FRET- FLIM

In common laser scanning fluorescence microscopes a large amount of information is lost because only averaged information of a number of photons, e.g. the intensity, is recorded. In contrast, Time-Correlated Single Photon Counting offers the possibility to overcome these limitations, because it allows to store spatial, timing and spectral information for every detected photon individually. Traditional acquisition techniques like imaging (including FRET and FLIM) and sophisticated point measurements (e.g. FCS) can now be implemented based on this ultimately flexible data collection scheme called Time-Tagged Time-Resolved (TTTR) mode. All possible analysis methods can thereby be applied to the same raw data set. The optical design of the presented system (MicroTime 200) is based on a standard inverted microscope equipped with a three dimensional piezo scanning device which allows either for sample or probe (objective) scanning, which also permits to investigate heavy and/or bulky samp...

A global fit assumes that one or several of the lifetimes of a multi-exponential decay are constant in all pixels of a FLIM image. By using this a priori knowledge, the decay parameters can be obtained at a better
accuracy than by a multi-exponential fit with all decay parameters floating. However, a global fit is enormously computation-intensive. Processing times with standard CPU processing are therefore unacceptably long. We
present a Global Fit algorithm based on GPU processing that reduces the calculation time from previously hours to a few minutes and less.

We demonstrate Z stack imaging of a fly (Musca domestica) with 289 Z planes of 2.5 μm Z step width, 1024 x 1024 pixels in X and Y, and 1024
channels in time by using existing functions of the Becker & Hickl DCS-120 confocal FLIM system. Doubleexponential decay analysis was performed by the batch processing function of Becker & Hickl SPCImage NG. 3D reconstructions on the basis of the SPCImage results were performed by ImageJ / FIJI.

We demonstrate two-photon FLIM with a FemtoFibre smart 780 laser of Toptica Photonics AG in combination with a Becker & Hickl DCS-120 MP TCSPC / FLIM system. The laser delivers femtosecond pulses at 783 nm, 80 MHz repetition rate, and 125 mW average power. We show that the laser power is sufficient to obtain highresolution images in all the commonly performed FLIM applications. We demonstrate the performance of the system for label-free (autofluorescence) imaging of tissue, label-free imaging of cells, FLIM of ultrafast fluorescence decay in biological material, and FLIM of fluorophore-labelled cells and tissues.

αB-crystallin (heat shock protein β5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A ...

Förster resonance energy transfer (FRET) imaging is an essential analytical method in biomedical research. The limited photon-budget experimentally available, however, imposes compromises between spatiotemporal and biochemical resolutions, photodamage and phototoxicity. The study of photon-statistics in biochemical imaging is thus important in guiding the efficient design of instrumentation and assays. Here, we show a comparative analysis of photon-statistics in FRET imaging demonstrating how the precision of FRET imaging varies vastly with imaging parameters. Therefore, we provide analytical and numerical tools for assay optimization. Fluorescence lifetime imaging microscopy (FLIM) is a very robust technique with excellent photon-efficiencies. However, we show that also intensity-based FRET imaging can reach high precision by utilizing information from both donor and acceptor fluorophores.

Time-of-Flight (ToF) technologies are developed mainly for range estimations in industrial applications or consumer products. Recently, it was realized that ToF sensors could also be used for the detection of fluorescence and of the minute changes in the nanosecond-lived electronic states of fluorescent molecules. This capability can be exploited to report on the biochemical processes occurring within living organisms. ToF technologies, therefore, provide new opportunities in molecular and cell biology, diagnostics, and drug discovery. In this short communication, the convergence of the engineering and biomedical communities onto ToF technologies and its potential impact on basic, applied and translational sciences are discussed.

The intracellular N-terminus of human ether-ago go related gene (HERG) potassium channels constitutes a key determinant of activation and deactivation characteristics and is necessary for hormone-induced modifications of gating properties. However, the general organization of the long amino and carboxy HERG terminals remains unknown. In this study we performed fluorescence resonance energy transfer (FRET) microscopy with a library of fluorescent HERG fusion proteins obtained combining site-directed and transposon-based random insertion of GFP variants into multiple sites of HERG. Determinations of FRET efficiencies with functional HERG channels labeled in different combinations localize the fluorophores, introduced in the amino and carboxy ends, in two quadratic planes of 7.8 and 8.6 nm lateral size, showing a vertical separation of nearly 8 nm without major angular torsion between the planes. Similar analysis using labels at positions 345 and 905 of the amino and carboxy terminals, located them slightly above the planes delimited by the amino and carboxy end labels, respectively. Our data also indicate an almost vertical arrangement of the fluorophores introduced in the NH 2 and COOH ends and at position 905, but a near 45°angular rotation between the planes delimited by these labels and the 345-located fluorophores. Systematic triangulation using interfluorophore distances coming from multiply labeled channels provides an initial constraint on the overall in vivo arrangement of the HERG cytoplasmic domains, suggesting that the C-linker/CNBD region of HERG hangs centrally below the transmembrane core, with the initial portion of the amino terminus around its top and side surfaces directed towards the gating machinery.

Directional and circularly polarised emission from quantum dots coupled with chiral Ag nanostructures has been demonstrated. Semiconductor quantum dots were deposited onto the chiral structures using the Layer-by-Layer deposition technique. Time-resolved photoluminescence measurements demonstrated a lifetime quenching efficiency of 82%, confirming the strong interaction between the emitters and the antennas. Angle- and polarisation-resolved photoluminescence measurements revealed that the chiral nanostructures influence the far-field emission properties of the quantum emitters through dipole coupling. The far-field emission from the quantum dots coupled to the chiral nanostructures displays directionality in the emission pattern and more than 17% circular polarisation, following the handedness of the chiral structure.

The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two nonnative IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners.

Movies have held a mesmerizing grip on humanity ever since the Lumière brothers presented the first motion picture to a stunned audience in 1895. FMOVIES From the silent era's poignant expressions to today's immersive digital experiences, the art of filmmaking has continually evolved, shaping and reflecting our culture, aspirations, and imaginations. Early Days: Birth of a Medium In the early 20th century, silent films dominated the cinematic landscape. Icons like Charlie Chaplin and Buster Keaton brought laughter and tears without uttering a word, relying solely on their physical prowess and emotive expressions. These pioneers laid the foundation for storytelling through moving images, demonstrating the profound impact of visuals in communicating narratives.

Xavier Michalet received an Engineering Degree from Ecole Polytechnique (France), before getting acquainted with research in soft condensed matter under the supervision of David Bensimon at the Ecole Normale Supérieure. After his doctorate, he joined the group of Aaron Bensimon, with whom he developed genetic applications of DNA molecular combing. He joined Shimon Weiss's group at the Lawrence Berkeley National Laboratory, working on quantum dots applications to biology. He is now a project scientist at UCLA, working in the Weiss group on single-molecule studies of receptor diffusion, protein folding, mapping of transcription factor binding sites on whole genomes, and developing various techniques and detectors for single-molecule spectroscopy and microscopy. Shimon Weiss is a Professor in the Department of Chemistry and Biochemistry and in the Department of Physiology at UCLA. He was previously a staff scientist at Lawrence Berkeley Labs, where, since 1991, he has been involved in nanoscale science and single-molecule spectroscopy. He pioneered research on single-molecule spectroscopy and its application to biology. His team was the first to detect FRET between a single donor and a single acceptor molecule, a technique that is now widely used to study the structure and conformational dynamics of macromolecules. He also pioneered the use of fluorescent semiconductor nanocrystals, or quantum dots, as biological probes, revolutionizing the use of fluorescence in modern biotechnology and cellular imaging. His group is currently involved in research on molecular imaging with quantum dots and single-molecule studies of transcription initiation, membrane receptor dynamic, and protein folding, among many others.

In the realm of cinematic storytelling, the evolution of technology has played a significant role in shaping narratives and driving innovation. From the advent of sound in the 1920s to the rise of CGI in the modern era, filmmakers have continuously embraced new tools and techniques to bring their stories to life on the silver screen. However, amidst the digital revolution, there exists a nostalgic fascination with analog communication devices, such as the humble beeper. In this article, we delve into the world of "beeper movies," exploring how these devices have been portrayed and utilized in cinema, and the cultural significance they hold in an increasingly digital age.

Conformational changes in biomolecules underpin all biological processes and being able to quantify these structural changes in solution is crucial to understand biological function. By carefully positioning two fluorophores within the biomolecule, it is possible to use fluorescence resonance energy transfer (FRET) as a molecular ruler to measure the desired distance. In current FRET assays, the biomolecule needs to be labelled with two different chemical fluorophores acting as a donor-acceptor FRET pair. Incorporation of these two chemically different species at specific positions within the biomolecule is challenging due to limited chemical labelling strategies. Here, we present a radically different strategy for measuring distances in biomolecules. We have developed the concept of TWIN-FRET which removes the need for two different fluorophores attached to the biomolecule by chemically encoding the FRET pair within the structure of the fluorophore itself. We have designed and synthesised a fluorescent molecule to prove this concept. The fluorophore (FH) has an acid-base equilibrium with a ground state pKa$8.9. We have derivatized the fluorophore to its succinimide ester, and used this derivative, to label a duplex DNA with two molecules of the same fluorophore at specific positions. Our results demonstrate the transfer of non-radiative energy from the neutral (FH) to the anionic (F À) state of the fluorescent molecule. We further demonstrate the use of TWIN-FRET to measure nanometer-size distances within the DNA duplex, and we obtained distance values similar to those obtained using a conventional FRET pair (Alexa488-Cy3). By removing the need to introduce two different chemical structures within the biomolecule, we greatly simplified the methodology to measure nanometer-size distances in biomolecules. We expect this technique to be widely used in structural and biophysical studies of nucleic acids, proteins and interactions between them.

In this paper, the detection of a thyroid cancer market (Thyroglobulin, Tg) protein has demonstrated by using a hybrid plasmonic bimetallic nanoshell-microsphere sensor based on the reactive sensing principal (RSP). Tunable optical properties and plasmon resonances at three frequencies made a bimetallic nanoshell more special for sensing purposes. The rough surface of the bimetallic nanoshell affixed to the microsphere surface locally enhanced the electric field within the microcavity without significantly degrading a Q-factor (7 .59 × 10 5), at near infrared wavelength. The rough surface of the bimetallic nanoshell effectively enhanced a wavelength shift of the hybrid microcavity by the factor of~300. At lower energy mode, the electric field enhancement of the hybrid plasmonic microsphere resonator increases by~863% at the surface of the bump in comparison with a smooth nanoshell. This enhancement occurs in rough bimetallic nanoshell as localized surface Plasmon resonance (LSPR), which increases the Raman signal through the surface Raman scattering (SERS) effect in hybrid microcavity. Calculated results demonstrate that the hybrid plasmonic microsphere resonator can generate very strong SERS enhancement factor at a visible wavelength (~1.5 × 10 7).

A formation of pseudoisocyanine (PIC) dye Jaggregates in the polyelectrolyte film by the layer-by-layer (LbL) assembly method has been studied. It has been shown that this process leads to significant J-band widening and fluorescence quenching as a result of increasing static disorder. To enhance the J-aggregate fluorescence properties, the effect of J-aggregate interaction with plasmon resonances of gold nanoparticles has been used. It is found that the maximal 8-fold fluorescence enhancement for PIC J-aggregates in the LbL films could be achieved at 16 nm distance between Au nanoparticles (NPs) and the J-aggregates. Plasmon influence on the J-aggregate fluorescence has been analyzed using a two-level system in the local plasmon field approximation. The model gives a good correlation with the experimental results and could be used for further studying the exciton−plasmon interaction in J-aggregates.

Epidermal growth factor receptor (EGFR) is frequently mutated in non-small cell lung cancer (NSCLC) where it has been successfully targeted with tyrosine kinase inhibitors, but these are eventually overcome by drug resistance. Overcoming this therapeutic challenge has been hindered by the poor understanding of how wild type (WT)-EGFR elicits ligand-independent signals essential for cell homeostasis, which are then hijacked by cancer mutations. Ligand-independent signaling cannot be explained by the autoinhibited ligand-free EGFR dimer and oligomer structures so far available. Here we show that EGFR achieves ligand-independent phosphorylation by assembling dimer conformers into obligate ligand-free hetero-conformational oligomers. We reveal the structure of these ligand-free oligomers and their mechanisms of assembly. We also show how these mechanisms are exploited by drug-resistant T766M and Ex20ins EGFR mutants, revealing oncogenic functions for hitherto orphan transmembrane and ki...

Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. Th...

We have developed a model-based, parallel procedure to estimate fluorescence lifetimes. Multiple frequencies are present in the excitation signal. Modeling the entire fluorescence and measurement process produces an analytical ratio of polynomials in the lifetime variable τ. A non-linear model-fitting procedure is then used to estimate τ. We have analyzed this model-based approach by simulating a 10 µM fluorescein solution (τ = 4 ns) and all relevant noise sources. We have used real LED data to drive the simulation. Using 240 µs of data, we estimate τ = 3.99 ns. Preliminary experiments on real fluorescent images taken from fluorescein solutions (measured τ = 4.1 ns), green plastic test slides (measured τ = 3.0 ns), and GFP in U2OS (osteosarcoma) cells (measured τ = 2.1 ns) demonstrate that this model-based measurement technique works.

Fast frame rate complementary metal-oxide-semiconductor cameras in combination with photon counting image intensifiers can be used for microsecond resolution wide-field fluorescence lifetime imaging with single photon sensitivity, but the time resolution is limited by the camera exposure time. We show here how the image intensifierʼs P20 phosphor afterglow can be exploited for accurate timing of photon arrival well below the camera exposure time. By taking ratios of the intensity of the photon events in two subsequent frames, photon arrival times were determined with 300 ns precision with 18.5 μs frame exposure time (54 kHz camera frame rate). Decays of ruthenium and iridiumcontaining compounds with around 1 μs lifetimes were mapped with this technique, including in living HeLa cells, using excitation powers below 0.5 μW. Details of the implementation to calculate the arrival time from the photon event intensity ratio are discussed, and we speculate that by using an image intensifier with a faster phosphor decay to match a higher camera frame rate, photon arrival time measurements on the nanosecond time scale could be possible.

We present the results of experiments designed to create and detect Surface Plasmon Polaritons (SPP) using low intensity light such that SPP excitation is achieved under extremely low intensity condition. This is accomplished using an experimental apparatus which utilizes a laser-intensity varying system, a PMMA on gold on glass sample, and a Single Photon Counting Module (SPCM). We image the characteristic SPP propagation ring using a typical CCD camera and find the corresponding peak intensities using low intensity pump methods and the SPCM.

Time-Correlated Single Photon Counting (TCSPC) is a very efficient technique for measuring weak and fast optical signals, but it is mainly limited by the relatively “long” measurement time. Multichannel systems have been developed in recent years aiming to overcome this limitation by managing several detectors or TCSPC devices in parallel. Nevertheless, if we look at state-of-the-art systems, there is still a strong trade-off between the parallelism level and performance: the higher the number of channels, the poorer the performance. In 2013, we presented a complete and compact 32 × 1 TCSPC system, composed of an array of 32 single-photon avalanche diodes connected to 32 time-to-amplitude converters, which showed that it was possible to overcome the existing trade-off. In this paper, we present an evolution of the previous work that is conceived for high-throughput fluorescence lifetime imaging microscopy. This application can be addressed by the new system thanks to a centralized l...

Different-sized, 3-mercaptopropionic acid (MPA) stabilized CdTe quantum dots (QDs) have been prepared in aqueous solution, and potential cosensitization of such QDs in ZnO nanorod (NR)-based dye-sensitized solar cells (DSSCs) has been established. The results presented in this study highlight two major pathways by which CdTe QDs may contribute to the net photocurrent in a DSSC: (1) a direct injection of charge carriers from QDs to ZnO semiconductor via photoinduced electron transfer (PET) and (2) an indirect excitation of the sensitizing dye (SD) N719 molecules by funneling harvested light via Forster resonance energy transfer (FRET). The steady-state and picosecond-resolved luminescence measurements were combined to clarify the process of PET and FRET from the excited QDs to ZnO NR and SD N719, respectively. On the basis of these advantages, the short-circuit current density and the photoconductivity of the QD-assembled DSSCs with distinct architectures are found to be much higher than DSSCs fabricated with N719 sensitizer only.

The engineered coupling between a guest moiety (molecule, nanoparticle) and the host photonic nanostructure may provide a great enhancement of the guest optical response, leading to many attractive applications. In this article, we describe briefly the basic concept and some recent progress considering the coupling of a single nanoparticle into a photonic structure. Different kinds of nanoparticles of great interest including quantum dots and nitrogen-vacancy centers in nanodiamond for single photon source, nonlinear nanoparticles for efficient nonlinear effect and sensors, magnetic nanoparticles for Kerr magneto-optical effect, and plasmonic nanoparticles for ultrafast optical switching and sensors, are briefly reviewed. We focus further on the coupling of plasmonic gold nanoparticles and polymeric photonic structures by optimizing theoretically the photonic structures and developing efficient way to realize desired hybrid structures. The simple and low-cost fabrication technique, the optical enhancement of the fluorescent nanoparticles induced by the photonic structure, as well as the limitations, challenges and appealing prospects are discussed in details.

Epidermal growth factor receptor (EGFR) is frequently mutated in non-small cell lung cancer (NSCLC) where it has been successfully targeted with tyrosine kinase inhibitors, but these are eventually overcome by drug resistance. Overcoming this therapeutic challenge has been hindered by the poor understanding of how wild type (WT)-EGFR elicits ligand-independent signals essential for cell homeostasis, which are then hijacked by cancer mutations. Ligand-independent signaling cannot be explained by the autoinhibited ligand-free EGFR dimer and oligomer structures so far available. Here we show that EGFR achieves ligand-independent phosphorylation by assembling dimer conformers into obligate ligand-free hetero-conformational oligomers. We reveal the structure of these ligand-free oligomers and their mechanisms of assembly. We also show how these mechanisms are exploited by drug-resistant T766M and Ex20ins EGFR mutants, revealing oncogenic functions for hitherto orphan transmembrane and ki...

The engineered coupling between a guest moiety (molecule, nanoparticle) and the host photonic nanostructure may provide a great enhancement of the guest optical response, leading to many attractive applications. In this article, we describe briefly the basic concept and some recent progress considering the coupling of a single nanoparticle into a photonic structure. Different kinds of nanoparticles of great interest including quantum dots and nitrogen-vacancy centers in nanodiamond for single photon source, nonlinear nanoparticles for efficient nonlinear effect and sensors, magnetic nanoparticles for Kerr magneto-optical effect, and plasmonic nanoparticles for ultrafast optical switching and sensors, are briefly reviewed. We focus further on the coupling of plasmonic gold nanoparticles and polymeric photonic structures by optimizing theoretically the photonic structures and developing efficient way to realize desired hybrid structures. The simple and low-cost fabrication technique, the optical enhancement of the fluorescent nanoparticles induced by the photonic structure, as well as the limitations, challenges and appealing prospects are discussed in details.

Förster resonance energy transfer (FRET) is a distance-dependent process that transfers excited state energy from a donor molecule to an acceptor molecule without the emission of a photon. The FRET rate is determined by the proximity between the donor and the acceptor molecules; it becomes significant only when the proximity is within several nanometers. Therefore, FRET has been applied to visualize interactions and conformational changes of biomolecules, such as proteins, lipids, and nucleic acids that cannot be resolved by optical microscopy. Here, we report photoacoustic tomography of FRET efficiency at a 1-cm depth in chicken breast tissue, whereas conventional high-resolution fluorescence imaging is limited to <0.1 cm. Photoacoustic tomography is expected to facilitate the examination of FRET phenomena in living organisms.

While methods for the determination of protein structure in vitro are highly advanced, the question always remains as to whether the observations are representative of the molecule in its native environment. Fluorescence resonance energy transfer (FRET) is widely used to probe protein structure both in vitro and in vivo. Because of their ease of use, in vivo FRET measurements frequently rely on the cyan and yellow fluorescent proteins (i.e. CFP donor and YFP acceptor). The severe spectral overlap and significant direct excitation of the acceptor complicate extraction of true FRET efficiency. Furthermore, the large size and poor photophysical properties of fluorescent proteins (FPs) make them a less than ideal FRET pair. To benchmark the reliability of FP-FRET as a probe of protein conformation in vivo, we characterized a series of CFP-YFP tandems with varying linker lengths. Ensemble and single molecule fluorescence were used to measure FRET in vitro. We then measured FRET in live cells with total internal reflection fluorescence microscopy. Only by using a single filter cube with a ratiometric image splitter, were we able to get agreement between in vitro and in vivo measurements. However, measurements of the radius of hydration, using analytical ultracentrifugation and chromatography, revealed a much different size distribution than was suggested by the FRET dependence on the linker length between CFP and YFP. Our studies suggest that the conformation of our protein test set is the same in live cells and in solution. In vitro and in vivo measurements for each construct were in good agreement. Unfortunately, distances derived from FP-FRET appear to misrepresent the actual interchromophore distance.

Spectral unmixing is the process of breaking down data from a sample into its basic components and their abundances. Previous work has been focused on blind unmixing of multi-spectral fluorescence lifetime imaging microscopy (m-FLIM) datasets under a linear mixture model and quadratic approximations. This method provides a fast linear decomposition and can work without a limitation in the maximum number of components or end-members. Hence this work presents an interactive software which implements our blind end-member and abundance extraction (BEAE) and quadratic blind linear unmixing (QBLU) algorithms in Matlab. The options and capabilities of our proposed software are described in detail. When the number of components is known, our software can estimate the constitutive end-members and their abundances. When no prior knowledge is available, the software can provide a completely blind solution to estimate the number of components, the end-members and their abundances. The characterization of three case studies validates the performance of the new software: ex-vivo human coronary arteries, human breast cancer cell samples, and in-vivo hamster oral mucosa. The software is freely available in a hosted webpage by one of the developing institutions, and allows the user a quick, easy-to-use and efficient tool for multi/hyper-spectral data decomposition.

In some applications of biomedical imaging, a linear mixture model can represent the constitutive elements (end-members) and their contributions (abundances) per pixel of the image. In this work, the extended blind end-member and abundance extraction (EBEAE) methodology is mathematically formulated to address the blind linear unmixing (BLU) problem subject to positivity constraints in optical measurements. The EBEAE algorithm is based on a constrained quadratic optimization and an alternated least-squares strategy to jointly estimate end-members and their abundances. In our proposal, a local approach is used to estimate the abundances of each end-member by maximizing their entropy, and a global technique is adopted to iteratively identify the end-members by reducing the similarity among them. All the cost functions are normalized, and four initialization approaches are suggested for the end-members matrix. Synthetic datasets are used first for the EBEAE validation at different noise types and levels, and its performance is compared to state-of-the-art algorithms in BLU. In a second stage, three experimental biomedical imaging applications are addressed with EBEAE: m-FLIM for chemometric analysis in oral cavity samples, OCT for macrophages identification in post-mortem artery samples, and hyper-spectral images for in-vivo brain tissue classification and tumor identification. In our evaluations, EBEAE was able to provide a quantitative analysis of the samples with none or minimal a priori information. INDEX TERMS Blind linear unmixing, constrained optimization, fluorescence lifetime imaging microscopy, hyperspectral imaging, optical coherence tomography.

Conformational changes in biomolecules underpin all biological processes and being able to quantify these structural changes in solution is crucial to understand biological function. By carefully positioning two fluorophores within the biomolecule, it is possible to use fluorescence resonance energy transfer (FRET) as a molecular ruler to measure the desired distance. In current FRET assays, the biomolecule needs to be labelled with two different chemical fluorophores acting as a donor-acceptor FRET pair. Incorporation of these two chemically different species at specific positions within the biomolecule is challenging due to limited chemical labelling strategies. Here, we present a radically different strategy for measuring distances in biomolecules. We have developed the concept of TWIN-FRET which removes the need for two different fluorophores attached to the biomolecule by chemically encoding the FRET pair within the structure of the fluorophore itself. We have designed and synthesised a fluorescent molecule to prove this concept. The fluorophore (FH) has an acid-base equilibrium with a ground state pKa$8.9. We have derivatized the fluorophore to its succinimide ester, and used this derivative, to label a duplex DNA with two molecules of the same fluorophore at specific positions. Our results demonstrate the transfer of non-radiative energy from the neutral (FH) to the anionic (F À) state of the fluorescent molecule. We further demonstrate the use of TWIN-FRET to measure nanometer-size distances within the DNA duplex, and we obtained distance values similar to those obtained using a conventional FRET pair (Alexa488-Cy3). By removing the need to introduce two different chemical structures within the biomolecule, we greatly simplified the methodology to measure nanometer-size distances in biomolecules. We expect this technique to be widely used in structural and biophysical studies of nucleic acids, proteins and interactions between them.

Förster resonance energy transfer (FRET) is a distance-dependent process that transfers excited state energy from a donor molecule to an acceptor molecule without the emission of a photon. The FRET rate is determined by the proximity between the donor and the acceptor molecules; it becomes significant only when the proximity is within several nanometers. Therefore, FRET has been applied to visualize interactions and conformational changes of biomolecules, such as proteins, lipids, and nucleic acids that cannot be resolved by optical microscopy. Here, we report photoacoustic tomography of FRET efficiency at a 1-cm depth in chicken breast tissue, whereas conventional high-resolution fluorescence imaging is limited to <0.1 cm. Photoacoustic tomography is expected to facilitate the examination of FRET phenomena in living organisms.

Epidermal growth factor receptor (EGFR) is frequently mutated in non-small cell lung cancer (NSCLC) where it has been successfully targeted with tyrosine kinase inhibitors, but these are eventually overcome by drug resistance. Overcoming this therapeutic challenge has been hindered by the poor understanding of how wild type (WT)-EGFR elicits ligand-independent signals essential for cell homeostasis, which are then hijacked by cancer mutations. Ligand-independent signaling cannot be explained by the autoinhibited ligand-free EGFR dimer and oligomer structures so far available. Here we show that EGFR achieves ligand-independent phosphorylation by assembling dimer conformers into obligate ligand-free hetero-conformational oligomers. We reveal the structure of these ligandfree oligomers and their mechanisms of assembly. We also show how these mechanisms are exploited by drug-resistant T766M and Ex20ins EGFR mutants, revealing oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Our findings provide a framework for future drug discovery directed at tackling EGFR mutations in cancer by disabling oligomer-assembling interactions. Reagents Antibodies and reagents were purchased as follows: mAb-2E9

Introduction Single-molecule fluorescence imaging techniques are becoming popular tools for probing the structure and dynamic properties of living matter. The success of these techniques is dependent on detecting the weak fluorescence signal above background noise, and dealing with the single molecule effects of bleaching and blinking. For this reason, dyes are often selected for single molecule experiments based on their photophysical characteristics, such as quantum yield, photostability, and resistance to blinking. However, in an earlier publication we demonstrated that another important factor for single molecule experiments in cells is the propensity of dyes to bind nonspecifically to the substrate on which the cells are cultured. This non-specific binding results in a large number of immobile fluorescent molecules, skewing the results of any analysis of molecular mobility in cells. Here we describe the results of a systematic study of many dyes, and demonstrate that dye chemis...

This chapter reviews a part of the physics of fluorescence, Förster resonance energy transfer (FRET), and anisotropy measurements. See the book "Principles of Fluorescence Spectroscopy" 1 , written by J. R. Lakowicz, for additional details. This chapter also discusses the structure and function of the EGF-receptor. This is followed by a summary of previously done research on the EGF-receptor to show that the properties of fluorescent molecules can be used to learn about the clustering behavior of the EGF-receptor. 3.1 Physical properties of fluorescent molecules This part of the chapter covers the physical properties of fluorescent molecules. Firstly the fluorescence processes in the absence of FRET will be discussed. Secondly, the fluorescence energy transfer rate and its effect on the fluorescence processes will be discussed. Then the theoretical background on fluorescence anisotropy measurements and the effect of homo-FRET on anisotropy values will be discussed. Finally the effect of homo-FRET on the fluorescence lifetime is theoretically investigated. Also the theory on fluorescence lifetime measurements in the frequency domain will shortly be explained here.

A monomeric sensor, TagRFP-23-Ultramarine (TR-23-U), for effector caspase-3 was obtained. The overlap integrals of new pairs of red fluorescent protein TagRFP with four chromoproteins were calculated. The monomeric state of the Ultramarine protein and the TR-23-U sensor was confirmed by gel filtration chromatography and dynamic light scattering. Incubation with caspase-3 showed the possibility of using the new fusion protein as a FRET sensor for apoptosis detection.

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for ...

Conformational changes in biomolecules underpin all biological processes and being able to quantify these structural changes in solution is crucial to understand biological function. By carefully positioning two fluorophores within the biomolecule, it is possible to use fluorescence resonance energy transfer (FRET) as a molecular ruler to measure the desired distance. In current FRET assays, the biomolecule needs to be labelled with two different chemical fluorophores acting as a donor-acceptor FRET pair. Incorporation of these two chemically different species at specific positions within the biomolecule is challenging due to limited chemical labelling strategies. Here, we present a radically different strategy for measuring distances in biomolecules. We have developed the concept of TWIN-FRET which removes the need for two different fluorophores attached to the biomolecule by chemically encoding the FRET pair within the structure of the fluorophore itself. We have designed and synthesised a fluorescent molecule to prove this concept. The fluorophore (FH) has an acid-base equilibrium with a ground state pKa$8.9. We have derivatized the fluorophore to its succinimide ester, and used this derivative, to label a duplex DNA with two molecules of the same fluorophore at specific positions. Our results demonstrate the transfer of non-radiative energy from the neutral (FH) to the anionic (F À) state of the fluorescent molecule. We further demonstrate the use of TWIN-FRET to measure nanometer-size distances within the DNA duplex, and we obtained distance values similar to those obtained using a conventional FRET pair (Alexa488-Cy3). By removing the need to introduce two different chemical structures within the biomolecule, we greatly simplified the methodology to measure nanometer-size distances in biomolecules. We expect this technique to be widely used in structural and biophysical studies of nucleic acids, proteins and interactions between them.

Fluorescence lifetime imaging (FLIM) techniques for biological imaging have to unite several features, such as high photon efficiency, high lifetime accuracy, resolution of multi-exponential decay profiles, simultaneous recording in several wavelength intervals and optical sectioning capability. The combination of multi-dimensional time-correlated single photon counting (TCSPC) with confocal or two-photon laser scanning meets these requirements almost ideally. Multi-dimensional TCSPC is based on the excitation of the sample by a high repetition rate laser and the detection of single photons of the fluorescence signal. Each photon is characterised by its arrival time with respect to the laser pulse and the coordinates of the laser beam in the scanning area. The recording process builds up a photon distribution over these parameters. The result can be interpreted as an array of pixels, each containing a full fluorescence decay curve. More parameters can be added to the photon distribution, such as the wavelength of the photons, the time from a stimulation of the sample, or the time with respect to an additional modulation of the laser. In this review, the application of the technique will be described for the measurement of molecular environment parameters within a sample, protein interaction experiments by Förster resonance energy transfer (FRET), autofluorescence measurements of cells and tissue, and in-vivo imaging of human skin and the fundus of the human eye.

We describe a TCSPC FLIM system that uses 8 parallel TCSPC channels to record FLIM data at a peak count rate on the order of 5010 6 s-1. By using a polychromator for spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live plants.

The Zeiss LSM-510 NLO laser scanning microscope can be combined with a new TCSPC (time-correlated single photon counting) lifetime imaging technique developed by Becker & Hickl, Berlin. This technique is based on a three-dimensional histogramming process that records the photon density over the time within the fluorescence decay function and the x-y coordinates of the scanning area. The histogramming process avoids any time gating and therefore yields a counting efficiency close to one. Upgrading the LSM-510 for TCSPC imaging does not require changes in the microscope hardware or software. A fast detector is attached to the fibre output of the scanning head, and synchronisation of the TCSPC module with scanning is achieved via the user I/O of the scan controller. With an MCP-PMT as a detector, fluorescence decay components down to 30 ps can be resolved. The capability of the instrument is shown for the separation of chromphores by their fluorescence lifetime and for CFP/YFP FRET.

Nano-communication has gained significant attention in the last few years, as a means to establish information transfer between future nano-machines. Comparing with other communication techniques for nano-scale (calcium ions signaling, molecular or catalytic nanomotors, pheromones propagation, bacteria-based communication), the phenomenon called Förster Resonance Energy Transfer (FRET) offers significantly smaller propagation delays and high channel throughput. In this paper, we report our recent experiments on FRET-based nano-networks performed in the Laboratory of Cell Biophysics of the Jagiellonian University, Kraków. We propose to use Alexa Fluor dyes as nano transmitters and receivers, as they enable to create multiple-input multi-output (MIMO) FRET communication channels and thus enhance FRET efficiency. We measure FRET efficiency values, calculate bit error rates for the measured scenarios and extend the calculations to consider a general case of MIMO (n,m) FRET channels.

Live cell imaging has been greatly advanced by the recent development of new fluorescence microscopy-based methods such as multiphoton laser-scanning microscopy, which can noninvasively image deep into live specimens and generate images of extrinsic and intrinsic signals. Of recent interest has been the development of techniques that can harness properties of a fluorescence, other than intensity, such as the emission spectrum and ezcited state lifetime of a fluorophore. Spectra can be used to discriminate between fluorophores, and lifetime can be used to report on the microenvironment of fluorophores. We describe a novel technique—combined spectral and lifetime imaging—which combines the benefits of multiphoton microscopy, spectral discrimination, and lifetime analysis and allows for the simultaneous collection of all three dimensions of data along with spatial and temporal information.

In F€ orster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ 2 problem) indicates that, for short linkers and unknown local environments, additional κ 2-related uncertainties are clearly outweighed by better defined dye positions.

Emerging molecular studies have shown that the transcription factor NF-E2-related factor (Nrf2) plays an essential role in cancer chemoprevention. Here, we report the development of a molecular biosensor for rapid detection of antioxidant-responsive element (ARE)-bound Nrf2 protein. The development will provide a molecular assay for high-throughput screening of chemopreventive compounds. Specifically, a double-stranded DNA probe is designed based on the ARE sequence. One of the DNA strands is labeled with a fluorophore on the 5′ end and the complementary strand is labeled with a quencher on the 3′ end. A single-stranded DNA competitor is also designed. The existence of the Nrf2 stabilizes the fluorescent probes and delays the competitor from separating the fluorophore-quencher complex. Therefore, the concentration of the Nrf2 proteins can be measured quantitatively based on the fluorescence intensity. The molecular binding scheme was demonstrated using purified p50 and the detection...

Transient absorption results are presented on two types of GaSe nanoparticle aggregates. The results indicate that both types of aggregates exhibit larger emission oscillator strengths than the corresponding monomers, that is, superradiance. One type of GaSe nanoparticle is synthesized by conventional methods, and the other is synthesized at room temperature from very small GaSe particle nuclei. The latter type of particles form very strongly interacting aggregates, presumably because they have fewer edge-binding ligands. In both cases, the extent of superradiance is inferred from the stimulated emission band in the transient absorption spectrum. Oscillator strength ratios (n-aggregate/n monomers) are 1.5 and 1.8-2.0 for the conventional and cold-synthesized aggregates, respectively. These ratios can be quantitatively understood in terms of an electrostatic coupling model and random inhomogeneous broadening in the aggregates.

We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/ Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions.

In some applications of biomedical imaging, a linear mixture model can represent the constitutive elements (end-members) and their contributions (abundances) per pixel of the image. In this work, the extended blind end-member and abundance extraction (EBEAE) methodology is mathematically formulated to address the blind linear unmixing (BLU) problem subject to positivity constraints in optical measurements. The EBEAE algorithm is based on a constrained quadratic optimization and an alternated least-squares strategy to jointly estimate end-members and their abundances. In our proposal, a local approach is used to estimate the abundances of each end-member by maximizing their entropy, and a global technique is adopted to iteratively identify the end-members by reducing the similarity among them. All the cost functions are normalized, and four initialization approaches are suggested for the end-members matrix. Synthetic datasets are used first for the EBEAE validation at different noise types and levels, and its performance is compared to state-of-the-art algorithms in BLU. In a second stage, three experimental biomedical imaging applications are addressed with EBEAE: m-FLIM for chemometric analysis in oral cavity samples, OCT for macrophages identification in post-mortem artery samples, and hyper-spectral images for in-vivo brain tissue classification and tumor identification. In our evaluations, EBEAE was able to provide a quantitative analysis of the samples with none or minimal a priori information. INDEX TERMS Blind linear unmixing, constrained optimization, fluorescence lifetime imaging microscopy, hyperspectral imaging, optical coherence tomography.

How we see organisms and cells depends on the tools at our disposal. For over 150 years, biologists were forced to rely on fixed, dehydrated and stained specimens in order to guess how the living cells could function. It all changed abruptly during the last two decades when the rapid development of novel imaging techniques revolutionized the way scientists look at the structures of life alive.