Fluorescence lifetime tomography of live cells expressing enhanced green fluorescent protein embedded in a scattering medium exhibiting background autofluorescence

We present a novel fluorescence lifetime tomography system applied to a highly scattering autofluorescent phantom containing live cells expressing the fluorophore enhanced green fluorescent protein (EGFP). The fluorescence signal was excited using a fiber-laser-pumped supercontinuum source and detected using wide-field time gating imaging. To facilitate rapid 3D reconstruction of the fluorescence lifetime distribution, the time-resolved data were Fourier-transformed in time to give complex functions that formed a data set for the Fourier domain reconstruction. Initially the presence of an unspecified background autofluorescence signal impeded reconstruction of the lifetime distribution, but we show that this problem can be addressed using a simple iterative technique.     

Spectral imaging of time-resolved anisotropy: theory and experiment

Time-resolved fluorescence anisotropy on the nanosecond time scale is useful for the study of the rapid rotation of macromolecules. A system combining the capabilities of fluorescence spectral imaging with time-resolved fluorescence anisotropy and enabling the wide-field measurement of the spectroscopic parameters of fluorophores is discussed. The phasor approach is used to quantitatively analyze the time-resolved fluorescence anisotropy by transforming the polarized parallel and perpendicular components to the phasor space in the frequency domain, respectively, and a unique way to calculate the fluorescence rotational correlation time is put forward. Experimental results prove that the phasor approach is a proper model for the time-resolved fluorescence anisotropy.     

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.     

Fluorescence lifetime imaging in biosciences: technologies and applications

The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging techniques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved techniques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.     

Fluorescence lifetime imaging in biosciences: technologies and applications

The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging techniques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved techniques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.      

荧光分子层析中的全时间分辨图像重建法

在荧光分子层析(Fluorescence molecular tomography,FMT)中.全时间分辨(Time Resolved.TR)测量包含了最多的光子传输信息.基于有限元一有限差分扩散方程的正向模型和Newtown-Raphson的逆向模型,将全时间分辨方法用于时域荧光分子层析中.用模拟数据对算法在空间分辨率、定量性、重建尺寸和灰度的保真度以及噪声稳健性等方面进行了验证.结果表明,此方法能够实时重建荧光产率和荧光寿命图像.与以前发展的基于广义脉冲谱技术(Generalized pulse spectrum technique,GPST)的特征数据法进行图像重建相比较.整体上优于广义脉冲谱技术.     

Optically sectioned fluorescence lifetime imaging using a Nipkow disk microscope and a tunable ultrafast continuum excitation source

We demonstrate an optically sectioned fluorescence lifetime imaging microscope with a wide-field detector, using a convenient, continuously tunable (435-1150 nm) ultrafast source for fluorescence imaging applications that is derived from a visible supercontinuum generated in a microstructured fiber.     

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     

Femtosecond Kerr-gated wide-field fluorescence microscopy

We present a Kerr-gated microscope capable of collecting diffraction-limited 2D fluorescence images with sub-100 fs time resolution. The concept is based on the insertion of a solid-state optical Kerr gate into a wide-field microscope. In addition to the considerably improved temporal resolution, the wide-field design allows for simultaneous tracking of several objects and ultrafast fluorescence lifetime imaging of doped and heterogeneous surfaces. The ultrafast fluorescence dynamics of gold nanoparticles is presented as an example of the capabilities of the instrument.     

散斑照明宽场荧光层析显微成像技术研究

介绍了一种新的宽场荧光层析显微方法.在传统宽场显微镜中引入散斑图案照明样品,控制散斑图案的动态变化,利用CCD相机记录对应的一系列荧光图像.由于焦平面内强度变化远比焦平面外强度变化剧烈,通过合适的算法能够获得焦平面的层析分辨的荧光显微图像.标定了系统参数,并研究了不同的图像重建算法对系统性能的影响,获得了不同生物组织样品的层析图像.实验表明,该显微方法能用于组织光学切片成像,在临床医学中具有实际应用价值. 关键词： 荧光 散斑照明 荧光显微 层析     

Hybrid reflecting objectives for functional multiphoton microscopy in turbid media

Most multiphoton imaging of biological specimens is performed using microscope objectives optimized for high image quality under wide-field illumination. We present a class of objectives designed de novo without regard for these traditional constraints, driven exclusively by the needs of fast multiphoton imaging in turbid media: the delivery of femtosecond pulses without dispersion and the efficient collection of fluorescence. We model the performance of one such design optimized for a typical brain-imaging setup and show that it can greatly outperform objectives commonly used for this task.     

Time-Resolved FRET and FLIM of Four-way DNA Junctions

Conformational transitions in a 4-way DNA junction when titrated with ionic solutions are studied using time-resolved fluorescence resonance energy transfer. Parameters characterising the transition in terms of critical ion concentration (c _1/2) and the Hill coefficient for ion binding are obtained by fitting a simple two-state model using steady-state spectra. Data obtained from a fluorescence lifetime plate reader and analysed by fitting a single exponential to donor fluorescence lifetime decays are shown to be in good agreement with the parameters obtained from steady-state measurements. Fluorescence lifetimes, however, offer advantages, particularly in being independent of fluorophore concentration, output intensity, inhomogeneity in the excitation source and output wavelength. We demonstrate preliminary FRET-FLIM images of DNA junction solutions obtained using a picosecond gated CCD which are in agreement with results from a fluorescence lifetime plate reader. The results suggest that time-resolved FRET-FLIM is sensitive to subtle structural changes and may be useful in assays based on 4-way DNA junctions.     

Steady-State and Time-Resolved Fluorescence Polarization Behavior of Acenaphthene

Steady-state and time-resolved fluorescence polarization studies have been carried out on acenaphthene (ACE) in low-temperature glass solutions and at room temperature. In the low-temperature glass the fluorescence polarization values vary considerably with both emission and excitation wavelength. There is a time dependence (on the nanosecond time scale) of the fluorescence anisotropy, r(t), at 77 K, which has a strong dependence upon the excitation and emission wavelengths. Under these conditions, the time-dependent decay of the anisotropy is not attributable to chromophoric motion. The observations are consistent with emission from two closely lying and interconverting excited states. Rate constants for the photophysical processes involved have been determined by fitting the data using a model proposed by Fleming et. al. The results are discussed with particular reference to the care required in using dynamic fluorescence polarization measurements to determine energy transfer rates in systems containing this chromophore.     

Remote time-resolved filament-induced breakdown spectroscopy of biological materials

We report, for what we believe to be the first time, on the feasibility of remote time-resolved filament-induced breakdown spectroscopy (FIBS) of biological materials. The fluorescence from egg white and yeast powder, induced by femtosecond laser pulse filamentation in air, was detected in the backward direction with targets located 3.5 m away from the detection system. The remarkably distinct spectra of egg white and yeast allow us to propose that this technique, time-resolved FIBS, could be potentially useful for remote detection and identification of harmful biological agents.     

Emerging biomedical and advanced applications of time-resolved fluorescence spectroscopy

Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods, are resulting in the rapid migration of timeresolved fluorescence to the clinical chemistry lab, the patient's bedside, and even to the doctor's office and home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. Future horizons of state-of-the-art spectroscopy are also described. Two photon-induced fluorescence provides an increased information content to time-resolved data. Two photoninduced fluorescence, combined with fluorescence microscopy and time-resolved imaging, promises to provide detailed three-dimensional chemical imaging of cells. Additionally, it has recently been demonstrated that the pulses from modern picosecond lasers can be used to quench and/or modify the excited-state population by stimulated emission since the stimulated photons are directed along the quenching beam and are not observed. The phenomenon of light quenching should allow a new class of multipulse time-resolved fluorescence experiments, in which the excited-state population is modified by additional pulses to provide highly oriented systems.     

The concept of 2D gated imaging for particle sizing in a laminar diffusion flame

In this work, time-resolved laser-induced incandescence (TiRe LII) has been employed to measure primary particle diameters of soot in an atmospheric laminar ethylene diffusion flame. The generated data set complements existing data determined in one single location and takes advantage of the good spatial resolution of the ICCD detection. Time resolution is achieved by shifting the camera gate along the LII decay. One key input parameter for the analysis of time-resolved LII is the local flame temperature. This was determined on a grid throughout the flame by coherent anti-Stokes Raman scattering. The accurate temperature data, in combination with other published data from this flame, are well suited for soot model validation purposes while we showed feasibility of a shifted gate approach to deduce 2D particle sizes in the chosen standard flame.     

Detection of Volume Changes in Calcein-Stained Cells Using Confocal Microscopy

Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters – which correspond to relatively thick optical sections – fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285 μm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.     

Multiple-gate time domain diffuse fluorescence tomography allows more sparse tissue sampling without compromising image quality

Diffuse fluorescence tomography systems that employ highly sensitive photo-multiplier tubes for single-photon detection are pushing the sensitivity limits of the field. However, each of these detectors only offers a single data projection to be collected, implying these imaging systems either require many detectors or long scan times to collect full data sets for image reconstruction. This study presents a method of utilizing the time-resolved collection capabilities of time-correlated single-photon counting techniques to increase spatial resolution and to reduce the number of data projections to produce reliable fluorescence reconstructions. Experimental tissue phantom results demonstrate that using data at 10 time gates in the fluorescence reconstructions for only 40 data projections provided superior image accuracy when compared to reconstructions on 320 continuous-wave data projections.     

Study of Solid Transparent Nanocomposite Organic/Inorganic Matrices and Thin Films by Time-Resolved Fluorescence Techniques

This work focuses on the study of nanocomposite organic/inorganic materials, particularly, those made by the sol-gel method, by using time-resolved fluorescence techniques. A model of stretched exponentials is presented and used to fit fluorescence (luminescence) decay profiles for fluorescence quenching reactions obtained by energy-transfer or by diffusion or both. Various types of information on both bulk and thin-film nanocomposite materials can be obtained by such analysis: for example, determination of the percolation threshold for the organic subphase, localization or mobility of incorporated molecular species, and extraction of structural parameters.     

Quantitative Time-Resolved Fluorescence Spectrum of the Cortical Sarcoma and the Adjacent Normal Tissue

The difference in time-resolved fluorescence spectrum between the cortical sarcoma and the adjacent normal tissue was studied in both experimental and theoretical ways. The Clinical data were obtained in vivo using a time-resolved fluorescence spectrometer employing a single fiber-optic probe for excitation and detection. Tissue was modeled as s-180 sarcoma tumor surrounded with normal muscle and was mediated by the Palladium-porphyrin photosensitizer (Pd-TCPP). The emitted fluorescence was considered as arising from the tumor tissue or the normal muscle, due to the presence of the photosensitizer. A computational code which could simulating time-resolved fluorescence emission was presented and applied to comparing fluorescence decay of photosensitizer in different stages of tumor growth. In this code the different stages of the tumor was modeled through changing the time τ, the delay of the fluorescence photon emission and z _max, the thickness of the tumor. It was found in the in vivo experiment that the fluorescence from tumor tissue decayed more quickly than from the adjacent normal muscle. For the ten rats in the first experiment day, the mean decay constant of tumor T _s and normal tissue T _n were 554 and 526 μs, respectively. And T _s increased with the tumor growth, from 554 μs in the first day to 634 μs in the eighth day while T _s kept steady. It was believed that the more adequate oxygen supplied by the normal tissue can more effectively quench the fluorescence and in the normal tissue the photosensitizer lifetime is smaller. As a result the simulated time-resolved fluorescence spectrum of normal tissue showed more quickly decay. And the thickness of the tumor can also delay the fluorescence decay. Both the experimental and simulated results indicated that the germination of the tumor would increase the decay constant of the time-resolved fluorescence spectrum. So decay constant of the tumor tissue spectrum should be larger than that of adjacent normal tissue for the reason of hypoxia and overgrouth. This fact could be of use in the tumor diagnoses.