Fiber laser based two-photon FRET measurement of calmodulin and mCherry-E(0)GFP proteins

The speed and accuracy of Förster resonance energy transfer (FRET) measurements can be improved by rapidly alternating excitation wavelengths between the donor and acceptor fluorophore. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two‐photon excitation (TPE). This system offers the potential for rapid wavelength switching with the benefits of axial optical sectioning and improved penetration depth provided by TPE. Correction of FRET signals for cross excitation and cross emission was achieved by switching the excitation wavelength with an electrically controlled modulator. Measurement speed was primarily limited by integration times required to measure fluorescence. Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. In addition, we measured two‐photon induced FRET in an E⁰GFP‐mCherry protein construct. Results from one‐photon and two‐photon excitation are compared to validate the rapid wavelength switched two‐photon measurements. Microsc. Res. Tech. 75:837–843, 2012. © 2011 Wiley Periodicals, Inc.     

Issues in confocal microscopy for quantitative FRET analysis

Previously, we have carried out extensive quantitative analysis of F?rster (or fluorescence) resonance energy transfer (FRET) data to show that polymeric IgA receptors and their ligands cluster in endocytic membranes in the process of sorting and trafficking in polarized cells. Here, we use a similar technique to assay the organization and distribution of another membrane-bound receptor: transferrin receptor (TFR) and its ligand, holo-transferrin (Tfn), while explaining the step-by-step measures to be taken for successful quantitative analysis of the FRET data. In particular, methodological issues in FRET quantitative imaging, such as spectral bleed-through and background correction, optimal selection of regions of interest, how to deal with outliers and pooling data and statistical analysis of FRET data, are addressed. Our results indicating a clustered organization of TFR-Tfn complexes fit the well-known homodimeric structure of TFR. These quantitative approaches can be adapted for other biological applications of FRET.     

Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization

Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.     

Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency

A spectrograph with continuous wavelength resolution has been integrated into a frequency‐domain fluorescence lifetime‐resolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross‐talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral‐FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Förster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral‐FLIM data, the spectral dispersion of the acceptor signal can be used to derive the donor lifetime – and thereby the FRET efficiency – without iterative fitting. The lifetime relation between the donor and acceptor, in conjunction with spectral dispersion, is also used to separate the FRET pair signals from the donor alone signal. This method can be applied further to quantify the signals from separate FRET pairs, and provide information on the dynamics of the FRET pair between different states.     

Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

Frequency-domain fluorescence lifetime imaging microscopy (FLIM) has become a commonly used technique to measure lifetimes in biological systems. However, lifetime measurements are strongly dependent on numerous experimental parameters. Here, we describe a complete calibration and characterization of a FLIM system and suggest parameter optimization for minimizing measurement errors during acquisition. We used standard fluorescent molecules and reference biological samples, exhibiting both single and multiple lifetime components, to calibrate and evaluate our frequency domain FLIM system. We identify several sources of lifetime precision degradation that may occur in FLIM measurements. Following a rigorous calibration of the system and a careful optimization of the acquisition parameters, we demonstrate fluorescence lifetime measurements accuracy and reliability. In addition, we show its potential on living cells by visualizing FRET in CHO cells. The proposed calibration and optimization protocol is suitable for the measurement of multiple lifetime components sample and is applicable to any frequency domain FLIM system. Using this method on our FLIM microscope enabled us to obtain the best fluorescence lifetime precision accessible with such a system. Microsc. Res. Tech., 2009. © 2008 Wiley-Liss, Inc.     

Applying spectral fingerprinting to the analysis of FRET images

F?rster resonance energy transfer (FRET) allows one to study interactions between two fluorescently labeled molecules (donors and acceptors) at distances on the order of 5 nm. Many studies have described methods of how to measure the efficiency of FRET. However, few have addressed the question of how fluorescence from unpaired donors and acceptors can be determined in addition to that from FRET-pairs and how the signal-to-noise ratio (SNR) of such estimates depends on the presence of the partner species. Such knowledge, however, is essential for many biological applications, in which-after initial characterization of the spectral properties of a well-defined donor-acceptor complex-the in vivo affinity and stoichiometry of the complex is of interest. Here, we provide a theoretical analysis on how spectral fingerprinting can be applied to separate fluorescence of FRET pairs from that originating from unpaired donors and acceptors and how to select imaging parameters to optimize the SNR of the estimates. Thereby, we uncover a fundamental problem in this application and discuss ways to evade its adverse consequences. We compare the expected resolution of traditional FRET measures with that of optimized spectral fingerprinting and analyze the resolution of a method for FRET measurements that combines spectral with fluorescence lifetime information.     

Epifluorescence microscopy with a pulsed nitrogen tunable dye laser source

This paper describes an epifluorescence system that uses a pulsed laser source as an excitation source for quantitative fluorescence measurements. A 1 μm diameter portion of the sample can be illuminated with high intensity, short light pulses and the fluorescence can be measured with a fast detection system. Selective excitation is possible at 337 nm and from 357 to 710 nm by using different dyes in the laser system. The spectral bandwidth is 0·1–3 nm. Several advantages over the commonly used systems with continuum spectral sources are indicated, especially when measured intensities are low, as in the case of intrinsic fluorescence of the sample, and where reflection of unwanted excitation light may cause appreciable errors when using conventional light sources.     

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.     

Fluorescence lifetime imaging--techniques and applications

Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time‐domain FLIM by multidimensional time‐correlated single photon counting, time‐domain FLIM by gated image intensifiers, frequency‐domain FLIM by gain‐modulated image intensifiers, and frequency‐domain FLIM by gain‐modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein‐interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity‐based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM‐FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM‐based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM‐FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady‐state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady‐state fluorescence techniques.     

Inhibition of protein adsorption to muscovite mica by monovalent cations

Global analysis of fluorescence lifetime image microscopy (FLIM) data can be used to obtain an accurate fit of multi‐exponential fluorescence decays. In particular, it can be used to fit a bi‐exponential decay to single frequency FLIM data, which is not possible with conventional fitting techniques. Bi‐exponential fluorescence decay models can be used to analyse quantitatively single frequency FLIM data from samples that exhibit fluorescence resonance energy transfer (FRET). Global analysis algorithms simultaneously fit multiple measurements acquired under different experimental conditions to achieve higher accuracy. To demonstrate that bi‐exponential models can indeed be fitted to single frequency data, we derive an analytical solution for the special case of two measurements and use this solution to illustrate the properties of global analysis algorithms. We also derive a novel global analysis algorithm that is optimized for single frequency FLIM data, and demonstrate that it is superior to earlier algorithms in terms of computational requirements.     

Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing

Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein–protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed‐through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed‐through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)‐ and yellow fluorescent protein (YFP)‐tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well‐established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.     

Multi-dimensional fluorescence lifetime and FRET measurements

When and where proteins associate with each other in living cells are key questions in many biological research projects. One way to address these questions is to measure the extent of F?rster resonance energy transfer (FRET) between proteins that have been labeled with appropriate donor and acceptor fluorophores. When both proteins interact, donor and acceptor fluorophores are brought into close vicinity so that the donor can transmit a part of its excitation energy to the acceptor. As a result, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches to determine FRET efficiency: One is to detect changes in the intensity of donor and acceptor emission, the other is to measure changes in the lifetime of the donor molecule. One important advantage of the fluorescence lifetime approach is that it allows to distinguish between free and associated donor molecules. However, like intensity measurements it lacks an intrinsic control ensuring that changes in the measured parameters are only due to FRET and not other quenching processes. Here, we show how this limitation can be overcome by spectrally resolved fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting approach. Both approaches allow biologists to record both donor and acceptor fluorescence emitted by the sample in a single measurement.     

Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy

We present here the phasor approach to biosensor Förster resonance energy transfer (FRET) detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors, which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is transformed into a two‐dimensional coordinate system where independent mixtures of fluorophores can be distinguished from changes in lifetime due to FRET. This enables the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor to be quantified in each pixel of an image. The physical properties intrinsic to each biosensor design are also accurately characterized by the phasor analysis; thus, this method could be used to inform biosensor optimization at the developmental stage. We believe that as biosensors become more sophisticated and are multiplexed with other fluorescent molecular tools, biosensor FRET detection by the phasor approach to FLIM will not only become imperative to their use but also their advancement. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching

To study protein–protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor–acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein–protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP-YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV-induced single-strand DNA damage. ERCC1 and XPF form a heterodimeric 5' endonuclease in nucleotide excision repair. FRET between ERCC1-GFP and XPF-YFP occurs with an efficiency of 30%.     

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.     

Quantitative FRET measurement using emission‐spectral unmixing with independent excitation crosstalk correction

Quantification of fluorescence resonance energy transfer (FRET) needs at least two external samples, an acceptor‐only reference and a linked FRET reference, to calibrate fluorescence signal. Furthermore, all measurements for references and FRET samples must be performed under the same instrumental conditions. Based on a novel notion to predetermine the molar extinction coefficient ratio (R_C) of acceptor‐to‐donor for the correction of acceptor excitation crosstalk, we present here a robust and independent emission‐spectral unmixing FRET methodology, Iem‐spFRET, which can simultaneously measure the E and R_C of FRET sample without any external references, such that Iem‐spFRET circumvents the rigorous restriction of keeping the same imaging conditions for all FRET experiments and thus can be used for the direct measurement of FRET sample. We validate Iem‐spFRET by measuring the absolute E and R_C values of standard constructs with different acceptor‐to‐donor stoichiometry expressed in living cells. Our results demonstrate that Iem‐spFRET is a simple and powerful tool for real‐time monitoring the dynamic intermolecular interaction within single living cells.     

Recognition of Borehole Radar Cable-Related Effects Using Variable Offset Sounding

Conductive cables can influence borehole radar measurements and introduce artifacts into data and therefore must be considered during data analysis and interpretation. This study presents examples of some cable-related effects in data acquired with a radar system that relies on conductive cables for signal transmission. Data show that measurements can be affected when energy radiated from the transmitter antenna induces currents on conductive cables, which can function as an electromagnetic waveguide, allowing fields to propagate along cables and be detected by the receiver antenna. Additionally, periodic artifacts can result when currents traveling on cables reflect at system impedance mismatches.Variable offset soundings (VOS) are not typically conducted during borehole radar studies, but can be useful for recognizing cable-related effects on recorded data and studying propagation characteristics in a borehole. In addition to single-hole VOS measurements, VOS measurements made on the ground surface using E-Plane and H-Plane configurations are shown to have the potential for providing additional insight in regards to coupling mechanisms between borehole antennas and cables.     

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device

A novel compact illumination device in variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four‐layer model, as well as a Gaussian‐shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell–substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time‐resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).     

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.     

Multi-dimensional time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) to detect FRET in cells

We present a novel, multi‐dimensional, time‐correlated single photon counting (TCSPC) technique to perform fluorescence lifetime imaging with a laser‐scanning microscope operated at a pixel dwell‐time in the microsecond range. The unsurpassed temporal accuracy of this approach combined with a high detection efficiency was applied to measure the fluorescent lifetimes of enhanced cyan fluorescent protein (ECFP) in isolation and in tandem with EYFP (enhanced yellow fluorescent protein). This technique enables multi‐exponential decay analysis in a scanning microscope with high intrinsic time resolution, accuracy and counting efficiency, particularly at the low excitation levels required to maintain cell viability and avoid photobleaching. Using a construct encoding the two fluorescent proteins separated by a fixed‐distance amino acid spacer, we were able to measure the fluorescence resonance energy transfer (FRET) efficiency determined by the interchromophore distance. These data revealed that ECFP exhibits complex exponential fluorescence decays under both FRET and non‐FRET conditions, as previously reported. Two approaches to calculate the distance between donor and acceptor from the lifetime delivered values within a 10% error range. To confirm that this method can be used also to quantify intermolecular FRET, we labelled cultured neurones with the styryl dye FM1‐43, quantified the fluorescence lifetime, then quenched its fluorescence using FM4‐64, an efficient energy acceptor for FM1‐43 emission. These experiments confirmed directly for the first time that FRET occurs between these two chromophores, characterized the lifetimes of these probes, determined the interchromophore distance in the plasma membrane and provided high‐resolution two‐dimensional images of lifetime distributions in living neurones.