Excitation Path Research Articles

Dual-view light-sheet imaging through a tilted glass interface using a deformable mirror.

Light-sheet microscopy has become indispensable for imaging developing organisms, and imaging from multiple directions (views) is essential to improve its spatial resolution. We combine multi-view light-sheet microscopy with microfluidics using adaptive optics (deformable mirror) which corrects aberrations introduced by the 45o-tilted glass coverslip. The optimal shape of the deformable mirror is computed by an iterative algorithm that optimizes the point-spread function in two orthogonal views. Simultaneous correction in two optical arms is achieved via a knife-edge mirror that splits the excitation path and combines the detection paths. Our design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experiments and high-content screening.

Polarization-dependent laser resonance ionization of beryllium

Using TRIUMF's off-line laser ion source test stand with a system of tunable titanium sapphire lasers, the polarization dependence of laser resonance ionization has been investigated using beryllium. A significant polarization dependence was observed for the excitation path 1S0 → 1P1∘ → 1S0, which are typical transitions for alkaline-earth and alkaline-earth like elements. This polarization dependence was further verified on Be radioactive isotopes at TRIUMF's isotope separator and accelerator facility (ISAC). Laser polarization was proven to be an important parameter in operating resonance ionization laser ion sources (RILIS).The polarization spectroscopy was performed off-line both on the 2p2 1S0 autoionizing (AI) state and high-n Rydberg states of the 2sns1S0 and 2snd1D2 series. The energy of the 2p2 1S0 AI state and ionization potential (IP) of beryllium were extracted as 76,167(6) cm−1 and 75,192.59(3) cm−1. Polarization spectroscopy can be used to determine the J values of newly found states in in-source spectroscopy of the complex/radioactive alkaline-earth like elements such as Ra, Sm, Yb, Pu and No.

Excitation and emission dynamics of a single photon emitting InGaN quantum dot in a photonic horn structure

Abstract We present a detailed analysis of the second-order correlation behavior of photons emitted from an InGaN QD in a photonic horn structure in order to discuss the excitation, emission, and environmental dynamics that impact the emission. We report the observation of a clear saturation in the antibunching decay rate as the excitation power is increased. We describe the saturation with a simple model describing a saturating excitation path, possibly via inefficient carrier relaxation into the QD emitting state.

Wavefront correction for adaptive optics with reflected light and deep neural networks

Light scattering and aberrations limit optical microscopy in biological tissue, which motivates the development of adaptive optics techniques. Here, we develop a method for wavefront correction in adaptive optics with reflected light and deep neural networks compatible with an epi-detection configuration. Large datasets of sample aberrations which consist of excitation and detection path aberrations as well as the corresponding reflected focus images are generated. These datasets are used for training deep neural networks. After training, these networks can disentangle and independently correct excitation and detection aberrations based on reflected light images recorded from scattering samples. A similar deep learning approach is also demonstrated with scattering guide stars. The predicted aberration corrections are validated using two photon imaging.

Sector Piezoelectric Sensor Array Transmitter Beamforming MUSIC Algorithm Based Structure Damage Imaging Method.

Elastic-wave-based structural health monitoring technology has a broad application potential for its sensitivity and ability to achieve regional monitoring. For structures with large damping and specific shapes, the traditional damage monitoring method is limited by the sensor arrangement area and affected by low signal-to-noise ratios, so it is difficult to accurately locate the damage in a structure. To solve this problem, this paper proposed a damage monitoring method based on a sector piezoelectric sensor array for multiple signal classification algorithm. By arranging two sector piezoelectric sensor arrays that are suitable for a specific structure, the damage scattering array signal under the multi-excitation source was obtained and synthesized, the signal-to-noise ratios were improved, and the damage location accuracy was thus improved. The effectiveness of the method was verified by monitoring the damage in a circular bonded structure with a metal ring. Compared with the damage localization methods based on the traditional single excitation source multiple signal classification algorithm, path imaging and delay-sum imaging, this method can achieve better damage location and has a higher localization accuracy.

Precisely calibrated and spatially informed illumination for conventional fluorescence and improved PALM imaging applications

Spatial light modulation using cost efficient digital micromirror devices (DMD) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, precise illumination by DMDs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMD and camera coordinate systems caused by optical components in the excitation and emission path. Here we develop a fast and easy to implement calibration procedure that determines these distortions and matches the DMD and camera coordinate system with a precision below the optical diffraction limit. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to a rigid transformation allowed by manual alignment of the DMD. We first demonstrate the application of our precisely calibrated light modulation by performing a proof of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP in budding yeast (S. cerevisiae). Next, we develop a spatially informed photoactivation approach for SMLM in which only regions of the cell that contain photoactivatable fluorescent proteins are selected for photoactivation. The reduced exposure of the cells to 405 nm light increased the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in cell morphology. As a result, the mean number of reliable single-molecule localizations was also significantly increased by 28%. Since the localization precision and the ability for single-molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatially informed photoactivation significantly improves the quality of SMLM images and single-molecule tracking data. Our precise calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.

Investigating Polarisation Effects in a Confocal Total Internal Reflection-Supercritical Angle Fluorescence (TIR-SAF) Geometry with Sample Scanning

Total internal reflection microscopy is a technique ideal for imaging the cell membrane, as it allows for selective excitation of fluorophores at or near the coverglass. When a beam enters a high NA objective off-axis, it produces an evanescent field, the penetration depth of which is dependent on the refractive index difference between the glass substrate and sample. A pitfall of this method is that sample refractive index varies across a cell surface and organelles, causing unwanted light refraction and scattering. By introducing a Bertrand lens into the detection optics, the critical angle on the back focal plane can be imaged, which allows to calculate the sample refractive angle, and emission from near-membrane and deeper fluorophores can be separated, too. Here, we use a scanning TIR-SAF system to measure the local cellular refractive index across the cell's “footprint” region. The excitation path contains a radial polarizer, and two axicon lenses to form evanescent Bessel beam illumination, allowing for an excitation “needle”. Tightly focused radially and azimuthally polarized Bessel beams are desirable as they allow for a smaller excitation spot size. However, neither effects of azimuthally nor radially polarized excitation on evanescent field formation have been characterized. We built and characterized a confocal TIR-SAF system, and the effects of polarization were assessed.

RLS iOH: ExoMars Raman laser spectrometer optical head bread board to flight model design and performance evolutions

AbstractRaman Laser Spectrometer (RLS) is the Pasteur Payload instrument of the ExoMars mission that will perform Raman spectroscopy for the first time in a planetary space mission. RLS main units are: SPU (SPectrometer Unit), iOH (internal Optical Head), and ICEU (Instrument Control and Excitation Unit), that includes the laser for samples excitation purposes.The iOH focuses the excitation laser into the crushed samples (located at the ALD, Analytical Laboratory Drawer, carrousel) through the excitation path, and collects the Raman emission from the sample (collection path). Its original design presented a high laser trace reaching to the SPU detector, and although a certain level was required for instrument calibration, the found level was expected to be capable of degrading the acquired spectra confounding some Raman peaks. So, the iOH optical and opto‐mechanical designs were needed to be updated from the BB (Bread Board) to the engineering and qualification model (iOH EQM), in order to fix the desired amount of laser trace, and after the fabrication and the commitment of the commercial elements, the assembly and integration verification (AIV) process was carried out. Considering the results obtained during the EQM integration verification and the first functional tests, the RLS calibration target (CT) emission analysis, additional changes were found to be required for the Flight Model, FM.In this paper, the RLS iOH designs and functional tests evolutions for the different models are summarized, focusing on the iOH AIV process and emphasizing on the iOH performance evaluation (by using CT spectra) from the re‐design activities.

Dynamic Double-Slit Experiment in a Single Atom.

A single-atom "double-slit" experiment is realized by photoionizing rubidium atoms using two independent low power lasers. The photoelectron wave of well-defined energy recedes to the continuum either from the 5P or 6P states in the same atom, resulting in two-path interference imaged in the far field using a photoelectron detector. Even though the lasers are independent and not phase locked, the transitions within the atom impart the phase relationship necessary for interference. The experiment is designed so that either 5P or 6P states are excited by one laser, before ionization by the second beam. The measurement cannot determine which excitation path is taken, resulting in interference in wave-vector space analogous to Young's double-slit studies. As the lasers are tunable in both frequency and intensity, the individual excitation-ionization pathways can be varied, allowing dynamic control of the interference term. Since the electron wave recedes in the Coulomb potential of the residual ion, a quantum model is used to capture the dynamics. Excellent agreement is found between theory and experiment.

Compact integrated magnetometer based on nitrogen-vacancy centres in diamond

We demonstrate an integrated and miniaturised magnetic field sensor based on the negatively charged nitrogen-vacancy centres (NV−) in diamond. The compact device includes all optical components, both for the optical excitation path and for the detection of the emitted fluorescence signal. We experimentally verify that it enables optically detected magnetic resonance (ODMR) measurements and we specify noise and sensitivity of the magnetometer. The minimal detectable magnetic field of the device is ≈ 1 μT for a given integration time of 1 ms, which is approximately one order of magnitude larger than its photon shot-noise limit. It has the significant advantage over traditional setups using NV− centres (including a laser and a complex optical system) that the specific construction volume is about 2.9 cm3 with a total power consumption of ≈ 1.5 W, which enables the device for a wide range of industrial sensing applications.

Preparation of PbF2:Ho3+, Er3+, Yb3+ phosphors and its multi-wavelength sensitive upconversion luminescence mechanism

In this paper, PbF2: Ho3+, Er3+, Yb3+ phosphors are prepared by a high-temperature solid-state method. The crystal structure acquired by X ray powder diffractometer shows that the resultant phosphor is a pure cubic PbF2 crystal. The upconversion (UC) emission spectra of samples are performed by a fluorescence spectrophotometer coupled with lasers of 980 nm, 1064 nm and 1550 nm, presenting the green emission in the range of 541∼548 nm and red emission in the range of 653–667 nm, corresponding to the excitation path: Ho3+: 4F9/2→4I15/2, Er3+: 4S3/2→4I15/2, Er3+: 4F9/2→4I15/2 and Ho3+: 5F5→5I8. Finally, the UC luminescence mechanism is analyzed under excitation of different wavelengths. The result shows that the phosphors have a promising prospect for application in anti-counterfeiting technologies and solar cell field.

Surface plasmon-assisted microscope.

Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100- to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 µm2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

Summation of two illusions of extent

In the present visual psychophysical study, the Oppel‑Kundt and and Müller‑Lyer illusion magnitudes were measured separately (by single figures) and in combination (by two patterns superposed spatially). Data for 30 subjects revealed extensive variability both for the separate and combined illusion strength. Nevertheless, the effect of addition of the perceived length distortions was established. The combined illusions were significantly stronger than the separate ones. Dynamics of the misperceptions summation was studied by varying length of the Müller‑Lyer wings in the superposed stimuli. According to the experimental data obtained, the two misperceptions in length occurred and combined into sensory response varying in dependence on the spatial parameters of the superposed stimuli and on the individual experimental accomplishment. The data supported an explanation for the origin of the filled/unfilled illusion: overestimate of a filled interval length developed due to the spatiotemporal integration along a continuous excitation path elicited by the real or imaginary contours of the filling.

Physics of Efficiency Droop in GaN:Eu Light-Emitting Diodes

The internal quantum efficiency (IQE) of an electrically-driven GaN:Eu based device for red light emission is analyzed in the framework of a current injection efficiency model (CIE). The excitation path of the Eu+3 ion is decomposed in a multiple level system, which includes the carrier transport phenomena across the GaN/GaN:Eu/GaN active region of the device, and the interactions among traps, Eu+3 ions and the GaN host. The identification and analysis of the limiting factors of the IQE are accomplished through the CIE model. The CIE model provides a guidance for high IQE in the electrically-driven GaN:Eu based red light emitters.

Luminescence induced by electrons outside zinc oxide nanoparticles driven by intense terahertz pulse trains

We investigated the behaviours of electrons from ZnO nanoparticles via a strong terahertz field. Luminescence from ZnO nanoparticles and surrounding nitrogen molecules was observed when the nanoparticles were irradiated with a terahertz free-electron laser (FEL). These excitations arose from the collision of electrons released via field electron emission with the ZnO nanoparticles and neighbouring nitrogen molecules. The strong excitation frequency dependence of the luminescence reflected the kinetic energy and trajectory of electrons outside the nanoparticles. We also observed spectral changes in the luminescence during macropulses of the FEL, even though the carrier lifetime of the nanoparticles was shorter than the interval between the micropulses. These changes were caused by the nanoparticles becoming charged due to electron emission, resulting in the electrons being re-emitted outside the nanoparticles. The electrons outside the nanoparticles were accelerated more efficiently by the terahertz field than the electrons inside the nanoparticles, and thus the motion of these exterior electrons provided a new excitation path.

Advanced Absorption Correction for 3D Elemental Images Applied to the Analysis of Pearl Millet Seeds Obtained with a Laboratory Confocal Micro X-ray Fluorescence Spectrometer.

We present a novel absorption correction approach for elemental distribution images obtained with a laboratory confocal micro X-ray fluorescence spectrometer. The procedure is suited especially for biological samples, as a constant dark matrix with a varying minor or trace element distribution is assumed. The constant absorption in the sample is extracted from depth dependent measurements. By using the concept of an effective excitation energy, depth-dependent, and element-specific excitation energy values are calculated. For each voxel of a full 3D measurement, a correction is performed taking into account the actual number of voxels in the excitation and detection path. As proof of concept, the embryonic region of pearl millet seeds is investigated. Data are measured from the top and bottom side, resulting in a good agreement after the application of the absorption correction procedure. The distribution of elemental micronutrients is compared in seeds of two pearl millet genotypes. The corrected images illustrate different localization patterns of the micronutrient elements in pearl millet seed tissues.

Ultrasmall, water dispersible, TWEEN80 modified Yb:Er:NaGd(WO4)2 nanoparticles with record upconversion ratiometric thermal sensitivity and their internalization by mesenchymal stem cells

This work presents the synthesis by coprecipitation of diamond shaped Yb:Er:NaGd(WO4)2 crystalline nanoparticles (NPs) with diagonal dimensions in the 5–7 nm × 10–12 nm range which have been modified with TWEEN80 for their dispersion in water, and their interaction with mesenchymal stem cells (MSCs) proposed as cellular NP vehicles. These NPs belong to a large family of tetragonal Yb:Er:NaT(XO4)2 (T = Y, La, Gd, Lu; X = Mo, W) compounds with green (2H11/2 + 4S3/2 → 4I15/2) Er-related upconversion (UC) efficiency comparable to that of Yb:Er:β-NaYF4 reference compound, but with a ratiometric thermal sensitivity (S) 2.5–3.5 times larger than that of the fluoride. At the temperature range of interest for biomedical applications (∼293–317 K/20–44 °C) S = 108–118 × 10−4 K−1 for 20 at%Yb:5 at%Er:NaGd(WO4)2 NPs, being the largest values so far reported using the 2H11/2/4S3/2 Er intensity ratiometric method. Cultured MSCs, incubated with these water NP emulsions, internalize and accumulate the NPs enclosed in endosomes/lysosomes. Incubations with up to 10 μg of NPs per ml of culture medium maintain cellular metabolism at 72 h. A thermal assisted excitation path is discussed as responsible for the UC behavior of Yb:Er:NaT(XO4)2 compounds.

Calculation model of fluorescence yield index for excitation path system of crude oils

For the fluorescence measurement, the detailed study of the photophysical and hydrochemical processes had always been ignored. To address these issues, we derived a path equation, which is a probabilistic combination of fluorescence excitation mechanism and the absorption of excitation and emission intensity. Based on the equation, a characteristic parameter, fluorescence yield index (FYI), was deduced. The FYI is related to the light propagation paths and is well suited for physical modeling. Fluorescence emission spectra have been measured from two diverse crude oils by measuring the exciting light. The FYI of samples accurately described the sum of fluorescence yield of the measurement point, and can feedback the best experimental concentration of crude oils. For the same concentration, we can use the FYI to distinguish the different kinds of oils. The theoretical and experimental analyses verify that the path equation and FYI are the efficient way to physically model light–medium interactions.

Tuning the fields focused by a high NA lens using spirally polarized beams (Invited Paper)

We show the power of spirally polarized doughnut beams as a tool for tuning the field distribution in the focus of a high numerical aperture (NA) lens. Different and relevant states of polarization as well as field distributions can be created by the simple turning of a λ/2 retardation wave plate placed in the excitation path of a microscope. The realization of such a versatile excitation source can provide an essential tool for nanotechnology investigations and biomedical experiments.