Forster Resonance Energy Transfer Microscopy Research Articles

Förster resonance energy transfer efficiency measurements on vinculin tension sensors at focal adhesions using a simple and cost-effective setup.

Forces inside cells play a fundamental role in tissue growth, affecting important processes such as cancer cell migration or tissue repair after injury. Förster resonance energy transfer (FRET)-based tension sensors are a remarkable tool for studying these forces and should be made easier to use. We prove that absolute FRET efficiency can be measured on a simple setup, an order of magnitude more cost-effective than a standard FRET microscopy setup, by applying it to vinculin tension sensors (VinTS) at the focal adhesions of live CHO-K1 cells. Our setup located at Université Paris-Saclay acquires donor and acceptor fluorescence in parallel on two low-cost CMOS cameras and uses two LEDs for rapid switching of the excitation wavelength at a reduced cost. The calibration required to extract FRET efficiency was achieved using a single construct (TSMod). FRET efficiencies were measured for VinTS and the tail-less control VinTL, lacking the actin-binding domain of vinculin. Measurements were confirmed on the same cell type using a more standard intensity-based setup located at Rutgers University. The average FRET efficiency of VinTS () over more than 10,000 focal adhesions is significantly lower () than that of VinTL (), our control that is insensitive to force, in agreement with the force exerted on vinculin at focal adhesions. Attachment of the CHO-K1 cells on fibronectin decreases FRET efficiency, thus increasing the force, compared with poly-lysine. FRET efficiency for the VinTL control is consistent with all measurements currently available in the literature, confirming the validity of our measurements and hence of our simpler setup. Force measurements, resolved spatially inside a cell, can be achieved using FRET-based tension sensors with a cost effective intensity-based setup. This will facilitate combining FRET with techniques for applying controlled forces such as optical tweezers.

Histone FRET microscopy of live cell genome architecture.

Inside the nucleus of an intact cell, DNA is folded around histone proteins into nucleosomes and compacted into a multi-layered three-dimensional chromatin network. The nanometre spacing between nucleosomes positioned throughout this structural framework is known to locally modulate local DNA template access and regulate genome function. However, given that this structural feature occurs on a spatial scale well below the diffraction limit of optical microscopy, real time observation of nucleosome proximity in live cells has proven technically difficult, despite recent advances in live cell super resolution imaging. Thus, in recent work, we have been exploring a powerful alternative solution that is based on fluorescence lifetime and anisotropy imaging microscopy (FLIM and FAIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome. Here we present recent findings from application of this technology to the study of live cell chromatin network dynamics, which demonstrate local ensemble nucleosome proximity, spatiotemporally oscillates between different spacings that are ‘invisible’ to diffraction limited microscopy.

SCC-FRET: single-cell-based calibration of a FRET system

Reliable measurements of calibration parameters are crucial for quantitative three-cube Förster resonance energy transfer (FRET) measurements. Here we have developed a single-cell-based calibration method (SCC-FRET), which can simultaneously obtain spectral crosstalk correction parameters (β and δ) and calibration parameters (G and k) of a quantitative FRET system by imaging a cell expressing one kind of standard FRET plasmid with a known FRET efficiency (E) and the donor-to-acceptor concentration ratio (RC). We performed the SCC-FRET method on a three-cube FRET microscopy for the cells expressing C5V, and obtained β = 0.150 ± 0.000, δ = 0.610 ± 0.000, G = 2.840 ± 0.065, and k = 0.847 ± 0.013. These parameters were used to measure the E and RC values of C17V and C32V constructs in living cells and obtained EC17V = 0.382 ± 0.010 and EC32V = 0.311 ± 0.007, RC17V = 1.010 ± 0.023 and RC32V = 1.050 ± 0.022, consistent with the reported values, demonstrating the effectiveness of the the SCC-FRET method. We also performed the SCC-FRET method for the cells with different S/N levels (S/N > 10, 10 > S/N > 3, 3 > S/N > 1, respectively), and obtained consistent system calibration parameters under different S/N levels, indicating excellent robustness. SCC-FRET requires only imaging a cell expressing one kind of standard FRET plasmid for measuring all calibration parameters under identical imaging conditions, rendering the SCC-FRET method extremely convenient, accurate, and robust. The SCC-FRET provides strong support for expanding the biological application of quantitative FRET analysis in living cells.

(Invited) Towards a FRET Based DNA SAM Biosensor for Detection of Nucleic Acids

Rapid and sensitive detection of nucleic acids is an essential tool in many medical and clinical settings, that allows for fast diagnosis of bacterial and viral infections. Here, we present a DNA FRET-based biosensor to detect a hybridization event on a DNA SAM on gold surface. The monolayer was prepared by a potential assisted thiol exchange deposition, which provides better control over the surface coverage and its uniformity1. Our approach relies on a change in FRET (Förster Resonance Energy Transfer) signal caused by variations in distance between fluorophores. FRET imaging is a well-known, very sensitive technique, used commonly in many areas of chemistry and microbiology. FRET signal depends strongly on the distance between an acceptor and a donor fluorophore. It is an excellent tool to analyze small changes in DNA distance between adjacent strands and the surface2. Metal quenching also plays a major role, since fluorescence is strongly dependent on the distance from the metal surface3, which is an efficient quencher. Fluorophores, both acceptor and donor, are quenched significantly by the gold surface, this however, decreases substantially as the distance increases between the surface and fluorophore. As DNA is binding its complementary strands, the distance between the surface and the fluorophore labelled end increases.Using a self-assembly property of thiols on gold surface, we show that mixed ssDNA SAM composed of two strands of thiolated ssDNA, each labelled with a different FRET fluorophore, gives an 8-to-10-fold signal enhancement, upon DNA hybridization (see figure 1). Binding of a complementary strand rigidifies the adsorbed DNA structure, and as a result we observe the increase in signal due to weaker gold quenching and shorter distance between FRET participating fluorophores. Moreover, by utilizing a single crystal electrode, we can study different surface concentration regimes and fluorescence signal increases, based on surface crystallography and the electrode potential.(1) Leung, K. K.; Gaxiola, A. D.; Yu, H.-Z.; Bizzotto, D. Tailoring the DNA SAM Surface Density on Different Surface Crystallographic Features Using Potential Assisted Thiol Exchange. Electrochim. Acta 2018, 261, 188–197. https://doi.org/https://doi.org/10.1016/j.electacta.2017.12.114.(2) Verhaven, A.; Doneux, T.; Bizzotto, D. Application of FRET Microscopy to the Study of the Local Environment and Dynamics of DNA SAMs on Au Electrodes. Langmuir 2018, 34 (49), 14802–14810. https://doi.org/10.1021/acs.langmuir.8b02131.(3) Casanova-Moreno, J.; Yu, Z. L.; Massey-Allard, J.; Ditchburn, B.; Young, J. F.; Bizzotto, D. Luminescence in Electrochemistry; Springer International Publishing: Cham, 2017; pp 21– 77. Figure 1

Optical section structured illumination-based Förster resonance energy transfer imaging.

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were , and , and the measured acceptor-to-donor concentration ratios ( ) were , , and , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

Quantification and Imaging of Nanoscale Contact with Förster Resonance Energy Transfer.

Adhesion is caused by molecular interactions that only take place if the surfaces are in nanoscale contact (NSC); i.e., the distance between the surfaces is in the range of 0.1–0.4 nm. However, there are several difficulties measuring the NSC between surfaces, mainly because regions that appear to be in full contact at low magnification may show no NSC when observed at higher magnifications. Thus, the measurement area of NSC is very small with imaging techniques, and an experimental technique to evaluate NSC for large contact areas has not been available thus far. Here, we are proposing Förster resonance energy transfer (FRET) spectroscopy/microscopy for this purpose. We demonstrate that NSC in a distance range of 1–10 nm can be evaluated. Our experiments reveal that, for thin films pressed under different loads, NSC increases with the applied pressure, resulting in a higher FRET signal and a corresponding increase in adhesion force/energy when separating the films. Furthermore, we show that local variations in molecular contact can be visualized with FRET microscopy. Thus, we are introducing a spectroscopic technique for quantification (FRET spectroscopy) and imaging (FRET microscopy) of NSC between surfaces, demonstrated here for the application of surface adhesion. This could be of interest for all fields where adhesion or nanoscale surface contact are playing a role, for example, soft matter, biological materials, and polymers, but also engineering applications, like tribology, adhesives, and sealants.

Designing a Compact, Low-Cost FRET Microscopy Platform for the Undergraduate Classroom

ABSTRACT Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.

Comparison of spectral FRET microscopy approaches for single-cell analysis.

Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focus fluorescence, and Z-resolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET "controls" with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.

FRET Microscopy in Yeast.

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.

Reducing the Detrimental Effects of Saturation Phenomena in FRET Microscopy.

Although Förster resonance energy transfer (FRET) is one of the most widely used biophysical methods in biology, the effect of high excitation intensity, leading to donor and acceptor saturation, has not been addressed previously. Here, we present a formalism for the experimental determination of the FRET efficiency at high excitation intensity when saturation of both the donor and the acceptor significantly affect conventional FRET calculations. We show that the proposed methodology significantly reduces the dependence of the FRET efficiency on excitation intensity, which otherwise significantly distorts FRET calculations at high excitation intensities commonly used in experiments. The work presented here adds additional rigor to the FRET-based investigation of protein interactions and strengthens the device independence of such results.

Application of FRET Microscopy to the Study of the Local Environment and Dynamics of DNA SAMs on Au Electrodes.

Immobilized DNA probe strands self-assembled on an electrode surface are the bases of many electrochemically based biosensors. Control or measurement of the local environment around each DNA molecule tethered to the electrode surface is needed because the local environment can influence the binding or hybridization efficiency of the target in solution. Measurement of this local environment in buffer or under electrochemical control can be challenging. Here we demonstrate the use of fluorescence microscopy and a Förster resonance energy transfer (FRET) methodology to characterize multicomponent DNA SAMs. The DNA SAMs that were studied were composed of a series of mole fraction ratios of alkylthiol-modified DNA which was labeled with either AlexaFluor488 or AlexaFluor647, a FRET donor and acceptor, respectively. The DNA SAMs were hybridized before assembly onto the electrode surface. Wide-field filter-based FRET microscopy was used to study the assembly of DNA SAMs onto gold bead electrodes. These single-crystal gold bead electrodes contain many surface crystallographic regions which enable the comparison of the adsorbed DNA local environment. These surfaces show that most surface modifications are uniformly prepared, and the FRET efficiency can be explained through simple surface density considerations. The FRET efficiency for different compositions of the donor and acceptor for these regions is also explained through 2D FRET modeling. Not all surfaces were similar to the (111) and (110) regions showing deviations from the expected FRET behavior. Also demonstrated is FRET imaging using a confocal microscope. This approach proves useful in the analysis of a more dynamic system, such as the analysis of reductive desorption of the mixed-component DNA SAM. FRET microscopy is useful for surface analysis of the DNA local environment, enabling a measure of the surface modification, local density, and clustering and eventually a new detection modality.

Chasing the signaling run by tri-molecular time-lapse FRET microscopy

A feasible design is made to measure three protein/protein interactions to visualize signal pathways by time-lapse Förster resonance energy transfer (FRET) microscopy. When interacting proteins are in close proximity, excitation energy is provided to allow the energy flow from the first molecule to excite the second, followed by energy transfer to the third. By phorbol ester/calcium ionophore stimulation, for example, a real-time complex formation of ectopic IκBα/ERK/WWOX occurs as measured by FRET microscopy, indicative of an ongoing functional signaling. Hyaluronan induces membrane Hyal-2 signaling, which allows FRET measurement of the complex formation of ectopic Smad4/WWOX/Hyal-2 for causing bubbling cell death. If ectopic p53 is recruited to replace Hyal-2, the resulting ectopic Smad4/WWOX/p53 complex induces membrane blebbing without cell death. Together, in this perspective review article, we demonstrate the utilization of time-lapse FRET microscopy to visualize the signaling event via the tri-molecular protein complex formation and their biological outcomes. We show an initial two-protein binding to form the driving force to jumpstart the tri-molecular execution for the signal pathway.

FRET Image Correlation Spectroscopy Reveals RNAPII-Independent P-TEFb Recruitment on Chromatin

Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Förster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, EFRET, because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through. However, the way FRET signal is usually analyzed does not provide information about the areas where protein-protein interactions take place. In this work, we developed a method, dubbed FRET image correlation spectroscopy (FICS), which relied on FRET fluorescence lifetime imaging image acquisition and image correlation spectroscopy of EFRET clusters to quantify the spatial distribution of interaction clusters in the nucleus. The combination of high content FRET microscopy with batch image analysis allowed a robust statistical analysis. By applying FICS, we characterized the area and density of interaction clusters between P-TEFb and RNAPII or histone H2A in single living cells. The FICS method applied to cells expressing genetically engineered mutated proteins confirmed that the histidine-rich domain of P-TEFb is required for its interaction with RNAPII. Furthermore, it demonstrated that P-TEFb was also located in close vicinity to histone H2A, independently of its interactions with RNAPII. These results support the hypothesis that P-TEFb dynamics on chromatin regulate its recruitment on RNAPII.

Research Techniques Made Simple: Methodology and Applications of Förster Resonance Energy Transfer (FRET) Microscopy

Classical biochemical techniques have contributed a great deal to our understanding of the mechanisms regulating fundamental biological processes. However, these approaches are typically end-point, population-based assays and are often insufficient in examining transient molecular events. Förster resonance energy transfer (FRET) microscopy is a powerful technique capable of investigating dynamic interactions between proteins and a plethora of biochemical signaling events based on the development of specific biosensors. This technique exploits the principle that when FRET occurs, energy from a donor fluorophore is transferred to an acceptor fluorophore only when certain conditions are met. These include dependence on both distance and fluorophore orientation. In this article, applications of FRET microscopy to protein interactions and modifications are discussed, and examples are given of the types of biosensors that can be developed. There are a number of methods to measure FRET. The most common modalities and specific advantages and shortcomings for each are reviewed. Finally, general considerations and guidelines for choosing a method are discussed.

Quantitative dual-channel FRET microscopy.

Acceptor-sensitized quantitative Förster resonance energy transfer (FRET) measurement (E-FRET) is mainly impeded by donor emission crosstalk and acceptor direct excitation crosstalk. In this paper, we develop a novel E-FRET approach (Lux-E-FRET) based on linear unmixing (Lux) of the fluorescence intensity ratio between two detection channels with each excitation of two different wavelengths. The two detection channels need not to selectively collect the emission of donor or acceptor, and the excitation wavelengths need not to selectively excite donor or acceptor. For a tandem FRET sensor, Lux-E-FRET only needs single excitation wavelength. We performed Lux-E-FRET measurements on our dual-channel wide-field fluorescence microscope for FRET constructs in living cells, and obtained consistent FRET efficiencies with those measured by other methods. Collectively, Lux-E-FRET completely overcomes all spectral crosstalks and thus is applicable to the donor-acceptor pair with larger spectral overlapping.

FRET Microscopy for Real-Time Visualization of Second Messengers in Living Cells.

Förster Resonance Energy Transfer (FRET) microscopy is a useful tool in molecular biology and medical research to monitor and quantify real-time dynamics of protein-protein interactions and biochemical processes. Using this well-established technique, many novel signaling mechanisms can be investigated in intact cells or tissues and even in various subcellular compartments. Here, we describe how to perform FRET measurements in living cells expressing FRET-based biosensors and how to evaluate these data. This general protocol can be applied for FRET measurements with various fluorescent biosensors.

Visualization of Mechanical Forces within the Immunological Synapse

The interaction between the T-cell receptor (TCR) and the antigen-presenting cell (APC) has a central role in the adaptive immune system. It is of the utmost importance that this process is fast, specific, and highly sensitive. The T-cell is able to effectively screen through a sea of self- and harmless antigens in order to rapidly find those that are harmful. Dynamic signaling processes and membrane reorganization events such as the creation of the immunological synapse support this screening procedure. However, preliminary experiments indicate the involvement of mechanical forces in antigen discrimination. Therefore, we seek to characterize the TCR-imposed forces on the APC by directly introducing a force sensor within the immunological synapse. Well defined and calibrated digital, as well as analog, fluorescent molecular sensors will be attached to the peptide-presenting MHC complex (pMHCs). The distance between two introduced fluorophores can be precisely determined via Forster resonance energy transfer microscopy, and will serve as an exact measure for the involved pulling forces. By characterizing a statistically meaningful amount of the aforementioned events, a force map of the immunological synapse can be created. Downstream signaling will be measured via Calcium imaging, and correlated with the stimulatory potency of the involved pMHCs. The aim of this project is to develop a reliable tool to measure molecular forces between two adjacent cells, and to use the topological information to create force maps.

Influence of the Background in Single Molecule FRET TIRF Microscopy

Single-molecule total internal reflection fluorescence (TIRF) microscopy combined with Forster Resonance Energy Transfer (FRET) is a powerful tool that allows direct observation of structural dynamics and heterogeneities without the need for ensemble synchronization. The weak signals in single-molecule TIRF microscopy experiments imply that background determination can have a critical influence on the measured fluorescence intensity.Here we compare and evaluate the performance of different background estimators in single-molecule FRET experiments in samples having different molecular densities. We found that not all background estimators perform well in samples with high molecular densities and propose a new and simple background estimator, called the local statistical percentile (LSP), which is suited for single molecule studies in dense biological samples. This background estimator is implemented as a new toolkit in the single molecule FRET microscopy software package iSMS (1). We illustrate how the LSP background estimator provides consistent single-molecule information over a wide range of molecular densities.(1)S. Preus, S. L. Noer, L. L. Hildebrandt, D. Gudnason, and V. Birkedal. 2015. iSMS: single-molecule FRET microscopy software. Nat Methods 12:593-594.

Probing the Folding Dynamics of Human Telomeric G-Quadruplex with Single-Molecule FRET

Guanine-rich sequences consisting of several tandem repeats of TTAGGG are abundant in human telomeric DNA. They have been shown to fold into unique secondary structures under physiological conditions. One of these is the G-quadruplex structure that can undergo complex folding pathways [1-3]. Single-molecule fluorescence microscopy combined with Forster resonance energy transfer (FRET) allows for probing the conformation and dynamics of individual G-quadruplex molecules without time and population averaging and thus resolving their structural heterogeneity that is otherwise hidden in ensemble experiments [4,5].Here we utilize single-molecule FRET microscopy to probe the K+-induced folding dynamics of human telomeric G-quadruplex DNA. Our single-molecule experiments identify several distinct FRET states populated along the folding of G-quadruplexes. We find that initial dynamic interconversion occurs between three states - the unfolded state and two transient folded states, followed by transition into a dominant long-lived folded G-quadruplex conformation. Additional single-molecule FRET measurements, employing specific chemically modified G-quadruplex sequences, and advanced molecular modeling enabled linking the experimentally observed FRET states to particular G-quadruplex conformations. Our results thus allow uncovering a possible folding pathway of the human telomeric G-quadruplex structures.(1) Bochman, M. L.; Paeschke, K.; Zakian, V. A. Nature Reviews Genetics 2012, 13, 770.(2) Biffi, G.; Tannahill, D.; McCafferty, J.; Balasubramanian, S. Nature Chemistry 2013, 5, 182.(3) Gray, R. D.; Trent, J. O.; Chaires, J. B. J. Mol. Biol. 2014, 426, 1629.(4) Single-Molecule Techniques - A Laboratory Manual; Selvin, P. R.; Ha, T., Eds.; Cold Spring Harbor, NY, 2008.(5) Lee, J. Y.; Okumus, B.; Kim, D. S.; Ha, T. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18938.

Single-Molecule Confocal FRET Microscopy to Dissect Conformational Changes in the Catalytic Cycle of DNA Topoisomerases.

Molecular machines undergo large-scale conformational changes during their catalytic cycles that are linked to their biological functions. DNA topoisomerases are molecular machines that interconvert different DNA topoisomers and resolve torsional stress that is introduced during cellular processes that involve local DNA unwinding. DNA gyrase catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. During its catalytic cycle, gyrase undergoes large-scale conformational changes that drive the supercoiling reaction. These conformational changes can be followed by single-molecule Förster resonance energy transfer (FRET). Here, we use DNA gyrase from Bacillus subtilis as an illustrative example to present strategies for the investigation of conformational dynamics of multisubunit complexes. We provide a brief introduction into single-molecule FRET and confocal microscopy, with a focus on practical considerations in sample preparation and data analysis. Different strategies in the preparation of donor-acceptor-labeled molecules suitable for single-molecule FRET experiments are outlined. The insight into the mechanism of DNA supercoiling by gyrase gained from single-molecule FRET experiment is summarized. The general strategies described here can also be applied to investigate conformational changes and their link to biological function of other multisubunit molecular machines.