted fluorescence maxima, a characteristic of shortened conjugated systems. Two subpopulations of fibers with different optical properties were observed, and the fluorescence of one fiber population was red shifted. [ABSTRACT FROM AUTHOR]

"in vivo two-photon functional Ca2+ imaging" is a powerful tool to analyze neural circuits of the cerebral cortex in the physiological condition. To monitor activities of excitatory neurons, inhibitory (GABAergic) neurons and astrocytes, we applied this imaging method to visual cortex of transgenic mice, in which GABAergic neurons express fluorescent protein (EGFP or Venus). With this method, we can monitor the neural activities from hundreds of excitatory and GABAergic neurons (and also from astrocytes) in vivo. We found that the difference in response selectivity and ocular dominance plasticity between excitatory and GABAergic neuron in the mouse visual cortex. [ABSTRACT FROM AUTHOR]

In two recent studies, energy transfer was reported in certain phthalocyanine-carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S) and phthalocyanines (Pcs) in the direction Pc → Car S (Kloz et al., J Am Chem Soc 133:7007-7015, ) as well as in the direction Car S → Pc (Liao et al., J Phys Chem A 115:4082-4091, ). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole-carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs. [ABSTRACT FROM AUTHOR]

Conventional linear and time-resolved spectroscopic techniques are often not appropriate to elucidate specific pigment-pigment interactions in light-harvesting pigment-protein complexes (LHCs). Nonlinear (laser-) spectroscopic techniques, including nonlinear polarization spectroscopy in the frequency domain (NLPF) as well as step- wise (resonant) and simultaneous (non-resonant) two-photon excitation spectroscopies may be advantageous in this regard. Nonlinear spectroscopies have been used to elucidate substructure(s) of very complex spectra, including analyses of strong excitonic couplings between chlorophylls and of interactions between (bacterio)chlorophylls and 'optically dark' states of carotenoids in LHCs, including the major antenna complex of higher plants, LHC II. This article shortly reviews our previous study and outlines perspectives regarding the application of selected nonlinear laser-spectroscopic techniques to disentangle structure-function relationships in LHCs and other pigment-protein complexes. [ABSTRACT FROM AUTHOR]

Distributed temperature sensing based on time-correlated two-photon excited fluorescence (TPF) in doped optical fiber is described. Counter-propagating laser pulses generate a TPF flash at the position of their overlap which is scanned along the fiber by a variable relative time delay. The flash is transmitted to one end where it is detected and analyzed to yield the temperature from its thermal dependence. To identify suitable dopants, the two-photon excitation spectra of glass doped with various rare-earths were recorded. Preliminary results on TPF in praseodymium doped single-mode fiber are presented. [ABSTRACT FROM PUBLISHER]

Distributed sensing of temperature can be achieved by using time-correlated two-photon excited fluorescence (TPF). To assess the extension of this technique to single-crystal fibers for high-temperature applications, various aspects are considered including the two-photon absorption cross-section (δ), dopant density, and the geometry of single crystal fibers. By comparing the fluorescence yield for two-photon excitation with that for single-photon excitation of the same transition, δ for ruby was measured over the 0.8-1.2 μm range with maximum room temperature values of 5.9 × 10-3 GM for e-polarization and 4.6 × 10-3 GM for o-polarization at 840 nm. It is shown that values of this magnitude are adequate for a practical TPF-based crystal fiber sensor to be realized. [ABSTRACT FROM PUBLISHER]

Abstract: The photostability is an outstanding feature of quantum dots (QDs) used as fluorescence probes in biological staining and cell imaging. To find out the related factors in the QD photostability, the photobleaching of naked CdTe QDs and BSA coated CdSe/CdS/ZnS QDs in human hepatocellular carcinoma (QGY) cells and human nasopharynx carcinoma (KB) cells were studied under single photon excitation (SPE) and two-photon excitation (TPE). In these two cell lines the cellular QDs were irradiated by a 405nm continuous wave laser for SPE or an 800nm femto-second (fs) laser for TPE. The QD photobleaching with the irradiation time was found to fit a biexponential decay. The fast decay plays a dominant role in the bleaching course and thus can be used as the parameter to quantitatively evaluate the QD photostability. The TPE decreased the QD photobleaching as compared to SPE. The BSA coated core/shell QDs had improved the photostability up to 4–5 times than the naked QDs due to the shielding effect of the QD shell. Therefore, it is better to use core/shell structured QDs as the fluorescence probe combining with a TPE manner for those long-term monitoring studies. [Copyright &y& Elsevier]

Bovine serum albumin (BSA)-coated CdTe/ZnS quantum dots (BSA-QDs) were selected to conjugate with folic acid (FA), forming FA-BSA-QDs. This study aims to develop these small FA-BSA-QDs (less than 10 nm) for the diagnosis of cancers in which the FA receptor (FR) is overexpressed. The enhancement of cellular uptake in FR-positive human nasopharyngeal carcinoma cells (KB cells) for FA-BSA-QDs was found by means of confocal fluorescence microscopy under single-photon and two-photon excitation. The uptake enhancement for FA-BSA-QDs was further evaluated by flow-cytometric analysis in 10 KB cells, and was about 3 times higher than for BSA-QDs on average. The uptake enhancement was suppressed when KB cells had been pretreated with excess FA, reflecting that the enhancement was mediated by the association of FR at cell membranes with FA-BSA-QDs. When human embryonic kidney cells (293T) (FR-negative cells) and KB cells, respectively, were incubated with FA-BSA-QDs (1 μM) for 40 min, the FA-BSA-QD uptake by 293T cells was much weaker than that by KB cells, demonstrating that FA-BSA-QDs could undergo preferential binding on FR-positive cancer cells. These characteristics suggest that FA-BSA-QDs are potential candidates for cancer diagnosis. [ABSTRACT FROM AUTHOR]

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur. [ABSTRACT FROM AUTHOR]

Abstract: The time-resolved photophysical characterisation of a set of fluorescent conjugates of neurotransmitter amino acids (glutamic acid, tyrosine and β-alanine) based on a methoxylated 5,6-benzocoumarin fluorophore was performed. Both one- and two-photon excitation were employed to excite the fluorophore and the cross-section with wavelength for the two-photon process determined. The photophysical characterisation was supported using a molecular modelling study, which helped elucidate the molecular transitions (by a ZINDO calculation) and differences in the electron density between the HOMO and LUMO in geometry (PM3) optimised structures. The effect of prolonged irradiation of the conjugates was monitored via fluorimetry and differences in the kinetics of the process observed were found to depend on the amino acid employed. [ABSTRACT FROM AUTHOR]

Lipophilic fluorescent dyes have been used to trace neuronal connections because of their ability to diffuse laterally within nerve cell membranes. Given the hundreds to thousands of connections that a typical neuron makes with its neighbours, a diffusion-matched set of spectrally distinct dyes is desirable. To extend a set of these dyes to obtain six independent labels, we have characterized the properties of novel violet and near-infrared candidates. By combining two-photon and confocal microscopy all of these candidates can be imaged using a single Titanium Sapphire laser. Here we present measurements of the two-photon action cross-sections and diffusion properties of the dyes, using either the relative diffusion distance or fluorescence recovery after photobleaching techniques, and demonstrate six-colour neuronal tracing within the spinal cord and brain tissue. [ABSTRACT FROM AUTHOR]

Abstract: The aggregation dependent correlation between fluorescence quenching and the electronic carotenoid–chlorophyll interactions, , as measured by comparing chlorophyll fluorescence observed after two- and one-photon excitation, has been investigated using native LHC II samples as well as mutants lacking Chl 2 and Chl 13. For native LHC II the same linear correlation between and the fluorescence quenching was observed as previously reported for the pH and Zea-dependent quenching of LHC II . In order to elucidate which carotenoid–chlorophyll pair might dominate this correlation we also investigated the mutants lacking Chl 2 and Chl 13. However, also with these mutants the same linear correlation as for native LHC II was observed. This provides indication that these two chlorophylls play only a minor role for the observed effects. Nevertheless, we also conclude that this does not exclude that their neighboured carotenoids, lutein 1 and neoxanthin, might interact electronically with other chlorophylls close by. [Copyright &y& Elsevier]

Spectral-fluorescent properties of benzothiazole styryl monomer (Bos-3) and homodimer (DBos-21) dyes in presence of DNA were studied. The dyes enhance their fluorescence intensity in 2–3 orders of magnitude upon interaction with DNA. Studied styrylcyanines in DNA presence demonstrate rather high values of two-photon absorption (TPA) cross-section, which are comparable with the values of TPA cross section of the rhodamine dyes. An applicability of the styrylcyanines as probes for the fluorescence microscopy of living cells was studied. It was shown that both dyes are cell-permeable but homodimer dye DBos-21 produces noticeably brighter staining of HeLa cells comparing with monomer dye Bos-3. Molecules of DBos-21 initially bind to the nucleic acids- containing cell organelles (presumable mitochondria) and are able to penetrate into the cell nucleus. Thus, homodimer styryl DBos-21 dye is viewed as efficient stain for single-photon and two-photon excitation fluorescence imaging of living cells. [ABSTRACT FROM AUTHOR]

We here discuss a problem of time-resolved fluorescence polarization spectroscopy at two-photon excitations of macroscopically isotropic molecular media, that can be organized locally on a nano-scale. The simplifying assumptions on the hydrodynamical shape of fluorophores and on the form of the two-photon absorption tensor, are considered and displayed on the corresponding examples of the emission anisotropy synthetic decays. These assumptions reduce the number of the model parameters making the theoretical description of the discussed spectroscopic technique applicable in the experimental practice. Because the two-photon excitations of fluorescence require the application of the exciting light of appropriately high intensity, and that may have a negative influence on the quality of the experimental data collected, we discuss the application of the wide-angular detection-aperture technique in such experiments. Furthermore, we consider the application of the symmetry adapted callibration (SAC) method enabling one to accurately analyze the experimental data even if the collecting optics employed is not ideal and is affected by several unexpected technical imperfections that are hard to be accounted for analytically. [ABSTRACT FROM AUTHOR]

Abstract: We introduce a new rhodamine-based fluorescent chemosensor, FD8 which exhibits a distinct two-photon excited fluorescence (TPEF) on/off characteristic upon binding Cr3+ ions. By coordination with metal cation, conformation of FD8 changes from spirocyclic to open-ring, resulting in remarkable enhancement of absorption and fluorescence both in one- and two-photon excitations. As a result, a 29-fold enhancement of two-photon excited fluorescent intensity was observed when 10eq. Cr3+ was added to the FD8 solution. The detection limit of Cr3+ cation concentration down to 1μM (0.01eq. of FD8) was achieved under our experimental condition. Besides the excitation within ultraviolet regime by fluorescence resonance energy transfer (FRET) mechanism, the TPEF on/off behavior further extends the excitation to near infrared regime (the biological optimal window of 700–1200nm), and shows more effective sensitivity. The broad excitation wavelength, on/off fluorescence and high selectivity to Cr3+ enable FD8 to be a powerful Cr3+ cation sensor with potential application, especially in biological detection. To the best of our knowledge, this is the first report about two-photon fluorescent sensor for Cr3+ ions. [Copyright &y& Elsevier]

Abstract: We demonstrate that DiI and rhodamine B, which are not easily distinguishable to one-photon measurements, can be differentiated and in fact quantified in mixture via tailored two-photon excitation pulses found by a genetic algorithm (GA). A nearly three-fold difference in the ratio of two-photon fluorescence of the two dyes is achieved, without a drop in signal of the favored fluorophore. Implementing an acousto-optic interferometer, we were able to prove that the mechanism of discrimination is second-harmonic tuning by the phase-shaped pulses to the relative maxima and minima of these cross-sections. [Copyright &y& Elsevier]

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S1, shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S1) and chlorophyll (Chl) states. ɸCar S1-ChlCoupling, correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both ChI fluorescence quenching and ɸCar S1-ChlCoupling with the intensity of red-shifted bands in the Cht Qy and carotenoid absorption was also observed. These results suggest quenching excitonic Car S1-Chl states as origin for the observed effects. Furthermore, real time measurements of the light-dependent downand up-regulation of the photosynthetic activity and ɸCar S1-ChlCoupling in wild-type and mutant (npq1, npq2, npq4, lut2 and WT+PsbS) Arabidopsis thaliana plants reveal that also in vivo the quenching parameter NPQ correlates always linearly with the extent of electronic Car S1-Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, ɸCar S1-ChlCoupling depends on the presence of PsbS and zeaxanthin (Zea) in an almost identical way as NPQ. In summary, these results provide clear evidence for a very close link between electronic Car S1-Chl interactions and the regulation of photosynthesis. These findings support a photophysical mechanism in which short-living, low excitonic carotenoidchlorophyll states serve as traps and dissipation valves for excess excitation energy. [ABSTRACT FROM AUTHOR]

Two types of peripheral light-harvesting complexes LH2 (B800–850) from photosynthetic purple bacterium Allochromatium minutissimum were studied. First type containing carotenoids was prepared from wild type cells. The other one was obtained from carotenoid depleted cells grown with diphenylamine. We have shown that under laser femtosecond excitation within absorption 1200–1500 nm wavelength range the two-photon excitation of LH2 complexes takes place. This can be observed as fluorescence of bacteriochlorophyll (BChl) spectral form B850 (BChl molecules of circular aggregate with strong exciton interaction in 850 nm spectral domain). LH2 fluorescence excitation spectra under two-photon excitation are the same for carotenoid-containing and carotenoidless preparations. In both cases the broad band with peak near 1350 (675) nm (FWHM ∼ 240 (120) nm) was found. It is concluded that the broad band with peak near 1350 (675) nm in two-photon excitation spectra of LH2 complexes from Allochromatium minutissimum cannot be interpreted as two-photon excitation band of the optically forbidden S0 → S1 transition of carotenoids (rhodopin). Possible nature of this band is discussed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Bromine atoms have been detected by vacuum UV emission after two-photon excitation. • Twenty lines of 25 allowed two-photon transitions were observed. • Five missing lines did not show infrared emission. • Detection limit of the number density of Br atoms is ≈109 cm−3. • The present method can be applied to the measurements under high pressure conditions. Two ultraviolet photons (250–282 nm) excited atomic bromine, Br (4 p 5 2 P J ; J = 1 / 2 , 3 / 2) , to the terms built from the 4p45p electronic configuration. Through visible and infrared (VIS–IR) transitions and/or collisions with ambient gases, the terms transfer to the 2 , 4 D J and 2 , 4 P J states in the 4p44d and 4p45s electronic configurations. The vacuum ultraviolet (VUV) emission (125–163 nm) from the 4p44d and 4p45s states to the 4 p 5 2 P J state was detected. Twenty in 25 allowed two-photon transitions were observed; however, no VUV or VIS–IR emission from the other five transitions was detected. The findings differ from those by the previous reports on the two-photon resonance-enhanced ionization. [ABSTRACT FROM AUTHOR]

We report a newly synthesized polymer of a star-shaped porphyrin compound (TPA-FxP) with four oligofluorene arms at its meso positions with the pronounced enhancement of the two-photon properties and the generation of singlet oxygen by utilizing the two-photon excited fluorescence resonance energy transfer. The steady-state spectra and transient triplet-triplet absorption spectra give evidence that the enhanced two-photon absorption cross section results from not only the through-space energy transfer (Förster) but also the through-bond energy transfer between conjugated peripheral oligofluorene arms and the porphyrin core. The two-photon absorption cross section at 780 nm up to 3360 GM (1 GM = 10−50 cm4·s/photon) of TPA-FxP was obtained, which is comparable to the highest values reported from other similar chemically modified porphyrin core compounds. Furthermore, the enhanced production of singlet oxygen under two-photon absorption conditions is also reported. [ABSTRACT FROM AUTHOR]

Anisotropic noble metal nanoparticles supporting more than one localized surface plasmon resonance can be tailored for efficient dual-mode fluorescence enhancement by ensuring an adequate coupling to both absorption and emission bands of fluorophores. This approach is naturally extended to two-photon excitation fluorescence, where a molecule is excited by simultaneous nonlinear absorption of two photons. However, the relative impact of plasmon coupling to excitation and emission on the overall fluorescence enhancement can be very different in this case. Here, by using the finite-difference time-domain method, we study the two-photon excitation fluorescence of near-infrared fluorescent protein (NirFP) eqFP670, which is the most red-shifted NirFP to date, in proximity to a silver nanobar. By optimizing the length and aspect ratio of the particle, we reach a fluorescence enhancement factor of 10 3 . We show that the single mode coupling regime with highly tuned near-field significantly outperforms the dual-mode coupling enhancement. The plasmon-induced amplification of the fluorophore's excitation rate becomes of utmost importance due to its quadratic dependence on light intensity, defining the fluorescence enhancement upon two-photon excitation. Our results can be used for the rational design of hybrid nanosystems based on NirFP and plasmonic nanoparticles with greatly improved brightness important for developing whole-body imaging techniques. [ABSTRACT FROM AUTHOR]