Time-wavelength two-dimensional femtosecond fluorescence imaging

We report a new femtosecond time-resolved fluorescence spectrometer that enables us to observe fluorescence intensity as a time-wavelength two-dimensional image in a single measurement. This method utilizes a time-to-space conversion technique and fluorescence sum-frequency mixing with a femtosecond gate pulse. It provides a fluorescence image covering temporal and spectral spans of approximately 2 ps and approximately 60 nm, respectively. Calibration of the time and intensity axes of the image is made by use of a long-lived dye fluorescence. The two-dimensional fluorescence image of beta-carotene obtained demonstrates the high potential of this method for quantitative studies of ultrafast excited-state dynamics.     

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.     

Fluorescence lifetime imaging of mixing dynamics in continuous-flow microdroplet reactors

Water-in-oil microdroplets within fluidic channels have the potential to serve as isolated reaction compartments for monitoring real-time dynamics with high efficiency and repeatability. Droplets, usually generated from aqueous and oil solutions using standard microfluidic formats, can be produced at frequencies in excess of 1 kHz. Although mixing within such microdroplets is normally enhanced by chaotic advection, the mixing pattern from droplet to droplet is almost identical and reproducible in form. Herein, we demonstrate that fluorescence lifetime imaging can be used to reconstruct mixing patterns within a droplet with a time resolution of 5 micros.     

Enhancement of rapid lifetime determination for time-resolved fluorescence imaging in forensic examination

An enhancement method of rapid lifetime determination is proposed for time-resolved fluorescence imaging to discriminate substances with approximate fluorescence lifetime in forensic examination. In the method, an image-exclusive-OR treatment with filter threshold adaptively chosen is presented to extract the region of interest from dual-gated fluorescence intensity images, and then the fluorescence lifetime image is reconstructed based on the rapid lifetime determination algorithm. Furthermore, a maximum and minimum threshold filtering is developed to automatically realize visualization enhancement of the lifetime image. In proof experiments, compared with traditional fluorescence intensity imaging and rapid lifetime determination method, the proposed method automatically distinguishes altered and obliterated documents written by two brands of highlighters with the same color and close fluorescence lifetime.     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

Fluorescence lifetime imaging of oxygen in living cells

The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence lifetime imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can be described by the Stem-Volmer equation. This shows that fluorescence quenching by oxygen is a dynamic quenching process. In addition, it was demonstrated that the fluorescence lifetime of RTDP is insensitive to pH, ion concentration, and cellular contents. This implies that a simple calibration procedure in buffers can be used to quantify oxygen concentrations within cells. First fluorescence imaging experiments on J774 macrophages show a nonuniform fluorescence intensity and a uniform fluorescence lifetime image. This indicates that the RTDP is heterogeneously partitioned throughout the cells, while the oxygen concentration is constant.     

Frequency-domain lifetime imaging methods at unilever research

Fluorescence lifetime imaging methodology has been successfully implemented at Unilever Research in a frequency-domain manner. The experimental rig constructed comprises a wide-bandwidth electrooptic modulator operating on a CW argon-ion laser. The modulated excitation with a typical upper modulation frequency limit of 200 MHz falls on macroscopic samples and the resultant scattered light or fluorescence emission is then imaged onto a custom gain-modulatable image intensifier and slow-scan CCD camera combination. Phase adjustment of the image intensifier relative to the laser modulator is achieved by the RF function generator driving the intensifier. Both homodyne and heterodyne (500-Hz) strobing modes are employed to generate a double image stack (scattered light reference and fluorescence emission) comprising an image sequence as a function of instrumental phase difference. These image stacks are analyzed by Fourier least-squares methods to yield lifetime images by both phase delay and normalized demodulation. Correct operation of the apparatus is deduced from the direct imaging of a quencher-induced lifetime variation of BODIPY disulfonate over a range of concentrations. A typical industrially relevant sample, comprising an investigation of the lifetime aspects of human dental enamel autofluorescence at 50MHz modulation frequency, is given. This shows that there are real emission lifetime decreases of about 0.5 nsec in white-spot lesion areas compared to the surrounding sound enamel.     

Fluorescence lifetime imaging with picosecond resolution for biomedical applications

We describe a novel whole-field fluorescence lifetime imaging system, based on a time-gated image intensifier and a solid-state laser oscillator-amplifier, that images lifetime differences of less than 10 ps. This system was successfully applied to discrimination between biological tissue constituents.     

Carrier dynamics in GaN at extremely low excited carrier densities

Carrier dynamics in GaN was studied using fluorescence lifetime measurement in the frequency domain technique in the temperature range from 8 to 300 K at very low and very high excitation levels. The study was performed in a high-quality GaN epilayer exhibiting a room-temperature nonequilibrium carrier lifetime of 2 ns, which was determined by a light-induced transient grating (four-wave mixing) technique. The results reveal the roles of donor–acceptor pair recombination and conduction band–acceptor recombination in yellow luminescence band formation.     

Time-resolved fluorescence anisotropy imaging applied to live cells

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.     

Time-gated biological imaging by use of colloidal quantum dots

The long (but not too long) fluorescence lifetime of CdSe semiconductor quantum dots was exploited to enhance fluorescence biological imaging contrast and sensitivity by time-gated detection. Significant and selective reduction of the autofluorescence contribution to the overall image was achieved, and enhancement of the signal-to-background ratio by more than an order of magnitude was demonstrated.     

Laser-induced fluorescence imaging of acetone inside evaporating and burning fuel droplets

Laser-induced fluorescence was used to visualize acetone fields inside individual droplets of pure acetone as well as droplets composed of methanol or 1-propanol initially mixed with acetone. Droplets were supported on a horizontal wire and two vaporization conditions were investigated: (1) slow evaporation in room air and (2) droplet combustion, which leads to substantially faster droplet surface regression rates. Acetone was preferentially gasified, causing its concentration in droplets to drop in time with resultant decreases in acetone fluorescence intensities. Slowly vaporizing droplets did not exhibit large spatial variations of fluorescence within droplets, indicating that these droplets were relatively well mixed. Ignition of droplets led to significant variations in fluorescence intensities within droplets, indicating that these droplets were not well mixed. Ignited droplets composed of mixtures of 1-propanol and acetone showed large time-varying changes in shapes for higher acetone concentrations, suggesting that bubble formation was occurring in these droplets.     

基于时间相关单光子计数的荧光寿命成像技术

采用时域法中的时间相关单光子计数方法记录荧光寿命,时间相关单光子计数采用多波长通道同时记录荧光光子数,可以提高计数效率和信息量,还可以在稳态图像中分离不同荧光团,形成4维图像。并采用多光子激发技术,利用长波长光源发出的两个或多个光子可以激发出一个短波长的光子。多个光子必须几乎同时到达激发点,才能提供被激发分子足够的能量以产生荧光。多光子激发波长较长,生物组织对其散射减小,因而可以穿透到更深层的组织,从而提高荧光成像深度和空间分辨力,并减少对活体样品的损伤。     

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochemical properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radio frequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is separated in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calculate in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.     

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.     

Sustained simultaneous high-speed imaging of scalar and velocity fields using a single laser

A high-speed technique that combines planar laser induced fluorescence (PLIF) detection of biacetyl and particle image velocimetry (PIV) for simultaneous imaging of scalar and velocity fields is demonstrated at a frame rate of 12 kHz for up to 32500 consecutive frames. A single diode-pumped, frequency-tripled Nd-YAG laser was used for excitation. Wavelength-separated recording was achieved for Mie scattering from silicone oil droplets with a CMOS camera and for the red-shifted fluorescence from biacetyl with an image-intensified CMOS camera. Interference between PIV and PLIF tracers was found to be negligible. Cross-talk between PIV and PLIF signals was low and a strategy to completely eliminate it was devised and is discussed. The signal-to-noise ratio is about 9 for single-shot scalar images. Example image sequences were recorded in an atmospheric pressure air jet at Re=2000. PACS 42.62.Fi; 33.50.Dq; 06.30.Gv; 06.60.Jn     

CVD grown 2D MoS2 layers: A photoluminescence and fluorescence lifetime imaging study

In this letter, we report on the fluorescence lifetime imaging and accompanying photoluminescence properties of a chemical vapour deposition (CVD) grown atomically thin material, MoS₂. µ‐Raman, µ‐photoluminescence (PL) and fluorescence lifetime imaging microscopy (FLIM) are utilized to probe the fluorescence lifetime and photoluminescence properties of individual flakes of MoS₂ films. Usage of these three techniques allows identification of the grown layers, grain boundaries, structural defects and their relative effects on the PL and fluorescence lifetime spectra. Our investigation on individual monolayer flakes reveals a clear increase of the fluorescence lifetime from 0.3 ns to 0.45 ns at the edges with respect to interior region. On the other hand, investigation of the film layer reveals quenching of PL intensity and lifetime at the grain boundaries. These results could be important for applications where the activity of edges is important such as in photocatalytic water splitting. Finally, it has been demonstrated that PL mapping and FLIM are viable techniques for the investigation of the grain‐boundaries.

     

Ag8 fluorescence in argon

The fluorescence of Ag8 in an argon matrix and in argon droplets is reported. This is the first unambiguous assignment of the fluorescence of a metal cluster larger than the tetramer, indicating that the excited state lifetime is longer than previously thought. It is discussed as a possible result of a matrix cage effect. The excitation spectrum is compared with two-photon-ionization measurements of Ag8 in helium droplets and to known absorption data. The agreement is excellent. We propose that the excited states relax rapidly through vibrational coupling to a long-lived state, from which the fluorescence occurs.     

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.     

一种实现显微荧光寿命图的测量方法

将脉宽120fs、重复率76MHz激光引入激光扫描显微镜的激发光路,利用其扫描系统对荧光标记样品激发扫描,将激发出的荧光从荧光探测光路引入备用的外部探测口;在探测口接一快速光电倍增管,将光电倍增管的信号送给时间相关单光子计数器,获得时间相关的荧光强度图;最后通过计算机处理获得荧光寿命图。应用此系统对青色荧光蛋白(CFP)、黄色荧光蛋白(YFP)荧光寿命进行了测量,并应用CFP、YFP实现荧光共振能量转移的测量。通过实验看出利用已有的激光扫描显微镜,配合较先进的寿命测量方法,可以很好地实现显微荧光寿命图的测量。