Whole-field optically sectioned fluorescence lifetime imaging

We describe a novel three-dimensional fluorescence lifetime imaging microscope that exploits structured illumination to achieve whole-field sectioned fluorescence lifetime images with a spatial resolution of a few micrometers.     

Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source

We present a time-gated, optically sectioned, hyperspectral fluorescence lifetime imaging (FLIM) microscope incorporating a tunable supercontinuum excitation source extending into the UV. The system is capable of resolving the excitation spectrum, emission spectrum, and fluorescence decays in an optically sectioned image.     

Terahertz single pixel imaging based on a Nipkow disk

We describe a terahertz single pixel imaging system based on a Nipkow disk. Nipkow disks have been used for fast scanning imaging systems since the first experimental television was invented in 1926. In our work, a Nipkow disk with 24 scanning lines was used to provide an axial resolution of 2 mm/pixel. We also show that by implementing a microscanning technique the axial resolution can be further improved to 0.5 mm/pixel. Imaging of several objects was demonstrated to show that this simple scanning system is promising for fast or real time terahertz imaging applications.     

Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Three-dimensional molecular mapping in a microfluidic mixing device using fluorescence lifetime imaging

Fluorescence lifetime imaging (FLIM) is used to quantitatively map the concentration of a small molecule in three dimensions in a microfluidic mixing device. The resulting experimental data are compared with computational fluid-dynamics (CFD) simulations. A line-scanning semiconfocal FLIM microscope allows the full mixing profile to be imaged in a single scan with submicrometer resolution over an arbitrary channel length from the point of confluence. Following experimental and CFD optimization, mixing times down to 1.3+/-0.4 ms were achieved with the single-layer microfluidic device.     

Quantitative imaging of mixing dynamics in microfluidic droplets using two-photon fluorescence lifetime imaging

Droplet-based microfluidic systems enable miniaturization of chemical reactions in femtoliter to picoliter volume compartments. Quantifying mixing dynamics of the reagents in droplets is critical to determine the system performance. In this Letter, we developed a two-photon excitation fluorescence lifetime imaging technique to quantitatively image the mixing dynamics in micro?uidic droplets. A cross/autocorrelation method was used to reconstruct a high-quality fluorescence lifetime image of the droplet. The fluorescence decay was analyzed for accurate determination of the mixing ratio at each pixel of the image.     

Construction and characterization of a frequency-domain fluorescence lifetime imaging microscopy system

The construction of a homodyne frequency domain fluorescence lifetime imaging microscope is described. The system consists of (i) an intensity-modulated laser excitation source, (ii) an epifluorescence microscope, (iii) a gain-modulated microchannel plate (MCP) image intensifier, and (iv) a slow-scan CCD camera. The phase and modulation homogeneity of the MCP image intensifier were determined at frequencies of 40, 100, 160, and 240 MHz. The detected modulation depths were 65, 52, 32, and 23%, respectively, and were highly homogeneously distributed. The phasedistribution image revealed iris effects at frequencies of 160 and 240 MHz but was homogeneous at lower frequencies. Lifetime imaging of a solution of the fluorescent flavoprotein lipoamide dehydrogenase demonstrated (i) the accuracy of the determined lifetimes (< 60 ps), (ii) the time resolution of the instrument (< 50 ps), and (iii) the average precision for single pixel fluorescence lifetimes (50 ps is feasible). The imaging of tiny fluorescent microspheres revealed that even in a volume of 0.3 x 10^-15 L, the standard error in the lifetimes can be as low as 79 ps. The spatial resolution of the instrument is estimated to be < 400 nm in the object plane at a 100 x magnification.     

Magnetic-grating free-induction decay and magnetic-grating echo using ultrafast excitation pulses

The interaction of atoms with ultrafast, counterpropagating optical fields is considered. The magnetic degeneracy and hyperfine splitting of the atomic levels are included in the calculations, which are carried out for arbitrary polarizations of the incident fields. The counterpropagating fields produce spatial harmonics in the ground state density matrix (gratings) which can be monitored by backscattering of a traveling wave probe pulse. Two types of excitation schemes are analyzed. The Magnetic-Grating Free-Induction Decay (MGFID) consists of excitation with a single counterpropagating wave field, while the Magnetic-Grating Echo (MGE) involves excitation by two counterpropagating wave fields, separated in time by T. The atomic response to the probe pulse is calculated in lowest-order perturbation theory for atoms cooled below the Doppler limit of laser cooling. Both the MGFID and MGE signals consist of pulses having a duration of order of the excited state lifetime, modulated at frequencies corresponding to the various hyperfine transitions. As a function of the delay between pulses, the signals oscillate at frequencies determined by the ground state hyperfine splittings. General expressions for the MGFID and MGE signals are derived and specific results are presented for the D₂ line in Na.     

Phase-modulation fluorometer using a phase-modulated excitation light source

We propose a phase-modulation fluorometer (PMF) with a light-emitting diode (LED) or a laser diode (LD) used as an excitation light source (ELS) that is driven in the phase-modulation (PM) mode. The PM-ELS generates many frequency sidebands that spread in the vicinity of carrier frequency f _c with the interval of modulation frequency f _m depending on the maximum phase deviation Δφ. The scheme enables us to derive fluorescence lifetime values of a multicomponent sample at one time. We show a typical numerical simulation result for explaining the principle of operation. To demonstrate the effectiveness of the proposed PMF, we have measured fluorescence lifetimes of three kinds of inorganic fluorescent glasses and that of a mixture solution of 1 × 10^?6M rhodamine 6G and 1 × 10^?6 M coumarin 152 in ethanol with a volume ratio of 1: 1.     

In vivo imaging of mice auricle vessels using adaptive optical confocal fluorescence microscope

Facilitated with stochastic parallel gradient descent(SPGD) algorithm for wavefront sensorless correcting aberrations, an adaptive optics(AO) confocal fluorescence microscopy is developed and used to record fluorescent signals in vivo. Vessels of mice auricle at 80, 100 and 120 μm depth are obtained, and image contrast and fluorescence intensity are significantly improved with AO correction. The typical 10%–90% rise-time of the metric value measured is 5.0 s for a measured close-loop bandwidth of 0.2 Hz. Therefore, the AO confocal microscopy implemented with SPGD algorithm for robust AO corrections will be a powerful tool for study of vascular dynamics in future.     

Highly sensitive fluorescence microscope system for single biological cell using laser excitation and digital image processing and display

A novel type of laser-excited fluorescence microscope system is reported, which, for the first time, realizes excellent sensitivity and spatial resolution in biological and medical applications using an ultra-high sensitivity image camera and digital image processing technique for three-dimensional display of the detected fluorescence intensity across a single cell. Experimental studies are also presented on the fluorescence intensity distribution of Rhodamine 6G adsorbed in an OH gel as a simple cell model and also of a hematoporphyrin-derivative (HpD) taken up by a cultured esophagus cancer cell.     

Compact soft x-ray microscope using a gas-discharge light source

We report on a soft x-ray microscope using a gas-discharge plasma with pseudo spark-like electrode geometry as a light source. The source produces a radiant intensity of 4 x 10(13) photons/(sr pulse) for the 2.88 nm emission line of helium-like nitrogen. At a demonstrated 1 kHz repetition rate a brilliance of 4.3 x 10(9) photons/(microm2 sr s) is obtained for the 2.88 nm line. Ray-tracing simulations show that, employing an adequate grazing incidence collector, a photon flux of 1 x 10(7) photons/(microm2 s) can be achieved with the current source. The applicability of the presented pinch plasma concept to soft x-ray microscopy is demonstrated in a proof-of-principle experiment.     

A CTRW-based model of time-resolved fluorescence lifetime imaging in a turbid medium

We develop an analytic model of time-resolved fluorescent imaging of photons migrating through a semi-infinite turbid medium bounded by an infinite plane in the presence of a single stationary point fluorophore embedded in the medium. In contrast to earlier models of fluorescent imaging in which photon motion is assumed to be some form of continuous diffusion process, the present analysis is based on a continuous-time random walk (CTRW) on a simple cubic lattice, the objective being to estimate the position and lifetime of the fluorophore. This can provide information related to local variations in pH and temperature with potential medical significance. Aspects of the theory were tested using time-resolved measurements of the fluorescence from small inclusions inside tissue-like phantoms. The experimental results were found to be in good agreement with theoretical predictions provided that the fluorophore was not located too close to the planar boundary, a common problem in many diffusive systems.     

Hard disk magnetic domain nano-spatial resolution imaging by using a near-field scanning microwave microscope with an AFM probe tip

A near-field scanning microwave microscope (NSMM) incorporating an atomic force microscope (AFM) probe tip was used for the direct imaging of magnetic domains of a hard disk under an external magnetic field. We directly imaged the magnetic domain changes by measuring the change of reflection coefficient S₁₁ of the NSMM at an operating frequency near 4.4 GHz. Comparison was made to the magnetic force microscope (MFM) image. Using the AFM probe tip coupled to the tuning fork distance control system enabled nano-spatial resolution. The NSMM incorporating an AFM tip offers a reliable means for quantitative measurement of magnetic domains with nano-scale resolution and high sensitivity.     

Improving the precision of fluorescence lifetime measurement using a streak camera

Streak camera has high temporal resolution and high sensitivity, and is a powerful tool in biomedical study to measure fluorescence lifetime and perform fluorescence lifetime imaging. However, nonuniformity of the gain in the streak tube and nonlinearity of the sweeping speed limit the precision of fluorescence lifetime measurement, particularly when fluorescence lifetimes are short. We have constructed a two-photon excitation fluorescence lifetime measurement system that is based on a synchroscan streak camera and have developed accordingly a method to correct the effect of gain nonuniformity and nonlinearity of sweeping speed on the measurement precision. A continuous-wave laser of high stability is used to calibrate the gain of the streak camera, and a Fabry-Perot etalon is used to calibrate the nonlinearity of the sweeping speed. Fitting algorithms are used to correct the gain of the streak camera and nonlinearity of the sweeping speed respectively, which significantly improves the measurement precision of the system, as characterized through the fluorescence lifetime of the short-lived fluorescence dye, Rose Bengal. Experimental results show that the measurement fluctuation of the lifetime has been improved from more than 10% to 2% after correcting the effects of gain nonuniformity and sweeping speed nonlinearity.     

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.     

Planar imaging of rayleigh and fluorescence light from H2-air combustion inside a bomb using tunable 193 nm light

Rayleigh scattering and planar laser induced predissociative fluorescence are used to obtain 2-D images of total and specific densities inside a combustion bomb. The experimental arrangement is the same for both methods except for the selection of laser wavelength and the filtering of the radiated light. The former method yields a distribution of total densities while the latter provides densities of hot O₂, i.e., those with v=2, J=17 and 33. Hydrogen-air mixtures of various compositions are used. Because the thermodynamics are well known, a bomb may serve as a reference device for diagnostics for high temperature species, and the results are in accord with calculations. Additional Rayleigh experiments are described which yield a) scattering cross sections at 193 nm, b) a 2-D temperature distribution in a hot air stream, and c) a 2-D temperature distribution, of limited precision, inside the bomb.     

Phase imaging flow cytometry using a focus-stack collecting microscope

This letter introduces a fluidics-based focus-stack collecting microscope. A microfluidic device transports cells through the focal plane of a microscope, resulting in an efficient method to collect focus stacks of large collections of single cells. Images from the focus stacks are used to reconstruct the quantitative phase of cells with the transport-of-intensity-equation method. Using the phase imaging flow cytometer, we measure three-dimensional shape variations of red blood and leukemia cells.     

Optically probing spin and charge interactions in a tunable artificial molecule

We optically probe and electrically control a single artificial molecule containing a well defined number of electrons. Charge and spin dependent interdot quantum couplings are probed optically by adding a single electron-hole pair and detecting the emission from negatively charged exciton states. Coulomb- and Pauli-blockade effects are directly observed, and tunnel coupling and electrostatic charging energies are independently measured. The interdot quantum coupling is shown to be mediated by electron tunneling. Our results are in excellent accord with calculations that provide a complete picture of negative excitons and few-electron states in quantum dot molecules.     

Fast XANES fluorescence imaging using a Maia detector

A new fast X‐ray absorption spectroscopy scanning method was recently implemented at the Hard X‐ray Microprobe endstation P06, PETRA III, DESY, utilizing a Maia detector. Spectromicroscopy maps were acquired with spectra for X‐ray absorption near‐edge structure (XANES) acquisition in the sub‐second regime. The method combines XANES measurements with raster‐scanning of the sample through the focused beam. The order of the scanning sequence of the axes, one beam energy axis and two (or more) spatial axes, is a variable experimental parameter and, depending on it, the dwell at each location can be either single and continuous (if the energy axis is the inner loop) or in shorter discontinuous intervals (if a spatial axis is innermost). The combination of improved spatial and temporal resolution may be necessary for rapidly changing samples, e.g. for following in operando chemical reactions or samples highly susceptible to beam damage where the rapid collection of single XANES spectra avoids issues with the emergence of chemical changes developing from latent damage. This paper compares data sets collected on a specially designed test pattern and a geological thin‐section scanning the energy as inner, middle and outer axis in the sequence. The XANES data of all three scanning schemes is found to show excellent agreement down to the single‐pixel level.