Multiphoton Excitation Research Articles

Detection by fluorescence microscopy of N-aminopeptidases in bacteria using an ICT sensor with multiphoton excitation: Usefulness for super-resolution microscopy

Bacterial proteases are relevant in pathological processes such as the survival, growth and development of microorganisms. In particular alanine amino peptidase (pepN), which is present in Gram (−) bacteria, is the only peptidase responsible for the ATP-independent degradation of cytosolic proteins in E. coli. These peptidases, including pepN, might be useful targets to fight bacterial infections, which are difficult to treat due to the increase in antibiotic resistance, as well as for diagnosis. In this work, we propose a new methodology for the identification of pepN-expressing bacteria by using a specific substrate, namely, DCM−NH−Ala. Substrate hydrolysis by pepN produces a sharp increase in the fluorescence band with peak at 662 nm when excited by a single photon at 480 nm or by two NIR photons (at approximately 800 nm). The emission kinetics are dependent on the intracellular pepN concentrations, which provide a powerful tool for detecting diverse virulent bacteria in a few minutes and with the inherent advantages of two-photon excitation. We resolved the enzymatic kinetics, obtained the Michaelis-Menten parameters (e.g., KM, vmax, and kcatalytic) and studied the photophysics of the released fluorophore DCM–NH2. In addition, DCM–NH2 meets the requirements for use in super-resolution microscopy. In bacteria with high pepN activity, the probe quickly initiates an enzymatic reaction at specific sites located on the bacterial membrane and some structures inside the bacterial body.

Planar Airy beam light-sheet for two-photon microscopy.

We demonstrate the first planar Airy light-sheet microscope. Fluorescence light-sheet microscopy has become the method of choice to study large biological samples with cellular or sub-cellular resolution. The propagation-invariant Airy beam enables a ten-fold increase in field-of-view with single-photon excitation; however, the characteristic asymmetry of the light-sheet limits its potential for multi-photon excitation. Here we show how a planar light-sheet can be formed from the curved propagation-invariant Airy beam. The resulting symmetric light sheet excites two-photon fluorescence uniformly across an extended field-of-view without the need for deconvolution. We demonstrate the method for rapid two-photon imaging of large volumes of neuronal tissue.

Fluorescence Anisotropy Sensor Comprising a Dual Hollow-Core Antiresonant Fiber Polarization Beam Splitter.

Fluorescence anisotropy imaging and sensing is a widely recognized method for studying molecular orientation and mobility. However, introducing this technique to in vivo systems is a challenging task, especially when one considers multiphoton excitation methods. Past two decades have brought a possible solution to this issue in the form of hollow-core antiresonant fibers (HC-ARFs). The continuous development of their fabrication technology has resulted in the appearance of more and more sophisticated structures. One of the most promising concepts concerns dual hollow-core antiresonant fibers (DHC-ARFs), which can be used to split and combine optical signals, effectively working as optical fiber couplers. In this paper, the design of a fluorescence anisotropy sensor based on a DHC-ARF structure is presented. The main purpose of the proposed DHC-ARF is multiphoton-excited fluorescence spectroscopy; however, other applications are also possible.

Multiphoton Excitation Microscopy for Identification and Operational Control of Extracellular Matrix Components of Body Tissues

The process of extraction of the extracellular matrix (ECM) requires operational monitoring, since there is a risk of destruction of the main proteins of the cell matrix at non-optimal parameters of the process. Using multiphoton microscopy the structure of collagen, the integrity of the cell membrane and the content of elastin in the process of isolation of ECM from various tissues of the rat body (brain, dermis, and muscle) has been studied. It has been shown that multiphoton microscopy is an effective tool for monitoring the quality of ECM extraction.

Sarcomere Lengths Become More Uniform Over Time in Intact Muscle-Tendon Unit During Isometric Contractions

The seemingly uniform striation pattern of skeletal muscles, quantified in terms of sarcomere lengths (SLs), is inherently non-uniform across all hierarchical levels. The SL non-uniformity theory has been used to explain the force creep in isometric contractions, force depression following shortening of activated muscle, and residual force enhancement following lengthening of activated muscle. Our understanding of sarcomere contraction dynamics has been derived primarily from in vitro experiments using regular bright-field light microscopy or laser diffraction techniques to measure striation/diffraction patterns in isolated muscle fibers or myofibrils. However, the collagenous extracellular matrices present around the muscle fibers, as well as the complex architecture in the whole muscles may lead to different contraction dynamics of sarcomeres than seen in the in vitro studies. Here, we used multi-photon excitation microscopy to visualize in situ individual sarcomeres in intact muscle tendon units (MTUs) of mouse tibialis anterior (TA), and quantified the temporal changes of SL distribution as a function of SLs in relaxed and maximally activated muscles for quasi-steady state, fixed-end isometric conditions. The corresponding muscle forces were simultaneously measured using a force transducer. We found that SL non-uniformity, quantified by the coefficient of variation (CV) of SLs, decreased at a rate of 1.9–3.1%/s in the activated muscles, but remained constant in the relaxed muscles. The force loss during the quasi-steady state likely did not play a role in the decrease of SL non-uniformity, as similar force losses were found in the activated and relaxed muscles, but the CV of SLs in the relaxed muscles underwent negligible change over time. We conclude that sarcomeres in the mid-belly of maximally contracting whole muscles constantly re-organize their lengths into a more uniform pattern over time. The molecular mechanisms accounting for SL non-uniformity appear to differ in active and passive muscles, and need further elucidation, as do the functional implications of the SL non-uniformity.

In-Depth Analysis of Egg-Tempera Paint Layers by Multiphoton Excitation Fluorescence Microscopy

The non-invasive depth-resolved imaging of pictorial layers in paintings by means of linear optical techniques represents a challenge in the field of Cultural Heritage (CH). The presence of opaque and/or highly-scattering materials may obstruct the penetration of the radiation probe, thus impeding the visualization of the stratigraphy of paintings. Nonlinear Optical Microscopy (NLOM), which makes use of tightly-focused femtosecond pulsed lasers as illumination sources, is an emerging technique for the analysis of painted objects enabling micrometric three-dimensional (3D) resolution with good penetration capability in semi-transparent materials. In this work, we evaluated the potential of NLOM, specifically in the modality of Multi-Photon Excitation Fluorescence (MPEF), to probe the stratigraphy of egg-tempera mock-up paintings. A multi-analytical non-invasive approach, involving ultraviolet-visible-near infrared (UV-Vis-NIR) Fiber Optics Reflectance Spectroscopy, Vis-NIR photoluminescence, and Laser Induced Fluorescence, yielded key-information for the characterization of the constituting materials and for the interpretation of the nonlinear results. Furthermore, the use of three nonlinear optical systems allowed evaluation of the response of the analyzed paints to different excitation wavelengths and photon doses, which proved useful for the definition of the most suitable measurement conditions. The micrometric thickness of the paint layers, which was not measurable by means of Optical Coherence Tomography (OCT), was instead assessed by MPEF, thus demonstrating the effectiveness of this nonlinear modality in probing highly-scattering media, while ensuring the minimal photochemical disturbance to the examined materials.

(Invited) Lanthanide Doped Nanoparticles and Their Applications

Since approximately 2000, lanthanide doped nanoparticles have received significant attention due to their interesting luminescent properties. At the core of this interest is their ability to convert near-infrared excitation light (typically 980 or 800 nm) to higher energies spanning the ultraviolet, visible and near-infrared regions of the spectrum through a multiphoton process known as upconversion. Upconversion differs from conventional multiphoton excitation in other materials where no real intermediate states are present necessitating the use of ultrafast lasers (in the femtosecond regime) for simultaneous excitation to the upper emitting state. The lanthanide ions possess a multitude of 4f electronic states that have long lifetimes (micro- to millisecond) thus act as population reservoirs in the upconversion process. Hence, upconversion occurs through real, long-lived intermediate states through a sequential photon absorption process. This eliminates the need for ultrafast excitation and as a result, upconversion can be observed using inexpensive, continuous wave diode lasers. Upconversion luminescence can be exploited for a number of applications in nanomedicine, theranostics, photovoltaics, photocatalysis, as well as many others.While their upconversion luminescence has been studied in great detail, lanthanide doped nanoparticles can also emit in the near-infrared through a direct Stokes (down-shifted) luminescence process. Of particular importance is that these near-infrared emissions lie within the biological windows where biological tissues are optically transparent. For obvious reasons, a great deal of the work on the nanomedicine and theranostics applications of lanthanide doped nanoparticles has shifted to their near-infrared luminescence properties.In this presentation, we will demonstrate the synthesis of these nanoparticles, establish how changing their nanoscale architecture can affect their luminescence properties (both upconversion and near-infrared), and discuss their potential applications, particularly in nanomedicine.

Advantage of multi-step vibrational excitation in laser assisted aerodynamic separation of sulfur isotopes and a novel method of estimating the separation efficiency of the process

The advantage of a red-shifted laser source in furthering the obtainable enrichment factor by laser assisted aerodynamic separation process has been reported. The enhancement owes its origin to the efficient multi-photon excitation possible here due to the red-shifted nature of the incident radiation. This has been experimentally validated in sulfur wherein the emission of a line tunable CW-CO2 laser interacted with SF6-Ar binary jet. Anharmonic splitting of the vibrational levels coupled with Coriolis rotational splitting facilitates such multi-photon up-pumping. Further, a novel spectroscopic method for estimation of relative concentration of isotopes following the above route is reported for the first time. The method exploits the resonant nature of the absorption on 10P(36) and 10P(20) lines of the CO2 laser by 34SF6 and 32SF6 isotopomers respectively. This method renders knowledge of absorption cross-sections irrelevant and facilitates precise measurement of separation factor. The details of the methodology are discussed in appendix section.

Enhanced photon blockade in an optomechanical system with parametric amplification.

We propose a scheme to enhance the single- and two-photon blockade effects significantly in a nonlinear hybrid optomechanical system with optical parametric amplification (OPA). The scheme does not rely on strong single-photon optomechanical coupling and can eliminate the disadvantages of suppressing multi-photon excitation incompletely. Through analyzing the single-photon blockade (1PB) mechanism and optimizing the system parameters, we obtain a perfect 1PB with a high occupancy probability of single-photon excitation, which means that a high-quality and efficient single-photon source can be generated. Moreover, we find that not only the two-photon blockade (2PB) effect is significantly enhanced, but also the region where 2PB occurs is widened when OPA exists, where we also derive the optimal parameter condition to maximize two-photon emission and higher photon excitations intensely suppressed at the same time.

Down- and up-conversion of femtosecond light pulses into Pr3+ luminescence in LiTaO3:Pr3+ single crystal

Spectra and decay curves of luminescence induced in a single crystal of LiTaO3:Pr3+ by femtosecond light pulses at different wavelengths from the UV-Blue and NIR regions were recorded and analyzed. It was found that excited levels involved in the broad UV absorption band of LiTaO3: Pr3+ are not able to relax via radiative transitions but they feed nonradiatively excited states of the ground Pr3+ configuration. Femtosecond pulse excitation in the NIR region results in an up-converted visible luminescence weakly affected by the pulse wavelength within the 1200 nm–1800 nm region. Spectra acquired imply that the supercontinuum (SC) generated does not promote single - or double-photon processes that may feed the 3P0 or 1D2 levels via transitions within the ground 4f2 configuration. It was concluded that the excitation mechanism is consistent with the multiphoton excitation (MPE) that feeds the 5d levels of Pr3+.

Feasibility of multimodal multiphoton microscopy to facilitate surgical margin assessment in pancreatic cancer.

Pancreatic cancer is a common cancer with poor odds of survival for the patient, with surgical resection offering the only hope of cure. Current surgical practice is time-consuming and, due to time constraints, does not sample the whole cut surface sufficiently to check for remaining cancer. Although microscopy with hematoxylin and eosin (H&E) stain is the gold standard for microscopic evaluation, multiphoton microscopy (MPM) has emerged as an alternative tool for imaging tissue architecture and cellular morphology without labels. We explored the use of multimodal MPM for the label-free identification of normal and cancerous tissue of the pancreas in a mouse model by comparing the images to H&E microscopy. Our early studies indicate that MPM using second-harmonic generation, third-harmonic generation, and multiphoton excitation of endogenous fluorescent proteins can each contribute to the label-free analysis of the pancreatic surgical margin.

High-harmonic generation at particle–hole multiphoton excitation in gapped bilayer graphene

A microscopic nonlinear quantum theory of the interaction of coherent electromagnetic radiation with gapped bilayer graphene is developed. The Liouville-von Neumann equation for the density matrix is solved numerically at the multiphoton excitation regime. The developed theory of interaction of charged carriers with the strong driving wave field is valid near the Dirac points of the Brillouin zone. We consider the harmonic generation process in the nonadiabatic regime of interaction when the Keldysh parameter is of the order of unity. On the basis of numerical solutions, we examine the rates of odd and even high harmonics at the particle–hole annihilation in the field of a strong pump wave of arbitrary polarization. The obtained results show that the gapped bilayer graphene can serve as an effective medium for the generation of even and odd high harmonics in the terahertz and far-infrared domains of frequencies.

Probing photoionization dichroism of excited electron ring currents by chiral photoelectron spectroscopy

Electron ring currents in atoms carry the nonzero azimuthal quantum number that determines their rotation velocities. Here we tune the rotation velocity of electron ring currents by multiphoton excitation with strong ellipticity-controlled laser fields, and then we probe the ionization dynamics of the ring currents in continuum states and excited states with a chiral light field. We obtain photoionization dichroism of the angular-momentum-varied ring currents, and we find that the ring currents with high angular momenta are more easily ionized by a corotating circularly polarized light field, which is different from multiphoton or tunneling ionization of ground-state ring currents. The ab initio solution of the time-dependent Schr\"odinger equation and the classical dynamics simulation agree with our measurements. This study may have implications for the control of the rotation velocity of electron ring currents and the extraction of chiral dynamical information in strong-field ionization.

Multi-Photon Excitation in Photoredox Catalysis: Concepts, Applications, Methods.

The energy of visible photons and the accessible redox potentials of common photocatalysts set thermodynamic limits to photochemical reactions that can be driven by traditional visible-light irradiation. UV excitation can be damaging and induce side reactions, hence visible or even near-IR light is usually preferable. Thus, photochemistry currently faces two divergent challenges, namely the desire to perform ever more thermodynamically demanding reactions with increasingly lower photon energies. The pooling of two low-energy photons can address both challenges simultaneously, and whilst multi-photon spectroscopy is well established, synthetic photoredox chemistry has only recently started to exploit multi-photon processes on the preparative scale. Herein, we have a critical look at currently developed reactions and mechanistic concepts, discuss pertinent experimental methods, and provide an outlook into possible future developments of this rapidly emerging area.

An excitation wavelength switching to enhance dual-color wide-field temporal-focusing multiphoton excitation fluorescence imaging

Dual-color two-photon excitation (TPE)-fluorescence imaging is used in conventional temporal-focusing multiphoton excitation microscopy (TFMPEM) to observe specimens with different fluorophore labels. However, concerns have been raised about the excitation efficiency and selectivity of the fluorophores under fixed-wavelength excitation. This study presents a wavelength-switching approach using a scanning mirror, beam expander, and diffraction grating in the TFMPEM to switch the excitation wavelengths and match the optimal absorption of the fluorophores to acquire dynamic dual-color TPE-fluorescence images. The presented TFMPEM system was demonstrated to have an axial excitation confinement of 2.3–5.0 μm for excitation wavelengths of 730–1000 nm, and was used to visualize three-dimensional images of the vasculature of a mouse brain. The TPE efficiencies of different fluorophores were evaluated through TFMPEM imaging with excitation wavelength scanning to obtain their TPE spectra. Consequently, time-lapsed dual-color TFMPEM imaging was performed on rhodamine 6G (R6G)–poly(lactic-co-glycolic acid) (PLGA) nanoparticles and enhanced-yellow-fluorescent protein (EYFP)-tagged clathrin using excitation wavelengths at the maximum TPEs of R6G and EYFP, respectively. Our results revealed the PLGA-nanoparticle uptake of live cells via long-lived clathrin-coated plaques in clathrin-mediated endocytosis.

Characterizing Multiphoton Excitation Using Time-Resolved X-ray Scattering

Molecular iodine was photoexcited by a strong 800 nm laser, driving several channels of multiphoton excitation. The motion following photoexcitation was probed using time-resolved X-ray scattering, which produces a scattering map $S(Q,\tau)$. Temporal Fourier transform methods were employed to obtain a frequency-resolved X-ray scattering signal $\tilde{S}(Q,\omega)$. Taken together, $S(Q,\tau)$ and $\tilde{S}(Q,\omega)$ separate different modes of motion, so that mode-specific nuclear oscillatory positions, oscillation amplitudes, directions of motions, and times may be measured accurately. Molecular dissociations likewise have a distinct signature, which may be used to identify both velocities and dissociation time shifts, and also can reveal laser-induced couplings among the molecular potentials.

Fluorescence of serotonin in the visible spectrum upon multiphotonic photoconversion.

The vital molecule serotonin modulates the functioning of the nervous system. The chemical characteristics of serotonin provide multiple advantages for its study in living or fixed tissue. Serotonin has the capacity to emit fluorescence directly and indirectly through chemical intermediates in response to mono- and multiphoton excitation. However, the fluorescent emissions are multifactorial and their dependence on the concentration, excitation wavelength and laser intensity still need a comprehensive study. Here we studied the fluorescence of serotonin excited multiphotonically with near-infrared light. Experiments were conducted in a custom-made multiphoton microscope coupled to a monochromator and a photomultiplier that collected the emissions. We show that the responses of serotonin to multiphoton stimulation are highly non-linear. The well-known violet emission having a 340 nm peak was accompanied by two other emissions in the visible spectrum. The best excitor wavelength to produce both emissions was 700 nm. A green emission with a ∼ 500 nm peak was similar to a previously described fluorescence in response to longer excitation wavelengths. A new blue emission with a ∼ 405 nm peak was originated from the photoconversion of serotonin to a relatively stable product. Such a reaction could be reproduced by irradiation of serotonin with high laser power for 30 minutes. The absorbance of the new compound expanded from ∼ 315 to ∼ 360 nm. Excitation of the irradiated solution monophotonically with 350 nm or biphotonically with 700 nm similarly generated the 405 nm blue emission. Our data are presented quantitatively through the design of a single geometric chart that combines the intensity of each emission in response to the serotonin concentration, excitation wavelengths and laser intensity. The autofluorescence of serotonin in addition to the formation of the two compounds emitting in the visible spectrum provides diverse possibilities for the quantitative study of the dynamics of serotonin in living tissue.

Multiphoton magnetic resonance in imaging: A classical description and implementation.

To develop a classical geometric interpretation of multiphoton excitation and apply it to MRI. To investigate ways in which multiphoton excitation can enable novel imaging techniques. We present a fully geometric view of multiphoton excitation by taking a particular rotating frame transformation. In this rotating frame, we find that multiphoton excitations appear just like single-photon excitations again, and therefore, we can readily generalize concepts already explored in standard single-photon excitation. With a homebuilt low frequency coil, we execute a standard slice selective pulse sequence with all of its excitations replaced by their equivalent two-photon versions. In the case of no extra hardware, we use oscillating gradients as a source of extra photons for excitation. Finally, with the multiphoton interpretation of oscillating gradients, we present a novel way to transform a standard slice selective adiabatic inversion pulse into a multiband version without modifying the RF pulse itself. The addition of oscillating gradients creates multiphoton resonances at multiple spatial locations and allows for adiabatic inversions at each location. With Bloch-Siegert shift corrections, analytical multiphoton excitation expressions match with Bloch equation simulations. Two-photon gradient-echo images of a lemon and a pork rib match with their single-photon counterparts. Frequency-offset RF combined with oscillating gradients generate excitation where the RF alone does not. The multiphoton interpretation presents new flexibilities for imaging. Excitation needs not be bound to the Larmor frequency, which opens doors to RF pulse design beyond the usual filter design and the potential for further imaging innovations.

Morphological characteristics of nanoholes induced by single-shot femtosecond laser ablation of borates and aluminate silicates

Single-shot femtosecond laser ablation experiments with linearly and circularly polarized light were conducted in order to investigate the morphological characteristics of surface nanostructures in lithium borate crystals and glasses, a strontium borate crystal, lanthanide containing borate crystals, and aluminate silicate crystals: Li2B4O7 (LTB) and LiB3O5 (LBO) crystals and Li2O⋅2B2O3 (LTB) and Li2O⋅3B2O3 (LBO) glasses; SrB4O7 (SBO) crystal; Li6Gd(BO3)3 (LGB) and LaSc3(BO3)4 (LSB) crystals; and Ca2Al2SiO7 (CAS) and CaSrAl2SiO7 (CSAS) crystals. In the present study, the material and laser polarization dependance of the morphology of nanoholes was examined in these crystals and glasses. A single nanohole or two holes (a primary hole and a secondary hole) were observed in the borate and aluminate silicate crystals. The size of the nanohole is not restricted by the diffraction limit but instead is dependent on the laser fluence and the materials. It is suggested that the formation of these secondary nanoholes in the studied crystals is attributed to a spontaneous reshaping of the incoming Gaussian pulse into a Gaussian–Bessel pulse. In the LTB and LBO crystals, nanoholes (both primary and secondary holes) with subwavelength sides exhibit a quadrilateral (approximately square or rectangular) morphology, regardless of linear or circular polarization. The sides of the quadrilateral nanoholes lie approximately in the {h h 0} planes on the LTB crystal and in the ({h 0 0} and {0 0 l}) planes on the LBO crystal. We found that the nanohole morphology did not reflect the spatial distribution of the laser intensity. These phenomena were the first observations on the anisotropic morphology of nanoholes. These morphologies do not correspond to the circular symmetric pattern of the Gaussian intensity distribution of the incoming laser beam. This is contrary to the expectations based on the generally accepted laser ablation mechanism. The quadrilateral nanoholes could be an inherent morphology in the LTB and LBO crystals. The morphology of the quadrilateral holes in the LTB and LBO crystals is considered to reflect the continuous BO33− and/or BO45− network structure in their respective tetragonal or orthorhombic unit cells, in which self-tapped excitons are formed in an initial process under multiphoton excitation. In contrast, the SBO, LGB, LSB, CAS, and CSAS crystals and the LTB and LBO glasses exhibit circular nanoholes with subwavelength diameters independent of the laser polarization, the structure, or the composition. The isotropic morphology of nanoholes in these samples reflects the circular pattern of the Gaussian intensity profile of the focused laser beam.

Photoacoustic effect applied on model membranes and living cells: direct observation with multiphoton excitation microscopy and long-term viability analysis

The photoacoustic effect is generated when a variable light interacts with a strongly light-absorbing material. In water, it may produce hot bubbles and shock waves that could affect the integrity of nearby cellular membranes, opening transient pores (photoporation). In this study, we have evaluated the effect of pulsed laser-irradiated carbon nanoparticles (cNP) on model membranes and on Chinese hamster ovary (CHO) cells. Fluorescence lifetime measurements of calcein-loaded liposomes support the notion that the photoacoustic effect causes transient openings in membranes, allowing diffusion fluxes driven by gradient concentrations. With CHO cells, we have shown that this effect can induce either intracellular delivery of calcein, or release of cellular compounds. The latter process has been recorded live with multiphoton excitation microscopy during pulsed infrared laser irradiation. Calcein loading and cell viability were assayed by flow cytometry, measuring necrotic cells as well as those in early apoptosis. To further assess long-term cell recovery after the rather harsh treatment, cells were reseeded and their behaviour recorded for 48 h. These extended studies on cell viability show that pulsed laser cNP photoporation may be considered an adequate intracellular delivery technique only if employed with soft irradiation conditions (below 50 mJ/cm2).