Processing of Fluorescence Lifetime Image Using Modified Phasor Approach: Homo-FRET from the Acceptor

As the hardware of FLIM technique becomes mature, the most important criterion for FLIM application is the correct interpretation of its data. In this research, first of all, a more orthogonal phasor approach, called as Modified Phasor Approach (MPA), is put forward. It is a way to calculate the lifetime of the complex fluorescent process, and a rule to measure how much the fluorescence process deviates from single exponential decay. Secondly, MPA is used to analysis the time-resolved fluorescence processes of the transfected CHO-K1 Cell lines expressing adenosine receptor A₁R tagged by CYP and YFP, measured in the channel of the acceptor. The image of the fluorescence lifetime and the multiplication of the fluorescence lifetime and deviation from single exponential decay reveal the details of the Homo-FRET. In one word, MPA provides the physical meaning in its whole modified phasor space, and broadens the way for the application of the fluorescence lifetime imaging.     

荧光寿命显微成像技术及应用的最新研究进展

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响,荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点,近年来已被广泛应用于生物医学等领域.然而,尽管FLIM的发明和发展已历经数十年时间,其在实际应用中仍然面临着许多挑战.例如,其成像分辨率受衍射极限限制,而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来,相关硬件和软件的快速发展及其与其他光学技术的结合,极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点,在此基础上概述了该技术的最新研究进展,包括其成像性能的提升和在生物医学应用中的研究现状,详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力,以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用,最后对其未来发展趋势进行了展望.     

Double-heterostructure ridge-waveguide GaAs/AlGaAs phase modulator for 780 nm lasers

Results of a GaAs/AlGaAs-based phase modulator designed for an operating wavelength of 780 nm are presented for the first time. The modulator is based on a P-p-i-n-N double heterostructure and features a waveguide with a vertical W-shaped index profile optimized for low propagation losses and a ridge waveguide for lateral index-guiding. A phase modulation efficiency of 12°/V mm is reported. The propagation losses are determined to about 1.4 dB/cm.     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca²⁺-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca²⁺ concentration image using anin vitro-determined calibration curve. The Ca²⁺ concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.     

Calcium imaging using fluorescence lifetimes and long-wavelength probes

We describe imaging of calcium concentrations using the long-wavelength Ca²⁺ indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.     

Diode-Side-Pumped Passively Q-Switched Mode-Locked 532 nm Laser with a Nd:YAG/Cr4+:YAG/YAG Composite Crystal

We present a diode-side-pumped Q-switched mode-locked (QML) laser operating at 532 nm for the first time employing a composite crystal Nd:YAG/Cr⁴⁺:YAG/YAG and a KTP. The experimental results show that, using a suitable cavity and crystals, one can obtain high-quality QML laser output at 532 nm with a side-pumped system. We measure and analyze the QML performance of the fundamental frequency laser and the green laser. Under a pump power of 127 W, we obtain a QML laser operating at 532 nm with an average power of 4.97 W and a repetition rate of 0.2 GHz for the mode-locked pulses; the corresponding depth of modulation is close to 100%.     

Different preparation methods and characterization of magnetic maghemite coated with chitosan

In this report, we investigated the so-called plasmonic platforms prepared to target ultra-short fluorescence and accurate instrumental response function in a time-domain spectroscopy and microscopy. The interaction of metallic nanoparticles with nearby fluorophores results in the increase of the dye fluorescence quantum yield, photostability and decrease of the lifetime parameter. The mentioned properties of platforms were applied to achieve a picosecond fluorescence lifetime (21 ps) of erythrosin B, used later as a better choice for deconvolution of fluorescence decays measured with “color” sensitive photo-detectors. The ultra-short fluorescence standard based on combination of thin layers of silver film, silver colloidal nanoparticles (about 60 nm in diameter), and top layer of erythrosin B embedded in 0.2 % poly(vinyl) alcohol. The response functions were monitored on two photo-detectors; microchannel plate photomultiplier and single photon avalanche photodiode as a Rayleigh scattering and ultra-short fluorescence. We demonstrated that use of the plasmonic base fluorescence standard as an instrumental response function results in the absence of systematic error in lifetime measurements and analysis.     

Time-lapse in situ fluorescence lifetime imaging of lipid droplets in differentiating 3T3-L1 preadipocytes with Nile Red

To study the mechanisms of and conditions for adipogenesis, an accurate in situ observation tool is necessary to monitor the quantity of intracellular neutral lipids in differentiating preadipocytes. Although conventional fluorescence intensity imaging is a powerful tool for observing the formation and growth of an individual lipid droplet, it suffers from photobleaching and ambiguous autofluorescence or background signals from cells. In this paper, we present a fluorescence lifetime imaging microscopy (FLIM) technique that has the potential to quantify the ratio of neutral to polar lipids in a cell. Measurement of time-lapse FLIM images of differentiating 3T3-L1 cells that contained the Nile Red (NR) probe showed that the average lifetime of NR decreased from 4 ns in preadipocytes to 3 ns in fully differentiated adipocytes after 10 days of differentiation. This large change in the lifetime of NR can be used to monitor the early stages of adipogenesis, even when the lipid droplet is too small to be identified with a conventional microscope.     

Nonadiabatic multi-step excitation for the blue–green light emission from dye grains induced by the near-infrared optical near-field

Blue–green light emission (wavelength range: 460–520 nm) from coumarin dye grains due to near-infrared excitation (CW, λ _ex=808 nm), based on the nonadiabatic excitation process induced by an optical near-field, was observed. A maximum frequency up-shift of 1.17 eV was confirmed. Based on the excitation intensity dependence, a light emission mechanism originating from a nonadiabatic three-step excitation was confirmed for the first time. The lifetime of the intermediate excited state was approximately 1.1–1.9 ps, and thus realization of frequency up-conversion with a rapid response can be expected.     

Mode-locked all-fiber ring laser based on broad bandwidth in-fiber acousto-optic modulator

Active mode-locking of long-cavity all-fiber ring laser based on broad bandwidth in-fiber acousto-optic modulator (AOM) is reported. The proposed AOM combines the advantages of intermodal coupling induced by standing flexural acoustic waves in a double-ended tapered fiber. We focus our attention on the effects of large tapered transitions to improve the bandwidth modulation. Our approach permits the implementation of broad modulation bandwidth (13 nm), high modulation depth (50 %), and low optical loss (1.1 dB) in an 80-μm configuration. The effects of the AOM in the laser performance are also investigated. Transform-limit optical pulses of 25 ps temporal width and 2.7 W peak power were obtained at 2.46 MHz repetition rate.     

Efficient electrochromic device based on sol–gel prepared WO<Subscript>3</Subscript> films

Tungsten oxide (WO₃) films were prepared on indium–tin oxide (ITO) glass by sol–gel method. The influence of annealing temperature on the structural, morphological, optical, electrochemical, and electrochromic properties has been investigated. The film annealed at 250 °C with an amorphous structure exhibits a noticeable electrochromic performance, such as the highest optical modulation of 58.5 % at 550 nm, high electrochemical stability, and excellent reversibility (Q _b/Q _c?=?96.3 %). An electrochromic (EC) device based on WO₃/NiO complementary structure shows improved performance. It exhibits high optical transmittance modulation of 62 % at 550 nm, excellent cycling stability, and relatively fast electrochromic response time (10 s for coloration and 19 s for bleaching).     

Enhancing the performance of thin film CdS/PbS photovoltaic solar cells

This work investigates dependence of the short-circuit current density, open-circuit voltage, fill factor and efficiency of a thin film CdS/PbS solar cell on thickness of transparent conductive oxide (TCO) layer, thickness of window layer (CdS), concentration of uncompensated acceptors (width of space-charge region), carrier lifetime in PbS and the reflectivity from metallic back contact. The effect of optical losses, front and rear recombination losses as well as the recombination losses on space-charge region are also considered in this study. As a result, by thinning the front contact layer indium tin oxide from 400 to 100 nm and window layer (CdS) from 200 to 100 nm it is possible to reduce the optical losses from 32 to 20%. The effect of electron lifetime on the internal and external quantum efficiency can be neglected at high width of the space-charge region. The maximum current density of 18.4 mA/cm² is achieved at wide space-charge region (concentration of uncompensated acceptors = 10¹⁵ cm^?3) and the longest lifetime (τ_n = 10^?6 s) where the optical and recombination losses are about 55%. The maximum efficiency of 5.17%, maximum open-circuit voltage of 417 mV and approximately fixed fill factor of 74% are yielded at optimum conditions such as: electron lifetime = 10^?6 s; concentration of uncompensated acceptors = 10¹⁶ cm^?3; thickness of TCO = 100 nm; thickness of CdS = 100 nm; velocity of surface and rear recombination = 10⁷ cm/s and thickness of absorber layer = 3 μm. When the reflectance from the back contact is 100%, the cell parameters improve and the cell efficiency records a value of 6.1% under the above conditions.     

Setup and performance of a streak camera apparatus for transient absorption measurements in the ns to ms range

We describe the setup and performance of an apparatus for simultaneous time- and spectrally resolved measurements of transient absorption. The key component in this apparatus is a streak camera, yielding, for every excitation of the sample, a two-dimensional data array with 512 × 512 data points. The apparatus covers the spectral range 350–750 nm and time windows ranging from 500 ns up to 10 ms. Due to the large dynamic range of the streak camera of 10,000:1 we obtain a multiplex factor of more than 100 compared with sequential measurements at individual wavelengths. This makes it possible to extract the maximum amount of information from small amounts of sample, e.g. light-sensitive proteins. We show that already a single pump probe pulse sequence can yield useful spectra in a 20-μs time range, and that 10 pump-probe pulse sequences yield good time constants from a global lifetime analysis. An iterative method is presented for the treatment of artifacts due to scattered excitation laser light or strong fluorescence. As an alternative to a global lifetime analysis we propose a maximum entropy-based inverse Laplace transform for analysis of the data. This results in a wavelength-dependent distribution of amplitudes p(k, λ) for all rate constants k accessible with a given time window. This analysis is model free and yields a direct visual evaluation of the uncertainties in the rate constants.     

A broad and tunable 250- to 430-nm source for microscopy and lifetime measurements by frequency doubling of a 78-MHz-picosecond white-light laser

Broadly tunable picosecond pulses in the UV for nonlinear microscopy and lifetime measurements are not yet readily available. Complex synchronously pumped optical parametric oscillators with subsequent frequency doubling are typically used. We show that direct second harmonic generation of a visible picosecond supercontinuum source at 78 MHz renders pulses easily tunable from 250 to 430 nm. We find that an unexpectedly large numerical aperture and the use of thick crystals increase the efficiency of the frequency doubling process dramatically. The observed spectral width and efficiency are nearly two orders of magnitude larger than predicted by conventional theory. With broadband achromatic doubling, a 130 nm wide spectrum is achieved. Pulse durations of 17–35 ps are found in the UV and an average power between 1 and 70 μW. This qualifies the setup for most UV-based microscopic investigations. As first application, the fluorescence lifetime of two differing conformations of 2-(2′-hydroxyphenyl) benzothiazole is measured.     

Small-angle neutron scattering study of the nano-sized features in an oxide dispersion-strengthened Fe12Cr alloy

Small-angle neutron scattering experiments, along with positron lifetime measurements and transmission electron microscopy observations, were performed on samples of an oxide dispersion-strengthened (ODS) Fe12Cr alloy and its non-ODS counterpart in order to characterize their nano-sized features. The nuclear and magnetic scattering data were analysed using the maximum entropy approach for obtaining the size distribution of the scattering centres in these materials. The positron annihilation results and the TEM information have made possible an interpretation of the volume distribution of the scattering centres having sizes below ~16 nm and their proper quantitative analyses. The smaller scattering centres in the ODS alloy exhibit distributions with modal values at ~6–7 and 12–14 nm. The peak at ~6–7 nm appears to be due to the overlapping of more than one type of scattering centres, while the one at ~12–14 nm can be exclusively attributed to the Y-rich centres. The quantitative analysis of the magnetic scattering data yields a volume fraction and number density of the Y-rich particles estimated in 0.70?±?0.03% and 0.77 × 10²² m^?3, respectively.     

A Feasible Add-On Upgrade on a Commercial Two-Photon FLIM Microscope for Optimal FLIM-FRET Imaging of CFP-YFP Pairs

Fluorescence lifetime imaging microscopy (FLIM) based on time-correlated single photon counting (TCSPC) is a widely used method for fluorescence resonance energy transfer (FRET). Here we report a feasible add-on approach to upgrade a commercial two-photon FLIM microscope into a single-photon FLIM microscope which provides optimal FLIM-FRET imaging of FRET pairs consisting of cyan fluorescent proteins (CFPs) as the donor and yellow fluorescent proteins (YFPs) as the acceptor. The capability of the upgraded system is evaluated and discussed, and the imaging performance of the system is demonstrated using FLIM-FRET experiments with a representative CFP-YFP FRET pair (mCerulean-mCitrine).     

Diode Laser‐Excited Optogalvanic and Absorption Measurements of Uranium in a Hollow Cathode Discharge

Optogalvanic spectra for fifty two transition lines of uranium in the wavelength ranges of 662–683, 774–792, and 834–862 nm were measured by using external‐cavity diode lasers. Among these transitions, 860.795 nm and 682.691 nm were chosen for a detailed investigation of the detection limit for uranium by wavelength modulation spectroscopy due to its stronger signal magnitudes. A detection limit of about 2 × 10^? 5 absorbance achieved at 860.795 nm is more sensitive than that obtained at 682.691 nm, but the absorption spectrum at 682.691 nm is preferable to determine the isotope ratio due to the narrower hyperfine structure as well as the larger isotope shift. A preliminary result for an isotope ratio determination in a depleted sample is presented.     

Fabrication and characterization of MoO3 clusters-coated TiO2 nanotubes showing charge-transferred photoluminescence

MoO₃ clusters-coated TiO₂ nanotubes were synthesized wet-chemically and characterized by measuring photoluminescence spectra and kinetic profiles as well as extinction spectra and electron microscope images. TiO₂ nanotubes having an average outer diameter of 30 nm and an average thickness of 8 nm are surrounded by MoO₃ clusters with an average thickness of 4 nm. The excitation of both the TiO₂ cores and the MoO₃ shells of the type-II nanocomposites suspended in water yields charge-transferred junction photoluminescence having a long lifetime of 2.3 ns at 460 nm.     

Dynamics of plastic and liquid cyclohexane in bulk and in porous glasses studied by NMR methods

Cyclohexane was investigated both in bulk and in porous glasses with pore diameters between 4 and 208 nm in the temperature range 136 K≤T≤300 K. The methods involved were field-cycling NMR relaxometry, field-gradient NMR diffusometry, transverse-relaxation spectroscopy, and differential scanning calorimetry (DSC). The field-cycling data for the bulk material can best be described assuming translational modulation of intermolecular dipole-dipole coupling. This interpretation is confirmed by experiments with different degrees of deuteration, and is in accordance with diffusion coefficients determined with the aid of field-gradient diffusometry. The confinement in pores produces substantial changes in the phase behaviour and in molecular dynamics. For pore diameters of 30 nm and above, a non-frozen two monolayers thick film on the surface retains a diffusivity about one order of magnitude lower than in the bulk liquid, but two orders of magnitude larger than in the bulk plastic phase. Experiments indicate an exchange mechanism between this layer and the crystallite inside the pore. In glass with a pore diameter of 4 nm, all applied methods corroborate DSC results of the virtual absence of a phase transition and reveal a continuously decreasing translational mobility down to temperatures more than 100 K below the bulk liquid/cubic phase transition temperature.