Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell

Dual-color fluorescence correlation spectroscopy is an interesting method to quantify protein interaction in living cells. But, when performing these experiments, one must compensate for a known spectral bleed through artifact that corrupts cross-correlation data. In this article, problems with crosstalk were overcome with an approach based on fluorescence lifetime correlation spectroscopy (FLCS). We show that FLCS applied to dual-color EGFP and mCherry cross-correlation allows the determination of protein-protein interactions in living cells without the need of spectral bleed through calibration. The methodology was validated by using EGFP-mCherry tandem in comparison with coexpressed EGFP and mCherry in live cell. The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during experiment is really very helpful to study quantitatively protein interactions in live sample.     

Correlated fluorescence lifetime and spectral measurements in living cells

Studies of proteins' interaction in cells by FRET can take benefit from two important photo-physical properties describing fluorescent proteins: fluorescence emission spectrum and fluorescence lifetime. These properties provide specific and complementary information about the tagged proteins and their environment. However, none of them taken individually can completely quantify the involved fluorophore characteristics due to their multiparametric dependency with molecular environment, experimental conditions, and interpretation complexity. A solution to get a better understanding of the biological process implied at the cellular level is to combine the spectral and temporal fluorescence data acquired simultaneously at every cell region under investigation. We present the SLiM-SPRC160, an original temporal/spectral acquisition system for simultaneous lifetime measurements in 16 spectral channels directly attached to the descanned port of a confocal microscope with two-photon excitation. It features improved light throughput, enabling low-level excitation and minimum invasivity in living cells studies. To guarantee a fairly good level of accuracy and reproducibility in the measurements of fluorescence lifetime and spectra on living cells, we propose a rigorous protocol for running experiments with this new equipment that preserves cell viability. The usefulness of SLiM approach for the precise determination of overlapping fluorophores is illustrated with the study of known solutions of rhodamine. Then, we describe reliable FRET experiments in imaging mode realized in living cells using this protocol. We also demonstrate the benefit of localized fluorescence spectrum-lifetime acquisitions for the dynamic study of fluorescent proteins. proteins.     

Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation

The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.     

基于时间相关单光子计数的非接触式荧光层析成像系统

基于时间相关单光子计数(TCSPC)技术原理,研究了一套面向早期乳腺癌检测的时域荧光层析成像系统,系统采用非接触式空间光扫描测量方式,相对于传统的光纤接触式测量方法,可增大空间采样量、减小测量误差、提高空间分辨率.通过仿体实验对系统的成像质量进行了验证,结果表明:单目标仿体实验能较好地对荧光产率与寿命进行重建;在双目标仿体荧光产率的重建中,可有效对中心距为20mm、边距为15mm的目标体进行分辨,且对不同浓度荧光剂目标的重建具有较好的线性.实验结果证明系统应用于乳腺检测的可行性,进一步发展可有望应用于临床乳腺成像中.     

Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MHC protein at an immunological synapse*

Fluorescence imaging of green fluorescent protein (GFP) may be used to locate proteins in live cells and fluorescence lifetime imaging (FLIM) may be employed to probe the local microenvironment of proteins. Here we apply FLIM to GFP-tagged proteins at the cell surface and at an inhibitory natural killer (NK) cell immunological synapse (IS). We present a novel quantitative analysis of fluorescence lifetime images that we believe is useful to determine whether apparent FLIM heterogeneity is statistically significant. We observe that, although the variation of observed fluorescence lifetime of GFP-tagged proteins at the cell surface is close to the expected statistical range, the lifetime of GFP-tagged proteins in cells is shorter than recombinant GFP in solution. Furthermore the lifetime of GFP-tagged major histocompatibility complex class I protein is shortened at the inhibitory NK cell IS compared with the unconjugated membrane. Following our previous work demonstrating the ability of FLIM to report the local refractive index of GFP in solution, we speculate that these lifetime variations may indicate local refractive index changes. This application of our method for detecting small but significant differences in fluorescence lifetimes shows how FLIM could be broadly useful in imaging discrete membrane environments for a given protein.     

共聚焦荧光寿命显微系统

提出一种集成了激光扫描共聚焦显微术和荧光寿命测量技术的共聚焦荧光寿命显微系统,用于观察生物细胞等样品微观结构以及对环境条件信息成像。通过配合使用空心角锥棱镜和平面反射镜简化光路调整,成功搭建系统。实验结果显示,系统的空间分辨率能达到约180nm,荧光寿命时间分辨率达到约20ps。本文系统能为生物细胞学、材料学等学科研究提供直观途径。     

基于多维TCSPC技术的心肌细胞Ca2+动力学研究

采用Fluo-4和Ca-orange标记细胞质及线粒体中的Ca2 ,通过多维时间相关单光子计数技术研究心肌收缩时Ca2 动力学.结果表明,在心肌收缩后10～110 ms的Fluo-4荧光强度明显强于心肌收缩后1 s的荧光强度,归一化光谱和细胞自发荧光一致;静止状态下,成分荧光寿命时间和相关振幅在峰值540nm波长处τ1=(0.42±0.01)ns(73±5)%,τ2=(2.74±0.67)ns(25±5)%,平均荧光衰减时间τmean=0.83 ns;Ca-orange荧光峰值560 nm,加入线粒体呼吸链阻断剂Rotenone后,10～110 ms的Ca-orange荧光强度显著降低.综合评估了心肌细胞收缩时细胞质和线粒体中荧光团的光谱及时间分辨荧光特性,该方法可为心肌收缩过程提供满意的光谱和时间分辨荧光记录,有助于理解细胞综合行为.     

The Gray Institute ‘open’ high‐content,fluorescence lifetime microscopes

We describe a microscopy design methodology and details of microscopes built to this ‘open’ design approach. These demonstrate the first implementation of time‐domain fluorescence microscopy in a flexible automated platform with the ability to ease the transition of this and other advanced microscopy techniques from development to use in routine biology applications. This approach allows easy expansion and modification of the platform capabilities, as it moves away from the use of a commercial, monolithic, microscope body to small, commercial off‐the‐shelf and custom made modular components. Drawings and diagrams of our microscopes have been made available under an open license for noncommercial use at http://users.ox.ac.uk/~atdgroup . Several automated high‐content fluorescence microscope implementations have been constructed with this design framework and optimized for specific applications with multiwell plates and tissue microarrays. In particular, three platforms incorporate time‐domain FLIM via time‐correlated single photon counting in an automated fashion. We also present data from experiments performed on these platforms highlighting their automated wide‐field and laser scanning capabilities designed for high‐content microscopy. Devices using these designs also form radiation‐beam ‘end‐stations’ at Oxford and Surrey Universities, showing the versatility and extendibility of this approach.     

First results of the LHC longitudinal density monitor

The Large Hadron Collider (LHC) at CERN is the world's largest particle accelerator. It is designed to accelerate and collide protons or heavy ions up to the center-of-mass energies of 14 TeV.Knowledge of the longitudinal distribution of particles is important for various aspects of accelerator operation, in particular to check the injection quality and to measure the proportion of charge outside the nominally filled bunches during the physics periods. In order to study this so-called ghost charge at levels very much smaller than the main bunches, a longitudinal profile measurement with a very high dynamic range is needed.A new detector, the LHC Longitudinal Density Monitor (LDM) is a single-photon counting system measuring synchrotron light by means of an avalanche photodiode detector. The unprecedented energies reached in the LHC allow synchrotron light diagnostics to be used with both protons and heavy ions.A prototype was installed during the 2010 LHC run and was able to longitudinally profile the whole ring with a resolution close to the target of 50 ps. On-line correction for the effects of the detector deadtime, pile-up and afterpulsing allow a dynamic range of 10⁵ to be achieved.First measurements with the LDM are presented here along with an analysis of its performance and an outlook for future upgrades.