Double-pulse fluorescence lifetime measurements

It is demonstrated that fluorescence lifetimes in the nanosecond and picosecond time-scale range can be observed with the recently proposed double-pulse fluorescence lifetime imaging technique (Müller et al. , 1995, Double-pulse fluorescence lifetime imaging in confocal microscopy. J. Microsc 177, 171–179).
A laser source with an optical parametric amplifier (OPA) system is used to obtain short pulse durations needed for high time resolution, wavelength tunability for selective excitation of specific fluorophores and high pulse energies to obtain (partial) saturation of the optical transition.
It is shown that fluorescence lifetimes can be determined correctly also with nonuniform saturation conditions over the observation area.
A correction scheme for the effect on the measurements of laser power fluctuations, which are inherently present in OPA systems, is presented. Measurements on bulk solutions of Rhodamine B and Rhodamine 6G in different solvents confirm the experimental feasibility of accessing short fluorescence lifetimes with this technique.
Because signal detection does not require fast electronics, the technique can be readily used for fluorescence lifetime imaging in confocal microscopy, especially when using bilateral scanning and cooled CCD detection.     

Fourier transform multipixel spectroscopy for quantitative cytology

A Fourier transform multipixel spectroscopy system was set up and applied to fluorescence microscopy of single living cells. Continuous fluorescence spectra for all pixels of the cell image were recorded simultaneously by the system. Multiple frames of data were first acquired and stored as a set of interferograms for each pixel of the image; they were then Fourier transformed and used as a spatially organized set of fluorescence spectra. Practical spectral resolution of 5 nm was achieved, typically, for 10⁴ pixels in a single cell. The net result was I ( x y ,λ), the fluorescence intensity ( I ) for each pixel of the image ( x y ), as function of wavelength (λ). The present study demonstrates that multipixel spectroscopy can reveal dynamic processes of the food-digestive cycle in the unicellular Paramecium vulgaris fed with algae. Spectral variability of fluorescence intensity at different cytoplasmic sites pinpointed the location of cellular deposits of chlorophyll (630 nm) and of pheophytin (695 nm), a digestive product of the chlorophyll. Localization of compartmental spectral changes was achieved using a 'similarity mapping' algorithm, followed by enhanced image construction. Similarity mapping based on the fluorescence spectrum of native chlorophyll revealed a highlighted image of the cell cytopharynx structure where algae were ingested. Phagolysosomes, migrating vacuoles and the cytoproct, each containing different ratios of pheophytin, were similarly imaged.     

A white light confocal microscope for spectrally resolved multidimensional imaging

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom‐built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto‐optic tunable filter to provide continuously tunable fluorescence excitation with a 1‐nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.     

AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers

Most treatments of frequency domain lifetime measurements indicate that a set of measurements must be made at multiple frequencies in order to determine the lifetimes of the components in a mixture. Although this is the case in general, under special conditions, single-frequency data can resolve multiple lifetimes. Here, data are presented showing several approaches to determining fluorescence lifetimes in two-component mixtures using single-frequency data. Common to all of the procedures presented is exploitation of variations in the relative contributions of a particular fluorophore to the total fluorescence from a mixture of fluorophores. This variation can be produced intentionally by observing a number of samples which contain different relative amounts of the fluorophores. It can be produced fortuitously by observing spatial variations in a mixture of fluorophores in a specimen or set of specimens observed with a lifetime imaging system. It can also be produced by examination of the lifetime spectrum obtained from a fluorophore mixture or by varying the concentration of a quencher in a fluorophore mixture, in which the two fluorophores have different rate constants for quenching. In many instances, the set of approaches presented here will be unsuitable for examination of arbitrary samples of unknown composition for which the multifrequency approach should be used. However, measurements produced using single-frequency methods may be applied to good effect for controlled experiments having defined fluorophores or sets of fluorophores, particularly in the case of biological lifetime imaging studies.     

Real-time cellular uptake of serotonin using fluorescence lifetime imaging with two-photon excitation

The real-time uptake of serotonin, a neurotransmitter, by rat leukemia mast cell line RBL-2H3 and 5-hydroxytryptophan by Chinese hamster V79 cells has been studied by fluorescence lifetime imaging microscopy (FLIM), monitoring ultraviolet (340 nm) fluorescence induced by two-photon subpicosecond 630 nm excitation. Comparison with two-photon excitation with 590 nm photons or by three-photon excitation at 740 nm shows that the use of 630 nm excitation provides optimal signal intensity and lowered background from auto-fluorescence of other cellular components. In intact cells, we observe using FLIM three distinct fluorescence lifetimes of serotonin and 5-hydroxytryptophan according to location. The normal fluorescence lifetimes of both serotonin (3.8 ns) and 5-hydroxytryptophan (3.5 ns) in solution are reduced to approximately 2.5 ns immediately on uptake into the cell cytosol. The lifetime of internalized serotonin in RBL-2H3 cells is further reduced to approximately 2.0 ns when stored within secretory vesicles.     

Optimizing imaging parameters for the separation of multiple labels in a fluorescence image

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal‐to‐noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal‐to‐noise ratio or optimize a newly defined ‘figure of merit’, which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.     

二氧化硅包覆对纳米多孔金增强荧光特性的调制

为了减弱金属基底对表面增强荧光的淬灭效应,设计了增强效果更好的荧光增强基底。采用化学生长二氧化硅的方法对纳米多孔金(NPG)表面进行修饰,避免荧光分子和NPG表面直接接触引起的淬灭效应,在SiO_2@NPG表面分别组装上罗丹明6G(R6G)和辐射中心波长为700 nm的量子点(QD 700)。通过探测分析荧光光谱,可以得出:二氧化硅包覆的基底可以使表面增强荧光得到显著的增强,并且二氧化硅厚度对荧光强度有调节作用;在基底增强量子点荧光信号的同时,量子点和NPG之间还出现非辐射的能量转移现象,二氧化硅的厚度对能量转移同样有调节作用,厚度约为5 nm时能量转移现象最显著。本实验为基于荧光能量转移的检测以及设计更好的荧光增强基底提供了参考。     

Fluorescence lifetime imaging microscopy using near-infrared contrast agents

Although single-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to image molecular processes using a wide range of excitation wavelengths, the captured emission of this technique is confined to the visible spectrum. Here, we explore the feasibility of utilizing near-infrared (NIR) fluorescent molecular probes with emission >700 nm for FLIM of live cells. The confocal microscope is equipped with a 785 nm laser diode, a red-enhanced photomultiplier tube, and a time-correlated single photon counting card. We demonstrate that our system reports the lifetime distributions of NIR fluorescent dyes, cypate and DTTCI, in cells. In cells labelled separately or jointly with these dyes, NIR FLIM successfully distinguishes their lifetimes, providing a method to sort different cell populations. In addition, lifetime distributions of cells co-incubated with these dyes allow estimate of the dyes' relative concentrations in complex cellular microenvironments. With the heightened interest in fluorescence lifetime-based small animal imaging using NIR fluorophores, this technique further serves as a bridge between in vitro spectroscopic characterization of new fluorophore lifetimes and in vivo tissue imaging.     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Two-photon excited lifetime imaging of autofluorescence in cells during UV A and NIR photostress

By monitoring coenzyme autofluorescence modifications. as an indicator of cell damage. the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UV A radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser. focused through highnumerical- aperture microscope optics. provided diffractionlimited mlcrobeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (_T-mapping). Upon the onset of UV or NIR exposure. Chinese hamster ovary cells exhibited blue/green autofluorescence witq a mean lifetime of 2·2 ns. which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 m W. 300 J cm^-2 ) resulted in oxidative stress correlated with increased autofluorescence intensity. onset of nuclear fluorescence. and a fluorescence lifetime decrease. The cellular response to femtosecond NIR micro beams depended significantly on peak power. Peak powers above a threshold value of about 0·5kW (average power: 6mW). 0·55kW (7mW) and 0·8kW (lOmW) at 730nm. 760nm and 800nm. respectively. resulted in the onset of short-lived luminescence with higher intensity (100x) than the intracellular NAD(P)H fluorescence. This luminescence. accompanied by destruction of cellular morphology. was localized and occurred in the mitochondrial region. In contrast. beams at a power of less than 0·5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.     

Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources

High brightness light emitting diodes are an inexpensive and versatile light source for wide‐field frequency‐domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio‐frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal‐to‐noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices (～40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal‐to‐noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.     

基于PCA的时间分辨油荧光光谱分析及优化

激光诱导荧光技术可广泛应用于油污染的监测中,然而普通的油荧光光谱技术只能实现油污染监测的粗分类,无法区分原油与燃料油的荧光特征。本文基于主成分分析方法(PCA)的时间分辨油荧光分类方法,实验测量了20种油样本的时间分辨荧光光谱特征,给出了对应的荧光寿命和时间分辨油荧光光谱的时序特征。在此基础上,利用前三个主成分构成的三维特征矢量空间,通过分析不同采集时刻下油样本矢量间相关距离的变化,对油样本的时间分辨荧光光谱进行聚类分析。为了体现油荧光变化的时序性,引入矢量距离的离散度参量,提出基于PCA进行时间分辨油荧光光谱分析的优化方法。实验结果表明,基于时间分辨油荧光光谱识别可实现原油与燃料油的光谱时序特征区分,具备良好的油荧光分类效果。     

A Self-Initiated Periodically Pulsed Light Source on Chlorine and Krypton Chloride Molecules

An electric-discharge light source, operating in the spectral range of 170–270 nm on a system of bands of Cl₂ ( = 200 and 257 nm) and KrCl ( = 222 nm) molecules is described. The radiator is pumped by a low-pressure volume discharge in a spherical anode-flat cathode system of electrodes with an interelectrode distance of 6 cm, so that the plasma has no contact with the quartz envelope of the lamp. The working mixtures are P(Kr)/P(Cl₂) = (40–640)/(40–280) Pa. When a dc voltage U 1 kV is applied to the discharge gap, a volume discharge exists only in a periodically pulsed mode (f = 0.1–50 kHz) and represents a source of short-wave radiation with a cylindrical working surface (1 cm in diameter and 6 cm long) and a mean radiation power of 3 W.     

确定气态饱和胺分子振子强度与荧光辐射速率常数的关系探讨

在此研究中,采用一系列具有相似结构的气态饱和单胺和二胺作为研究对象,应用Gaussian03计算化学软件,在DFT/B3LYP水平上,使用6-311+G(d)基组优化处于基态的胺分子结构并使用CIS/6-311+G(d)优化处于激发态胺分子的结构,计算激发能量、激发波长,以及S1←S0振子强度f。通过查得文献中已有的气态饱和胺的荧光寿命以及得到的荧光辐射速率常数实验数据,比较计算得出的振子强度f,得出结论:双胺的荧光寿命明显长于单胺;若f=0,表明跃迁禁阻,像DMPZ,DABCO,它们的荧光寿命很长,超过了770ns;若f>0,则允许跃迁,饱和胺分子荧光寿命小于60ns。可由计算得出的f值,初步判断荧光物质在UV辐射作用下的荧光寿命长短。并得到振子强度f与荧光辐射速率常数线性关系。与基态分子结构比较,激发态的饱和胺分子结构发生改变。     

Navigating transdermal diffusion with multiphoton fluorescence lifetime imaging

We demonstrate the potential of fluorescence lifetime imaging by time-correlated single-photon counting as a method for monitoring the transdermal diffusion pathway and diffusion rate of pharmaceuticals in human skin. The current application relies on observing subtle changes in the fluorescence lifetime of the intrinsic fluorophores present in the intracellular region between corneocytes of the stratum corneum. We have comprehensively characterized the measured fluorescence lifetimes from intracorneocyte junctions in three skin section types (dermatomed skin, epidermal membranes and stratum corneum) revealing statistically significant differences of the short lifetime component between each of the types, which we attribute to the sample preparation and imaging method. We show using epidermal membrane sections that application of a drug/solvent formulation consisting of ethinyl estradiol and spectroscopic grade ethanol to the surface gives rise to a slight but statistically significant shortening of the fluorescence lifetime of the long-lived emitting species present in the sample, from approximately 2.8 ns to 2.5 ns. The method may be useful for future studies where the kinetics and pathways of a variety of applied formulations could be investigated.     

Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation

We demonstrate the possibility to increase substantially the number of simultaneously detected fluorophores by utilizing both spectral and lifetime information. Using a two-detector confocal scanning laser microscope, experiments confirm that four different fluorophores can be detected with good channel separation. The signal-to-noise ratio (SNR) of the recorded images is investigated both theoretically and experimentally. It is found that in order to obtain a high SNR fluorophore lifetimes should differ by approximately an order of magnitude.     

Frequency domain lifetime and spectral imaging microscopy

In the femtoliter observation volume of a two-photon microscope, multiple fluorophores can be present and complex photophysics can take place. Combined detection of the fluorescence emission spectra and lifetimes can provide deeper insight into specimen properties than these two imaging modalities taken separately. Therefore, we have developed a detection scheme based on a frequency-modulated multichannel photomultiplier, which measures simultaneously the spectrum and the lifetime of the emitted fluorescence. Experimentally, the efficiency of the frequency domain lifetime measurement was compared to a time domain set-up. The performance of this spectrally and lifetime-resolved microscope was evaluated on reference specimens and living cells labeled with three different stains targeting the membrane, the mitochondria, and the nucleus.     

Simultaneous four-color imaging of single molecule fluorophores using dichroic mirrors and four charge-coupled devices

We developed a total-internal-reflection (TIR) fluorescence microscopy using three dichroic mirrors and four charge-coupled devices (CCDs) to detect simultaneously four colors of single-molecule (SM) fluorophores. Four spectrally distinct species of fluorophores (Alexa 488, Cy3, Cy5, or Cy5.5) were each immobilized on a different fused silica slide. A species of fluorophores on the slide was irradiated simultaneously, by two excitation beams from an Ar ion laser (488 and 514.5 nm) and a diode laser (642 nm) through TIR on the slide surface. Fluorescence emitted from the fluorophores was spectrally resolved into four components by the dichroic mirrors, and four images were generated from them simultaneously and continuously, with the four CCDs at a rate of 10 Hz. A series of images was thus obtained with each CCD. Fluorescence spots for a species were observed mainly in the series of images recorded by its respective-color CCD. In the first image in the series, we picked out the spots as continuous pixel regions that had the values greater than a threshold. Then we selected only those spots that exhibited single-step photobleaching and regarded them as SM fluorescence spots. Pixel values of SM fluorescence spots widely differed. Some SM fluorophores had pixel values smaller than the threshold, and were left unpicked. Assuming the pixel values of SM fluorescence spots differed with a Gaussian profile, we estimated the ratios of unpicked fluorophores to be less than 20% for all the species. Because of the spectral overlaps between species, we also observed cross-talk spots into CCDs other than the respective-color CCDs. These cross-talk SM fluorescence spots can be mistaken for correct species. We thus introduced the classification method and classified SM fluorescence spots into correct species in accordance with two kinds of four-dimensional signal vectors. The error rates of fluorophore classification were estimated to be less than 3.2% for all the species. Our system is suitable for the biological studies that desire to simultaneously monitor the four colors of SM fluorophores.     

Simultaneous two-photon fluorescence correlation spectroscopy and lifetime imaging of dye molecules in submicrometer fluidic structures

Fluorescence correlation spectroscopy (FCS) is a very sensitive technique that can be used, e.g., for the measurement of low concentrations and for the investigation of transport of fluorescent molecules. Fluorescence lifetime imaging (FLIM) provides spatially resolved information about molecular fluorescence lifetimes reflecting the interactions of the molecules with their environment. We have applied simultaneous two-photon FCS and FLIM to probe the behavior of fluorescent molecules diffusing in submicrometer silicon oxide channels. Our measurements reveal differences in fluorescence lifetimes compared to bulk solution that result from the effects of confinement and the presence of interfaces. Confinement also affects diffusional characteristics of fluorophores as reflected in fluorescence autocorrelation functions. These possible consequences of both spatial confinement and the presence of interfaces between media with different refractive indices on the diffusion and fluorescence lifetime of molecules in nanostructures are discussed in general.     

Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new,efficient upconverting fluorophore

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sap-phire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.