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.     

Graphical representation and multicomponent analysis of single-frequency fluorescence lifetime imaging microscopy data

Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of proper error‐weighting results in a non‐linear method that is mathematically identical to a global analysis algorithm that was recently derived. The graphical approach was applied to cellular data obtained from a lifetime‐based phosphorylation assay for the epidermal growth factor receptor and yielded results similar to those obtained by a global analysis algorithm.     

FRET microscopy: from principle to routine technology in cell biology

The phenomenon of resonance energy transfer first described by Theodor Förster presents the opportunity of retrieving information on molecular proximity, orientation and conformation on the nanometre scale from (living) samples with conventional fluorescence microscopes (or even macroscopic devices). During the past 10 years Förster (or fluorescence) resonance energy transfer (FRET) microscopy has been revolutionized by the vast progress in fluorescent protein and in situ fluorescent labelling technology as well as by the commercial availability of advanced quantitative microscopy instrumentation. FRET microscopy is now routinely used in modern cell biology research. This short review will guide the reader through the most established FRET microscopy techniques, their inherent strengths and limitations, potential pitfalls, and assist the reader in making an educated choice on the FRET microscopy method most suited for their specific application.     

Multiphoton fluorescence lifetime imaging of human hair

In vivo and in vitro multiphoton imaging was used to perform high resolution optical sectioning of human hair by nonlinear excitation of endogenous as well as exogenous fluorophores. Multiphoton fluorescence lifetime imaging (FLIM) based on time-resolved single photon counting and near-infrared femtosecond laser pulse excitation was employed to analyze the various fluorescent hair components. Time-resolved multiphoton imaging of intratissue pigments has the potential (i) to identify endogenous keratin and melanin, (ii) to obtain information on intrahair dye accumulation, (iii) to study bleaching effects, and (iv) to monitor the intratissue diffusion of pharmaceutical and cosmetical components along hair shafts.     

A practical implementation of multifrequency widefield frequency‐domain fluorescence lifetime imaging microscopy

Widefield frequency‐domain fluorescence lifetime imaging microscopy (FD‐FLIM) is a fast and accurate method to measure the fluorescence lifetime, especially in kinetic studies in biomedical researches. However, the small range of modulation frequencies available in commercial instruments makes this technique limited in its applications. Herein, we describe a practical implementation of multifrequency widefield FD‐FLIM using a pulsed supercontinuum laser and a direct digital synthesizer. In this instrument we use a pulse to modulate the image intensifier rather than the more conventional sine‐wave modulation. This allows parallel multifrequency FLIM measurement using the Fast Fourier Transform and the cross‐correlation technique, which permits precise and simultaneous isolation of individual frequencies. In addition, the pulse modulation at the cathode of image intensifier restores the loss of optical resolution caused by the defocusing effect when the cathode is sinusoidally modulated. Furthermore, in our implementation of this technique, data can be graphically analyzed by the phasor method while data are acquired, which allows easy fit‐free lifetime analysis of FLIM images. Here, our measurements of standard fluorescent samples and a Föster resonance energy transfer pair demonstrate that the widefield multifrequency FLIM system is a valuable and simple tool in fluorescence imaging studies. Microsc. Res. Tech. 76:282–289, 2013. © 2013 Wiley Periodicals, Inc.     

荧光共振能量转移显微术及其新进展

本文叙述荧光共振能量转移显微术及荧光寿命成像显微术的原理、方法及特点。同时介绍利用荧光共振能量转移显微术研究信号分子Rac蛋白在3T3成纤维细胞内的定位及活化过程,以及利用荧光共振能量转移—荧光寿命成像显微术研究转录因子CAATT/增强子结合蛋白α在小鼠垂体细胞内的二聚化现象。     

Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM)

Membrane type 1 matrix metalloproteinase (MT1‐MMP) is a membrane‐tethered collagenase primarily involved in the mechanical destruction of extracellular matrix proteins. MT1‐MMP has also been shown to be upregulated in several types of cancers. Many coordinated functions of MT1‐MMP during migration and invasion remain to be determined. In this paper, live cells from the invasive cell line HT‐1080 were imaged using an intracellular Förster resonance energy transfer‐based biosensor specific for MT1‐MMP; a substrate specific for MT1‐MMP was hybridized with the mOrange2 and mCherry fluorescent proteins to form the Förster resonance energy transfer‐based sensor. The configuration of the biosensor was determined with fluorescence lifetime‐resolved imaging microscopy using both a polar plot‐based analysis and a rapid data acquisition modality of fluorescence lifetime‐resolved imaging microscopy known as phase suppression. Both configurations of the biosensor (with or without cleavage by MT1‐MMP) were clearly resolvable in the same cell. Changes in the configuration of the MT1‐MMP biosensor were observed primarily along the edge of the cell following the removal of the MMP inhibitor GM6001. The intensities highlighted by phase suppression correlated well with the fractional intensities derived from the polar plot.     

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.     

The polarized AB plot for the frequency‐domain analysis and representation of fluorophore rotation and resonance energy homotransfer

The graphical representation of single‐frequency phase‐modulation fluorescence lifetime imaging data, referred to as the AB plot, is extended to take into account measurements of the polarized components of the fluorescence. For a hindered rotator model (characterized with a single excited‐state lifetime, a single rotational correlation time and limiting initial and final anisotropies) the rotational correlation time and the excited lifetime can be determined from the AB plot of any two of the following emission components: parallel, perpendicular, total emission or combinations thereof. A strategy for resolving the component hindered rotations and lifetimes for mixtures of two hindered rotators from measurements of the total, parallel and perpendicular components of the emission is developed. The analysis does not require prior knowledge of the initial limiting anisotropy or of the steady‐state anisotropy or of the excited state lifetime. Plots in polarized AB space derived for heterogeneous systems are constructed to aid interpretation of frequency‐domain dynamic depolarization imaging microscopy experiments. These plots can be used to distinguish spatially dependent rotational correlation time heterogeneity from heterogeneity in limiting anisotropies. The effects of noise and aperture depolarization are discussed. It is anticipated that the polarized AB plot will provide a useful adjunct to existing methods for visualizing and analysing dynamic polarization phenomena arising from molecular dynamics and homo‐energy transfer in single‐frequency microscopy applications.     

Improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime imaging microscopy data

The deconvolution of fluorescence lifetime imaging microscopy (FLIM) data that were processed with global analysis techniques is described. Global analysis of FLIM data enables the determination of relative numbers of molecules in different protein reaction states on a pixel-by-pixel basis in cells. The three-dimensional fluorescence distributions of each protein state can then be calculated and deconvolved. High-resolution maps of the relative concentrations of each state are then obtained from the deconvolved images. We applied these techniques to quantitatively image the phosphorylation state of ErbB1 receptors tagged with green fluorescent protein in MCF7 cells.     

Effects of donor and acceptor's fluorescence lifetimes on the method of applying Förster resonance energy transfer in STED microscopy

Förster resonance energy transfer (FRET) probes being used to improve the resolution of stimulated emission depletion (STED) microscopy are numerically discussed. Besides the FRET efficiency and the excitation intensity, the fluorescence lifetimes of donor and acceptor are found to be another key parameter for the resolution enhancement. Using samples of FRET pairs with shorter donor lifetime and longer acceptor lifetime enhances the nonlinearity of the donor fluorescence, which leads to an increased resolution. The numerical simulation shows that a double resolution improvement of STED microscopy can be achieved by using Cy3–Atto647N samples when compared with that of using standard Cy3‐only samples.