Fluorescence lifetime imaging and Fourier transform infrared spectroscopy of Michelangelo's David

We developed a combined procedure for the analysis of works of art based on a portable system for fluorescence imaging integrated with analytical measurements on microsamples. The method allows us to localize and identify organic and inorganic compounds present on the surface of artworks. The fluorescence apparatus measures the temporal and spectral features of the fluorescence emission, excited by ultraviolet (UV) laser pulses. The kinetic of the emission is studied through a fluorescence lifetime imaging system, while an optical multichannel analyzer measures the fluorescence spectra of selected points. The chemical characterization of the compounds present on the artistic surfaces is then performed by means of analytical measurements on microsamples collected with the assistance of the fluorescence maps. The previous concepts have been successfully applied to study the contaminants on the surface of Michelangelo's David. The fluorescence analysis combined with Fourier transform infrared (FT-IR) measurements revealed the presence of beeswax, which permeates most of the statue surface, and calcium oxalate deposits mainly arranged in vertical patterns and related to rain washing.     

Fluorescence lifetime imaging with near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.     

Spectral characterization of Yb-doped silica layers for high power laser applications

Thick silica layers, doped with rare-earth elements are required as active media for high power waveguide lasers and amplifiers. In this work, Yb/Al-codoped silica particles were deposited on pure silica wafers, followed by high temperature sintering and post-sinter laser annealing treatment. The optical properties of the layers were monitored at different stages of the process using transmission spectrometry in the near IR to UV range, micro-Raman spectroscopy, fluorescence spectrum, and decay measurements. Evolution of the Yb³⁺ ion fluorescence and stabilization of the Si:O bonds as a result of the sintering process were observed.Measurements of 30 μm thick layers showed high Yb absorption of 500 dB/m at 980 nm. The fluorescence lifetime was close to 1 ms and the propagation loss was less than 20 dB/m at 633 nm, currently limited by the measurement system. The results show that a potential material for high power applications has been achieved.     

Experimental verification of a theory for the time-resolved fluorescence spectroscopy of thick tissues

Fluorescence spectroscopy provides potential contrast enhancement for near-infrared tissue imaging and physiologically correlated spectroscopy. We present a fluorescence photon migration model and test its quantitative predictive capabilities with a frequency-domain measurement that involves a homogeneous multiple-scattering tissue phantom (with optical properties similar to those of tissue in the near infrared) that contains a fluorophore (rhodamine B). After demonstrating the validity of the model, we explore its ability to recover the fluorophore's spectral properties from within the multiple-scattering medium. The absolute quantum yield and the lifetime of the fluorophore are measured to within a few percent of the values measured independently in the absence of scattering. Both measurements are accomplished without the use of reference fluorophores. In addition, the model accurately predicts the fluorescence emission spectrum in the scattering medium. Implications of these absolute measurements of lifetime, quantum yield, concentration, and emission spectrum from within multiple-scattering media are discussed.     

Polarized spectroscopic properties of a potential self-frequency doubling crystal, Nd:BaCaBO3F

In this paper, the polarized absorption and fluorescence spectra of Nd³⁺ ion in the BaCaBO₃F non-linear optical crystal have been investigated at room temperature. The Judd-Ofelt theory, extended to anisotropic system, has been applied to calculate the phenomenological intensity parameters, spontaneous transition probabilities, branching ratios and radiative lifetimes. The maximum emission cross-sections have been calculated to be 7.22 × 10⁻²⁰ cm² and 15.21 × 10⁻²⁰ cm² at 1068 nm for π- and σ-polarization using the Füchtbauer-Ladenburg (F-L) equation. The fluorescence lifetime of ⁴F_3/2 manifold is equal to 57.7 μs, and the quantum efficiency is 26.8%, which is higher than those of most of other borates. The potential of Nd³⁺:BaCaBO₃F as a new self-frequency doubling laser crystal has also been evaluated preliminarily. Appreciable self-absorption at the second-harmonic wavelength of Nd³⁺:BaCaBO₃F crystal should be considered for further laser operations.     

Broadband amplified spontaneous emission fibre source near 2 μm using resonant in-band pumping

Broadband amplified spontaneous emission (ASE) at a peak wavelength of 1.85?μm from Tm:Ho-doped silica fibre sources is reported, using in-band pumping from a Yb:Er co-doped fibre laser operating at 1.61?μm. A maximum total output power from both ends of greater than 80?mW with slope efficiency >14% has been achieved. An ASE bandwidth at FWHM of near 70?nm was measured from the forward signal for the output power from 2–40?mW. The total fluorescence spectral width is >200?nm. This broadband ASE source has potential applications in optical metrology, fibre sensors, loss and dispersion tests on optical fibres, spectroscopy and medical imaging, including optical coherence tomography. A review of different pumping schemes in generating ASE around 1.9?μm in the Tm-doped fibre is given. Also the nature of three-level ASE and in-band pumping of Tm:Ho-doped silica fibre are also discussed. The results in this paper confirm that in-band pumped Tm-silica fibre is a route to achieve high power broadband output around 1.8?μm.     

Spectroscopic analysis and the effects of color centers on the laser performance of Nd: CaZn2Y2Ge3O12

Spectroscopic and EPR investigations of Nd³⁺-doped CaZn₂Y₂Ge₃O₁₂ (CAZGAR) have been performed. The absorption, fluorescence, excitation spectra and fluorescence lifetime have been measured at room temperature. The Judd-Ofelt theory has been applied to the measured optical absorption intensities to predict the radiative decay rates, branching ratios, and peak stimulated emission cross section from the metastable ⁴F_3/2 state to the ⁴I_9/2 manifold. The fluorescence lifetime of the ⁴F_3/2 level of Nd³⁺ at low concentration in this host was measured to be 285 ± 10 μs, which is longer than that for Nd³⁺: YAG. Color centers located at zinc octahedral sites have been produced in these crystals by ultraviolet irradiation and have been detected by EPR techniques. The effects of the color centers on the potential laser characteristics of this materials are discussed.     

Highly-sensitive multiplexed <Emphasis Type="Italic">in vivo</Emphasis> imaging using pegylated upconversion nanoparticles

Lanthanide-based upconversion nanoparticles (UCNPs) have been widely explored in various fields, including optical imaging, in recent years. Although earlier work has shown that UCNPs with different lanthanide (Ln³⁺) dopants exhibit various colors, multicolor-especially in vivo multiplexed biomedical imaging-using UCNPs has rarely been reported. In this work, we synthesize a series of UCNPs with different emission colors and functionalize them with an amphiphilic polymer to confer water solubility. Multicolor in vivo upconversion luminescence (UCL) imaging is demonstrated by imaging subcutaneously injected UCNPs and applied in multiplexed in vivo lymph node mapping. We also use UCNPs for multicolor cancer cell labeling and realize in vivo cell tracking by UCL imaging. Moreover, for the first time we compare the in vivo imaging sensitivity of quantum dot (QD)-based fluorescence imaging and UCNP-based UCL imaging side by side, and find the in vivo detection limit of UCNPs to be at least one order of magnitude lower than that of QDs in our current non-optimized imaging system. Our data suggest that, by virtue of their unique optical properties, UCNPs have great potential for use in highly-sensitive multiplexed biomedical imaging.      

Fabrication and characterisation of neodymium-doped silica glass by sol-gel process

 Neodymium-doped silica glass possesses many properties ideal for high-power laser applications. These include low thermal expansion coefficient, high temperature stability, and low nonlinear index of refraction. For the first time, the sol-gel process has been successfully employed to prepare highly doped neodymia silica glass, up to 5wt% Nd for homogeneous dopant distribution. The optical characteristics of the silica glass and its in-process gel, including the UV/VIS absorption spectra, infrared absorption spectra, fluorescence emission spectrum and the fluorescence lifetime, are measured and analyzed. The structure of the sample is also characterized by means of XRD and SEM. A porous gel is observed to have formed when the heat treatment temperature reaches 300^oC. Wavelength shifts in the absorption peak corresponding to the ⁴I_9/2 to ⁴F_5/2 transition have been observed during the densification process and for different Nd weig ht contents. The FTIR spectra have shown that high temperature heat treatment can greatly reduce the amount of OH groups and organic residue in the silica. We have also shown that a high OH content contributes to weak fluorescence intensity and short fluorescence lifetimes. Received: 3 March 1997 / Accepted: 27 May 1997     

Facile and Scalable Preparation of Fluorescent Carbon Dots for Multifunctional Applications

The synthesis of fluorescent nanomaterials has received considerable attention due to the great potential of these materials for a wide range of applications, from chemical sensing through bioimaging to optoelectronics. Herein, we report a facile and scalable approach to prepare fluorescent carbon dots (FCDs) via a one-pot reaction of citric acid with ethylenediamine at 150 °C under ambient air pressure. The resultant FCDs possess an optical bandgap of 3.4 eV and exhibit strong excitation-wavelength-independent blue emission (λ_Em = 450 nm) under either one- or two-photon excitation. Owing to their low cytotoxicity and long fluorescence lifetime, these FCDs were successfully used as internalized fluorescent probes in human cancer cell lines (HeLa cells) for two-photon excited imaging of cells by fluorescence lifetime imaging microscopy with a high-contrast resolution. They were also homogenously mixed with commercial inks and used to draw fluorescent patterns on normal papers and on many other substrates (e.g., certain flexible plastic films, textiles, and clothes). Thus, these nanomaterials are promising for use in solid-state fluorescent sensing, security labeling, and wearable optoelectronics.     

Superradiance and lasing properties of new two-photon absorption dye DEASPS

Pumped by picosecond pulses from a Nd:YAG laser, a new lasing dye, trans-4-[4′-(N,N-diethylamino)styryl]-N-methyl pyridinium methyl sulfate (abbreviated to DEASPS), shows both intense superradiance and strong lasing properties in benzyl alcohol solution. By using streak camera systems, the superradiance and lasing can be distinguished both spectrally and temporally. It has been found that the peak wavelength of lasing is at 620 nm with a red-shift of about 12 nm to the superradiance wavelength. The lasing pulse shows an oscillatory effect that it is not found in the superradiance pulse. The fluorescence lifetime is 529 ± 40 ps and the effective molecular two-photon absorption is (1.25 ± 0.1) × 10^?48 cm⁴ ·s·photon ^?1, measured using a nonlinear transmittance method. This dye shows effective optical limiting of the pumping wavelength.     

Digital micromirror device as a spatial illuminator for fluorescence lifetime and hyperspectral imaging

Time-domain fluorescence lifetime imaging (FLIM) and hyper-spectral imaging (HSI) are two advanced microscopy techniques widely used in biological studies. Typically both FLIM and HSI are performed with either a whole-field or raster-scanning approach, which often prove to be technically complex and expensive, requiring the user to accept a compromise among precision, speed, and spatial resolution. We propose the use of a digital micromirror device (DMD) as a spatial illuminator for time-domain FLIM and HSI with a laser diode excitation source. The rather unique features of the DMD allow both random and parallel access to regions of interest (ROIs) on the sample, in a very rapid and repeatable fashion. As a consequence both spectral and lifetime images can be acquired with a precision normally associated with single-point systems but with a high degree of flexibility in their spatial construction. In addition, the DMD system offers a very efficient way of implementing a global analysis approach for FLIM, where average fluorescence decay parameters are first acquired for a ROI and then used as initial estimates in determining their spatial distribution within the ROI. Experimental results obtained on phantoms employing fluorescent dyes clearly show how the DMD method supports both spectral and temporal separation for target identification in HSI and FLIM, respectively.     

Yb3+ ion in calcium niobium gallium garnet crystals: Nearest neighbor environment and optical spectra

We have studied inhomogeneously broadened optical spectra of the Yb³⁺ ion in calcium niobium gallium garnet crystals using selective laser excitation, time-resolved luminescence spectroscopy, and ² F _5/2 luminescence decay measurements under excitation in different portions of the absorption spectrum. We have identified spectral features due to individual optical centers differing in excitation and luminescence wavelengths and excited-state lifetime. Tentative structures of the optical centers responsible for the inhomogeneous broadening of the spectra are described.     

Three-dimensional imaging of objects embedded in turbid media with fluorescence and Raman spectroscopy

We present a single-ended technique for three-dimensional imaging of objects embedded in a turbid medium by the use of time-resolved fluorescence emission or Raman scattering. The technique uses the earliest arriving photons, which we show are not sensitive to the relatively long fluorescence lifetime, and thus can be used to extract the desired spatial information accurately, even at a distance equivalent to 100 mean free paths. The results also demonstrate the feasibility and the potential of one's combining time-resolved optical tomography with fluorescence or Raman spectroscopy to localize and identify the embedded objects. This technique may be valuable for the diagnosis of disease in highly scattering human tissue because it can provide spatial and biochemical information about the composition of embedded lesions.     

Spectroscopic characterization of red perylimide/surfactant nanocomposites

Novel photoluminescent materials formed by some selected surfactants, metal derivatives of bis(2-ethylhexyl) sulfosuccinate (M(AOT) _n ; M = Na⁺, Co²⁺, Er³⁺ and Yb³⁺), bis(2-ethylhexyl) amine (BEEA), bis(2-ethylhexy1) phosphoric acid (HDEHP) and a 1:1 BEEA/HDEHP mixture, doped with the red perylimide (ROT-300) have been prepared, and their optical properties have been tested by absorption spectroscopy and steady state and time-resolved fluorescence. Experimental results show spectral shifts of the typical ROT-300 absorption and fluorescence bands with respect to that in apolar solvent medium. Data analysis leads consistently to attribute this feature mainly to the freezing of the diffusive movement of the dye molecules confined in the nanodomains of the surfactant liquid crystals, whilst minor effects can be due to interaction with the surfactant polar groups. Potentialities of these novel luminescent nanostructured composites as dye lasers, optical amplifiers and solar concentrators have been highlighted. In particular, under optical pumping using a pulse laser, amplified spontaneous fluorescence emission of the ROT-300/HDEHP system above an excitation energy threshold value of about 725 mJ cm⁻² was observed.     

Two-photon-induced fluorescence from the phycoerythrin protein

Two-photon-induced fluorescence is observed from the photodynamic phycobiliprotein phycoerythrin. Temporal, spectral, and intensity-dependent properties of the two-photon-induced fluorescence emission from phycoerythrin excited by a 1.06-mum laser beam are reported. The measured two-photon absorption cross section of phycoerythrin is an order of magnitude larger than that of Rhodamine 6G. The potential applications of phycobiliproteins for two-photon-induced fluorescence for microscopy of three-dimensional biological samples and three-dimensional optical memory are discussed.     

Surface assessment of CaF2 deep-ultraviolet and vacuum-ultraviolet optical components by the quasi-Brewster angle technique

The requirements for optical components have drastically increased for the deep-ultraviolet and vacuum-ultraviolet spectral regions. Low optical loss, high laser damage threshold, and long lifetime fluoride optics are required for microlithographic applications. A nondestructive quasi-Brewster angle technique (qBAT) has been developed for evaluating the quality of optical surfaces including both top surface and subsurface information. By using effective medium approximation, the negative quasi-Brewster angle shift at wavelengths longer than 200 nm has been used to model the distribution of subsurface damage, whereas the positive quasi-Brewster angle shift for wavelengths shorter than 200 nm has been explained by subsurface contamination. The top surface roughness depicted by the qBAT is consistent with atomic force microscopy measurements. The depth and the microporous structure of the subsurface damage measured by the qBAT has been confirmed by magnetorheological finishing. The technique has been extended to evaluate both polished and antireflection-coated CaF(2) components.     

Crystal growth and spectral properties of Pr:La2(WO4)3

Pr³⁺-doped La₂(WO₄)₃ single crystal with dimensions up to Ø 20 mm × 35 mm has been grown by the Czochralski method. The structure of the Pr³⁺:La₂(WO₄)₃ crystal was determined by the X-ray powder diffraction and the Pr³⁺ concentration in this crystal was determined. The absorption and fluorescence spectra of Pr³⁺:La₂(WO₄)₃ crystal were measured at room temperature, and the fluorescence lifetime of main emission multiplets were estimated from the recorded decay curves. The spectral properties related to laser performance of the crystal were evaluated.     

Technique for measurement of fluorescence lifetime by use of stroboscopic excitation and continuous-wave detection

A study of the practicality a simple technique for obtaining time-domain information that uses continuous wave detection of fluorescence is presented. We show that this technique has potential for use in assays for which a change in the lifetime of an indicator occurs in reaction to an analyte, in fluorescence resonance energy transfer, for example, and could be particularly important when one is carrying out such measurements in the scaled-down environment of a lab on a chip (biochip). A rate-equation model is presented that allows an objective analysis to be made of the relative importance of the key measurement parameters: optical saturation of the fluorophore and period of the excitation pulse. An experimental demonstration of the technique that uses a cuvette-based analysis of a carbocyanine dye and for which the excitation source is a 650 nm wavelength, self-pulsing AlGaInP laser diode is compared with the model.