Single FRET Research Articles

FRET calibration by symmetrization on the cell surface

Maximum position of Shannon-information of FRET frequency distribution represented as a function of the α (or G) calibrating factor as calculated in the framework of dual-laser flow cytometric FRET method – also called 3-cube FRET – gives a fairly good approximation of the real α, using only a single FRET sample. The estimation is accurate for completely symmetric FRET distributions like Gaussian and uniform. For non-symmetric distributions, the committed error has been found to depend on the direction and degree of deviation from the complete symmetry. The maximum information is characterized also by zero skewness, and minima in kurtosis and in the χ-value, defined as the “absolute deviation from the Gaussian”, as other α- functions characterizing shape. As to practice, information maximum of FRET distribution can be used to determine α, whenever symmetry can be assumed. In other cases, the goodness of estimation can be inferred from the information content and shape parameters (skewness, kurtosis and “χ-value”) of the primarily measured FRET parameter A′, based on their inverse correlations with their counterparts computed with the target FRET distribution. Above a threshold in FRET efficiency or its coefficient of variation, resonant peaks (“Ω-curves”) in FRET information appear on the α-scale, with their positions stabilized around α values with reduced fluctuations dictated by symmetry of the target FRET distribution. The phenomenon is explained by a FRET-induced broadening of the A′ distribution reaching the domain containing A′ = −α.

Single day 14 serum hCG values allow prediction of viable pregnancy and are significantly higher in frozen as compared to fresh single blastocyst transfer

PurposeTo evaluate if single serum human chorionic gonadotropin (hCG) level measurements are sufficient for pregnancy monitoring after single embryo transfer (sET) and to compare the hCG levels between fresh (FRET) and frozen embryo transfers (FET) in medically assisted reproduction.MethodsThis was a retrospective exploratory cohort study including all patients who met the inclusion criteria, who received a single FRET (n = 249) or FET (n = 410) of a day five blastocyst at the IVF clinic at the Johannes Kepler University Linz between 2011 and 2020. hCG levels were measured on day 14 after embryo transfer. Threshold values for the viability of pregnancies were determined using receiver operating characteristic (ROC) curves.ResultsSignificantly higher hCG levels were found in those who received FET than in those who received FRET (1222.8 ± 946.7 mU/ml vs. 862.7 ± 572.9 mU/ml; p < 0.001). Optimal threshold values predicting a viable pregnancy were 368.5 mU/ml and 523 mU/ml in the FRET and FET groups, respectively.ConclusionsAfter FET, higher hCG values after 14 days of embryo transfer must be considered in pregnancy monitoring. Additionally, a single threshold hCG value seems to be sufficient for determining pregnancy viability. To exclude ectopic pregnancies, subsequent ultrasound examination is a mandatory requirement.

Salmonella typhimurium detection and ablation using OmpD specific aptamer with non-magnetic and magnetic graphene oxide

Foodborne diseases have increased in the last few years due to the increased consumption of packaged and contaminated food. Major foodborne bacteria cause diseases such as diarrhea, vomiting, and sometimes death. So, there is a need for early detection of foodborne bacteria as pre-existing detection techniques are time-taking and tedious. Aptamer has gained interest due to its high stability, specificity, and sensitivity. Here, aptamer has been developed against Salmonella Typhimurium through the Cell-Selex method, and to further find the reason for specificity and sensitivity, OmpD protein was isolated, and binding studies were done. Single molecular FRET experiment using aptamer and graphene oxide studies has also been done to understand the mechanism of FRET and subsequently used for target bacterial detection. Using this assay, Salmonella Typhimurium can be detected up to 10 CFU/mL. Further, Magnetic Graphene oxide was used to develop an assay to separate and ablate bacteria using 808 nm NIR where temperature increase was more than 60 °C within 30 s and has been shown by plating as well as a confocal live dead assay. Thus, using various techniques, bacteria can be detected and ablated using specific aptamer and Graphene oxide.

Conformational Dynamics of Classical and Atypical GPCRs, CXCR4 and ACKR3, by Single‐Molecule FRET

The G protein-coupled receptors (GPCRs) CXCR4 and ACKR3 both bind the same natural agonist CXCL12, yet CXCR4 signals through G proteins and arrestins, while ACKR3 only couples to the latter. This naturally occurring receptor-mediated signaling bias also manifests in mutations to CXCL12 that cause the agonist to no longer activate CXCR4, but still acts as an ACKR3 agonist. How does CXCL12 binding lead to the different activity of these related receptors? Here, we have investigated the conformational dynamics of the two receptors by single-molecule Förster resonance energy transfer (smFRET) using probes to detect translocation of transmembrane helix 6 (TM6) of purified receptors reconstituted in nanodiscs. Our results show a consistent picture of both CXCR4 and ACKR3 occupying multiple conformational states in the absence of ligands, which collapse to mostly a single FRET state with the addition of agonist. Additionally, the system responds as expected to antagonists and mutants of CXCL12 induce opposite conformational changes for CXCR4 and ACKR3. Additionally, our experimental setup is amenable to the addition of ancillary binders, such as G proteins and arrestins, and we observe further population shifts when these are included. In this way we are able to observe differences in activation of ACKR3 and CXCR4 by the same ligand. The optimization of our single-molecule system and investigations into the activation differences of CXCR4 and ACKR3 will be discussed along with potential caveats.

High-Resolution Single-Molecule FRET via DNA eXchange (FRET X).

Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures.

FRET-based ‘ratiometric’ molecular switch for multiple ions with efficacy towards real-time sampling and logic gate applications

Fluorescent nanoaggregates comprised of rhodamine based amphiphilic probe has been designed, which can simultaneously detect Al 3+ , Cr 3+ and F¯ ions in the aqueous medium (pH 7.4). Mechanistic investigation indicates that both the carbonyl oxygen of the spirolactam ring and the nitrogen center of indole participate in the coordination of metal ions. Consequently, metal ion-binding induces the opening of the spirolactam ring and allows the Förster energy transfer from indole to xanthene moiety. As a result, the colorless probe solution turns pink in presence of metal ions, with ratiometric fluorescence change from blue to yellow. Further, the addition of fluoride leads to dissociation of preformed Al 3+ -complex, resulting in the fading of pink color and reverting the fluorescence signal to the original. This not only serves as a tool to discriminate between Al 3+ and Cr 3+ ion but also acts as a strategy for detecting fluoride in the aqueous medium. Thus, using a single FRET probe, ‘ratiometric’, naked-eye sensing of multiple ions was achieved. Also, considering the differential response of probe molecule towards Cr(III) and Cr(VI), a colorimetric assay for biogenic thiols was developed. Simultaneously, a combinatorial molecular logic gate was designed based on the multiple ion sensing ability of the probe molecule. Further, the system showed real-time detection of Al 3+ and Cr 3+ in natural water samples, estimation of Al 3+ in antacid tablets and fluoride in mouthwash, etc. Lastly, low-cost, reusable paper strips were developed, which offers an alternative strategy for on-site detection of metal ions without engaging any sophisticated instruments or trained personnel. • Ratiometric sensing of Al 3+ , Cr 3+ and F − ions at nanomolar levels in water. • Assay for biogenic thiols based on differential response for Cr(III) and Cr(VI). • Design combinatorial logic gate based on multiple ion sensing. • Analysis of real-life samples: fluoride in mouthwash, Aluminium in antacid tablets. • Low-cost, reusable paper strips for onsite detection of ions.

Information theoretic FRET calibration on the cell surface

A new method of flow cytometric dual-laser FRET calibration is presented based on an information theoretical description of the FRET signals. This method belongs to the so called two-channel or ratiometric FRET methods, whose crucial and common element is the determination of a calibration factor called α (alpha- or G-factor), to place the detected FRET signal on the absolute scale of efficiency. Generally α is determined by extra cell samples labeled with only donor and only acceptor, where the donor-acceptor ratio is known. Alternatively, when the donor-acceptor ratio is not known, standard donor-acceptor FRET pairs of known FRET efficiency can also be used for determining α. In our approach, there is no need for extra information presented by the known acceptor-to-donor ratio or the standard FRET efficiency. Our approach proceeds solely on the signals of the interested FRET-pair applying only a single FRET sample, not necessitating extra ones. The missing information of extra cell samples is replaced by an information theoretic balance equation written for the actual FRET sample. Our method has been proven to be robust, inasmuch as both donor and acceptor appear in adequate levels in the FRET sample used for finding α.

Nanoscale control of single molecule Förster resonance energy transfer by a scanning photonic nanoantenna

Abstract Förster Resonance Energy Transfer (FRET) is a widely applied technique in biology to accurately measure intra- and inter-molecular interactions at the nanometre scale. FRET is based on near-field energy transfer from an excited donor to a ground state acceptor emitter. Photonic nanoantennas have been shown to modify the rate, efficiency and extent of FRET, a process that is highly dependent on the near-field gradient of the antenna field as felt by the emitters, and thus, on their relative distance. However, most of the experiments reported to date focus on fixed antennas where the emitters are either immobilized or diffusing in solution, so that the distance between the antenna and the emitters cannot be manipulated. Here, we use scanning photonic nanoantenna probes to directly modulate the FRET efficiency between individual FRET pairs with an unprecedented nanometric lateral precision of 2 nm on the antenna position. We find that the antenna acts as an independent acceptor element, competing with the FRET pair acceptor. We directly map the competition between FRET and donor-antenna transfer as a function of the relative position between the antenna and the FRET donor-acceptor pair. The experimental data are well-described by FDTD simulations, confirming that the modulation of FRET efficiency is due to the spatially dependent coupling of the single FRET pair to the photonic antenna.

Single-Measurement Multiplexed Quantification of MicroRNAs from Human Tissue Using Catalytic Hairpin Assembly and Förster Resonance Energy Transfer.

Absolute quantification of microRNAs (miRNAs) or other nucleic acid biomarkers is an important requirement for molecular and clinical biosensing. Emerging technologies with beneficial features concerning simplicity and multiplexing present an attractive route for advancing diagnostic tools toward rapid and low-cost bioanalysis. However, the actual translation into the clinic by miRNA quantification in human samples is often missing. Here, we show that implementing time-gated Förster resonance energy transfer (TG-FRET) into a catalytic hairpin assembly (CHA) can be used for the simultaneous quantification of two miRNAs with a single measurement from total RNA extracts of human tissues. A single terbium-dye FRET pair was conjugated at two specific distances within target-specific CHA hairpin probes, such that each miRNA resulted in distinct amplified photoluminescence (PL) decays that could be distinguished and quantified by TG PL intensity detection. Enzyme-free amplification in a separation-free assay format and the absence of autofluorescence background allowed for simple, specific, and sensitive detection of miR-21 and miR-20a with limits of detection down to 1.8 pM (250 amol). Reliable duplexed quantification of both miRNAs at low picomolar concentrations was confirmed by analyzing total RNA extracts from different colon and rectum tissues with single- and dual-target CHA-TG-FRET and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for comparison. These simple and multiplexed nucleic acid biomarker assays present a capable method for clinical diagnostics and biomolecular research.

The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis

Filament formation by enzymes has recently emerged as a new paradigm of enzyme regulation 1.Wide‐spread reversible self‐assembly of enzymes in cells has also been shown to occur in all branches of life and diverse metabolic and biological pathways. We use the model system, SgrAI, to investigate the filament forming enzyme mechanism.Filaments formed by SgrAI have been structurally characterized using cryo‐electron microscopy 2 and the full reaction pathway including all rate constants has been determined using single turnover enzyme reactions, FRET, and global kinetic modeling 3– 4.The structural studies uncover the mechanism of activation of enzyme activity in SgrAI by filament formation, as well as the origin of the unusual expansion of substrate specificity in SgrAI within the filament. A conformational change in the SgrAI dimer, stabilized by protein‐protein and protein‐DNA contacts in the filament, leads directly to a shift in the active site, hypothesized to lead to stronger binding of a second catalytic Mg2+ ion. DNA structure and energetics is hypothesized to control substrate specificity and filament formation by SgrAI such that the secondary class of DNA sequences are cleaved by SgrAI only upon joining a filament formed by SgrAI bound to a primary class of DNA sequences. The full kinetic model showed a slow second order step in filament formation which is shown to control enzyme activity such that only invading phage DNA is cleaved, leaving the host genome untouched. This model also shows that the filament forming mechanism is superior in fast activation of enzyme activity upon initial recognition of primary DNA sequences.Using the SgrAI endonuclease as a model system to study filament forming enzymes, we have discovered that the enzyme is activated by stabilization of an activated conformation by contacts made within the filament. Control of the equilibrium between a low activity, non‐filamentous state and the high activity, filamentous state by DNA structural energetics gives rise to the unusual apparent expansion of DNA sequence specificity of SgrAI by filament formation. Simulations using the full kinetic pathway reveal an advantage in the filament mechanism over other non‐filamentous mechanisms in the speed of activation in response to invading phage DNA, giving strong evolutionary advantage to this filament forming mechanism.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355.

Identification of guanine nucleotide exchange factors that increase Cdc42 activity in primary human endothelial cells

ABSTRACT The Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signalling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the overexpressed full-length GEFs. Our data reveal GEF dependent activation of Cdc42, with the most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and PREX1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may directly or indirectly activate Cdc42, which will be of great value for the field of vascular biology.

Simple, Amplified, and Multiplexed Detection of MicroRNAs Using Time-Gated FRET and Hybridization Chain Reaction.

The hybridization chain reaction (HCR) is a simple and sensitive method for quantifying nucleic acids. Current approaches cannot combine a washing-free sensing format with multiplexed target quantification at low concentrations, which would be highly desirable for detection both in solution and in situ. Here, we demonstrate the implementation of time-gated Förster resonance energy transfer (TG-FRET) between terbium donors and dye acceptors into HCR for multiplexed quantification of microRNAs (miR-20a and miR-21) and their DNA analogues. HCR-TG-FRET provided washing-free nucleic acid quantification with very low limits of detection down to 240 amol (1.7 pM) of microRNA and 123 amol (0.88 pM) of DNA. Efficient distinction from very homologous microRNAs demonstrated high target specificity. Multiplexing with a single measurement, a single excitation wavelength, and a single FRET pair allowed for a simultaneous quantification of miR-20a and miR-21 at concentrations between 30 and 300 pM from the same sample. HCR-TG-FRET showed similar performance for serum-free and serum-containing samples without the use of RNase inhibitors. Our results present a significant improvement in current HCR approaches regarding simplicity, sensitivity, and multiplexing. The versatile diagnostic performance of HCR-TG-FRET even in challenging biological environments presents an important advantage for advanced nucleic acid biosensing.

Generalized modeling of body response of acoustic guitar for all frets using a response for single fret

Analysis of music notes with their mathematical behavior has been the topic of interest since ages. This work is focused on the analysis of acoustic guitar notes to calculate and rather separate the guitar body response or impulse response and the excitation signal. The cepstral domain approach is used for estimation of guitar body response. The adaptive cepstral domain windowing (ACDW) is adopted to estimate the guitar body response. The guitar notes for two plucking styles, finger plucking, and plectrum plucking were recorded for all the twenty frets. This resulted in the database of two hundred and forty sound notes including both plucking styles. The complete analysis of the behavior of the guitar body on the coarse level, that is, string level and on fine level, that is, individual fret level, is carried out. This paper discusses the extraction of impulse response of acoustic guitar for all strings and all frets. This present work is extended to put forth a generalized mathematical model for the impulse response of acoustic guitar for all frets using a response for a single fret as a base. It discusses the formulation of a mathematical equation for finding the body response of one fret using the body response of the last fret. The body response so obtained is convolved with the excitation for the required fret using mathematical equation. The result is verified with the subjective listening test by the experts and with the objective measure as the correlation coefficient, the statistical parameter to indicate the degree of matching. It is observed that the synthesized fret note shows the correlation of more than 0.84 with the actual fret note. The listening tests results by the experts also give a score of more than 0.9 for the synthesized notes.

Perrin and Förster unified: Dual-laser triple-polarization FRET (3polFRET) for interactions at the Förster-distance and beyond

Dual laser flow cytometric energy transfer (FCET) – elaborated by Trón et al. in 1984 – is an efficient and rapid way of measuring FRET on large cell populations. FRET efficiency and the donor and acceptor concentrations are determined from one donor and two acceptor signals. In this communication this method is extended towards the domain of receptor dynamics by the detection of polarized components of the three intensities. By enabling a complete description of the proximity and dynamics of FRET-systems, the new measuring scheme allows a more refined description of both the structure and dynamics of cell surface receptor clusters at the nano-scale and beyond. Associated donor fraction, limiting anisotropy and rotational correlation time of the donor, acceptor anisotropy and cell-by-cell estimation of the orientation factor for FRET (κ2) are available in the steady state on a single FRET sample in a very rapid and statistically efficient way offered by flow cytometry. For a more sensitive detection of conformational changes the “polarized FRET indices” – quantities composed from FRET efficiency and anisotropies – are proposed. The method is illustrated by measurements on a FRET system with changing FRET-fraction and on a two donor-one acceptor-system, when the existence of receptor trimers are proven by the detection of “hetero-FRET induced homo-FRET relief”, i.e. the diminishing of homo-FRET between the two donors in the presence of a donor quencher. The method also offers higher sensitivity for assessing conformational changes at the nano-scale, due to its capability for the simultaneous detection of changes of proximity and relative orientations of the FRET donor and acceptor. Although the method has been introduced in the context of FRET, it is more general: It can be used for monitoring triple-anisotropy correlations also in those cases when FRET actually does not occur, e.g. for interactions occuring beyond the Förster-distance R0. Interpretation of κ2 has been extended.

The regulatory switch of F1-ATPase studied by single-molecule FRET in the ABEL Trap.

F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of 'non-productive' ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.

Rosiglitazone increases aquaporin 2 transcription and apical expression in renal cells: possible implication for treating nephrogenic diabetes insipidus? (892.10)

Aquaporin 2 (AQP2) is the vasopressin‐regulated water channel palying a crucial role in urine concentration in the kidney. Rosiglitazone (RGZ), an agonist of the peroxisome proliferator‐activated receptor gamma (PPARγ), increases AQP2 expression in normal rats. Here we tested whether RGZ could also modulate AQP2 intracellular trafficking in renal cells.Wild type C57BL‐6 mice were treated for 3 days with 20mg/Kg RGZ to confirm the transcriptional effect on AQP2 in mice. In parallel, we used mouse MCD4 renal collecting duct cells to study the effect of RGZ on AQP2 trafficking to the apical membrane. Apical surface biotinylation and confocal analysis were used to semiquantify the effect. Single cell live microfluorimetry and FRET were used to measure changes in Ca2+ and cAMP intracellular concentrations, respectively.In mice, RGZ induced a twofold increase of AQP2 compared to control mice.In MCD4 cells, stimulation with 50µM RGZ for 30 min robustly increased AQP2 trafficking to the apical plasma membrane to an extent comparable to that elicited by forskolin stimulation. Interestingly, RGZ was able to increasely AQP2 phosphorylation at Ser256 concomitant with intracellular Ca++ increase and without apparent intracellular cAMP elevation.Taken together these results suggest that RGZ might be useful to increase AQP2 transcription and apical trafficking in renal cells bypassing the stimulation of vasopressin receptor which is defective in pathological conditions as in Nephrogenic Diabetes Insipidus.Grant Funding Source: Telethon GGP12040/ AIFA MRAR08P011

Structural Dynamics Underlying Pip2 Modulation of Kir Channels Revealed by Single Molecule FRET

Ion channels are gated by a multitude of electrical, chemical and mechanical stimuli, and thereby play key roles in many physiological processes. Static crystal structures and dynamic electrophysiological studies have generated invaluable molecular insights into channel gating, but the time trajectory of conformational changes has been unattainable. KirBac1.1 is a model for one of the three main K channel families. In the present study, we have successfully (1) labeled purified tetrameric protein molecules with single FRET donor and acceptor fluorophore pairs at specific locations; (2) functionally reconstituted the proteins into liposomes; (3) examined the dynamics of channel structure in liposomes with single molecule FRET techniques. Our results indicate that the amphipathic 'slide helices' of KirBac1.1 that surround the channel gate immediately below the membrane, move toward each other to narrow the pore in the presence of PIP2, which closes the channel; the cytoplasmic domain resides at two major structural states, but demonstrates an overall shift away from the pore axis upon PIP2 inhibition; the extracellular loop of KirBac1.1, adjacent to the selectivity filter, is structurally rigid and does not move during PIP2 gating. Both slide helix and cytoplasmic domain structures become less dynamic when the channel is inhibited by PIP2, which implies that PIP2 acts as a 'lock' to confine the structural fluctuations of the channel protein. We further probed the relative motions of the KirBac1.1 transmembrane domain versus the cytoplasmic domain. The results indicate that the entire cytoplasmic domain moves away from the transmembrane domain during PIP2 inhibition, which may result from a rigid body motion coupled to TM2 bending. In summary, we provide direct observations of dynamic structures of an ion channel within a lipid membrane environment, revealing dynamic structural rearrangements KirBac1.1 upon PIP2 inhibition.

Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells

Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor–accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs – resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented.

A highly selective and efficient single molecular FRET based sensor for ratiometric detection of Fe3+ ions

A highly selective and efficient single molecular FRET based sensor has been developed for the ratiometric detection of Fe(3+) in aqueous samples and live cells.

Internalization of Fluorescent Biomolecules for Long-Lived Single-Molecule Observation in Living Bacteria

We present a complete toolbox for the internalization and single-molecule study of singly- or multiply-labeled fluorescent biomolecules (such as DNA and protein) into living E.coli cells. This technique allows use of organic fluorophores, which are much smaller, brighter and more photostable than genetically encoded fluorescent proteins (e.g. GFP), and provide better labeling flexibility (using in vitro site-specific labeling) and wider spectral range. As such, our methods enable experiments that have so far been precluded due to the inability to internalize fairly large molecules such as globular proteins through the cell membranes of micron-size bacterial cells.Our internalization method, based on electroporation, has allowed us to observe and track fluorescent molecules in living E.coli on the second-to-minute timescale providing >100-times longer observation spans compared to GFP. Aided by the quantized photobleaching of fluorophores, we have characterized the number of internalized molecules, which ranged from 1 to 1000, depending on the size and the amount of electroporated molecule. We also characterized the diffusion behaviour of the internalized molecules, obtaining information on diffusion coefficients, heterogeneity and paths.By internalising doubly-labeled DNA molecules, we observed single-molecule FRET in single bacteria for the first time. Systematic in vivo single-molecule FRET measurements with DNA standards (exhibiting FRET efficiencies from ∼20 to ∼90%) show all the hallmarks of single FRET pairs, with efficiencies that agree well with those obtained in vitro. We further demonstrated the technique with labeled proteins, successfully internalizing singly- and doubly-labeled proteins of different sizes. Currently we are extending this method to achieve live-cell super-resolution imaging. Our toolbox should be very helpful for addressing a wide range of questions regarding the structure, interactions and dynamics of bacterial systems in their natural context.