Available online at www.sciencedirect.com ScienceDirect Combining single-molecule manipulation and single-molecule detection 1 1 Juan Carlos Cordova , Dibyendu Kumar Das , 1 1,2 Harris W Manning and Matthew J Lang Single molecule force manipulation combined with of proteins and nucleic acid structures and general inves- fluorescence techniques offers much promise in revealing tigation of cell system machinery [1–4]. From the 8 nm mechanistic details of biomolecular machinery. Here, we step of kinesin [1], to time and spectrally resolved visual- review force-fluorescence microscopy, which combines the ization of enzymatic reactions [5], the advent of single best features of manipulation and detection techniques. Three molecule biophysics has advanced through an impressive of the mainstay manipulation methods (optical traps, magnetic series of milestones. Here we review recent progress in traps and atomic force microscopy) are discussed with respect the ability to not only ‘watch’, but also physically ‘manip- to milestones in combination developments, in addition to ulate’, individual molecules. Our ability to ‘watch’ with highlight recent contributions to the field. An overview of fluorescence includes fluorescence localization with additional strategies is discussed, including fluorescence spatial resolution of a few nm, angle, distance, spectral based force sensors for force measurement in vivo. Armed with changes, time resolved studies and simultaneous tracking recent exciting demonstrations of this technology, the field of of multiple molecules. Our ability to ‘manipulate’ with an combined single-molecule manipulation and single-molecule optical trap or atomic force microscope (AFM) is now detection is poised to provide unprecedented views of measured in angstroms, enabling the ability to track molecular machinery. moving molecules and scanning probe imaging. Appli- Addresses cation of force includes pick and place control over 1 Department of Chemical and Biomolecular Engineering, Vanderbilt molecular positioning, dynamic or clamped application University, Nashville, TN, United States of stresses and forces that reveal much about the system, 2 Department of Molecular Physiology and Biophysics, Vanderbilt to control over the reaction coordinate of interest. University School of Medicine, Nashville, TN, United States Corresponding author: Lang, Matthew J ([email protected]) Optical trapping combined with fluorescence Optical tweezers and single-molecule fluorescence are Current Opinion in Structural Biology 2014, 28:142–148 primary techniques in single-molecule biophysics. Given This review comes from a themed issue on Biophysical and the 4.1 pN nm magnitude of thermal motions, these molecular biological methods methods offer force and distance scales appropriate for studying biological motors and other molecular tran- Edited by David Millar and Jill Trewhella sitions. Integrating trapping and fluorescence correlates nanoscale structural changes with biomechanical tran- sitions, pinpointing their locations, magnitudes and tran- http://dx.doi.org/10.1016/j.sbi.2014.08.010 sition energies (Figures 1 and 2). 0959-440X/# 2014 Elsevier Ltd. All right reserved. Early efforts in combined optical trapping and single molecule fluorescence included dual functioning micro- scopes and spatially separated configurations, demon- strated by Yanagida and coworkers [6–8]. In order to reduce background fluorescence, prism type single-mol- Introduction ecule total internal reflection fluorescence (smTIRF) was The ability to watch, simultaneously manipulate and employed. TIRF offers localized excitation to a 1/e control individual molecules is a powerful tool for un- distance range from the glass-water interface. ‘Prism side’ derstanding the structure-functional–mechanical working methods excite molecules on the flow cell surface opposite of molecular machinery. Individual behavior is typically the objective, offering clean excitation and straightforward clouded by ensemble measurement, requiring specific alignment of incident angle and wide field of view. manipulation and detection strategies to reveal the prop- erties of isolated molecules. Single-molecule (sm) This combined trapping and fluorescence work used methods have been developed over the years driven by single-beam, and later dual-beam trapping with prism studies such as motility of motor proteins, RNA poly- side smTIRF to directly visualize nucleotide turnover merase, DNA repair enzymes, measurement of physical during kinesin walking [6], the force-generating step in properties of polymers and filaments, unfolding/refolding myosin’s mechanochemical cycle [7], and later to study Current Opinion in Structural Biology 2014, 28:142–148 www.sciencedirect.com Single molecule force fluorescence spectroscopy Cordova et al. 143 Figure 1 Manipulation method StrategyChallenges Demonstrations (a) (b) -Horizontal application of force -High photon flux of trap (A) damages fluoro- -Kinesin stepping (smFl) -Spatially separated trap and fluorescence beams phores, interlaced beams reduce photobleaching -DNA Hairpin unfolding (smFl, smFRET) impart controlled tension -Dual trap smTIRF (B) requires a pedestal, low -Holliday Junction (2 and 3 color smFRET) -Coincident trapping and fluoresescence (A) background assays can employ confocal or Epi- -Helicase driven DNA unwinding (smFl) beams have good position resolution illumniation -Myosin force generation(smFl) -Dual trap (B) assay for ultra high spatial resolution -DNA-protein interactions(smFl, STED, FIONA) -Fluorescence excitation through TIRF, confocal, -Force sensor calibration for in-vivo force measure- epifluorescence and superresolution ments (smFRET) -Surface coupled vertical application of force -High inherent cantilever stiffness -Active assembly of nano structures (cut and paste) -High spatial resolution -Scattering from cantilever reflection -Protein unfolding/refolding (smFRET) -Large force magnitude -Tip functionalization and tether placement -High speed AFM for probing dynamics in real time -TIRF or confocal fluorescence (a) (b) -Vertical and horizontal application of force -Lacks pinpoint steering -DNA packaging motor (polarization smFl) -Permanent (A) or electromagnet (B) -Sub-optimal position resolution in most setups -Helicase activity (smFRET) S -Surface coupled -DNA streching (smFRET) N -Torque/twisting applications -Multiplexed measurements -No adverse effects on sample from magnetic field - Simpler instrumental setup -Horizontal applications of force -Slow modulation of flow -DNA-protein interactions (smFl, smFRET) -Straightforward instrumental setup -Sub-optimal position resolution flow -Surface proximity optimal for TIRF -Molecule manipulation in one dimension -Multiplexed measurements -Larger beads required Current Opinion in Structural Biology Depiction of the experimental geometry for various single molecule manipulation and detection methods. Strategies, drawbacks, and achieved implementations are listed for each technique. RNA polymerase binding to DNA in a suspended fila- dyes [13]. Chu and coworkers used combined optical ment geometry. This work included a pedestal on the trapping and fluorescence with an actively stabilized slide surface to permit dumbbell trapping and constrain imaging system to resolve different colored dyes (Cy3 illumination to the DNA through TIRF [8]. In these cases and Alexa 647) bound to optically stretched DNA with fluorescence was spatially separated (SS) through a pair of subnanometer resolution [14]. traps, suspending 15–16 mm of DNA, a length that intro- duces compliance issues and compromises the ability to The next major combination incorporated fluorescence sense position. resonance energy transfer (smFRET), another powerful tool capable of revealing conformational/structural changes As high numerical aperture objectives became available, on the length scale of 2–8 nm (Figure 1). Combining ‘objective side’ smTIRF became possible. A simul- manipulation through force with short distance smFRET taneous and spatially coincident optical trapping and techniques enables direct localization of conformational single molecule fluorescence microscope was developed dynamics of loaded biomolecules providing unprecedent- by Block and coworkers to monitor strand separation of a ed mechanistic details of molecular machinery in real dye labeled 15 base pair region of dsDNA [9,10]. They time. Tarsa et al. developed this combination through witnessed the mechanical transitions corresponding to an interlaced trap and fluorescence method [15]. Their DNA hybrid rupture occurs simultaneously with changes coincident trapping and sm-FRET technique allowed in fluorescence emission. In spatially coincident exper- simultaneous observation of the mechanical transition of iments, enhanced photobleaching blocked use of favored bead position with smFRET changes between a donor single molecule dyes, which showed shortened lifetimes (Cy3) and acceptor (Alexa 647), during un/re-folding of a in the presence of the high photon flux of an optical trap DNA hairpin. This hairpin may act as a binary fluorescence [11]. A solution to this problem was provided by interla- based force sensor. cing the trapping and fluorescence lasers fast enough that the trapped bead behaved as if the trap were always on Later, Hohng et al. combined smFRET with a SS optical [12]. With interlaced methods, coincident trapping and trap to probe conformational dynamics of Holiday Junc- single molecule fluorescence was possible for a range of tion (HJ) molecules [16]. Here, the trap