Correlation Force Spectroscopy for Single Molecule Measurements


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The model in Figure 6 can explain all our observations. In this model, electrostatically mediated interactions between actin and myosin transition to the short-lived, stereo-specific state detected in our study. In this state, myosin can briefly bear force with an open actin binding cleft and P i bound.

Correlation force spectroscopy for single molecule measurements Milad Radiom

The working stroke of myosin occurs directly from this state and the closure of the cleft is tightly coupled to the stroke. Both the stroke and cleft-closure can be reversed under zero load, with a rate k int that increases with hindering load.

Phosphate release occurs after the stroke and rebinding of phosphate allows reversals of the stroke. The scheme of Figure 6 is fully consistent with a previously proposed model developed from muscle fiber experiments for skeletal myosin II Dantzig et al. That model included a force bearing state followed by reversible force generation the working stroke and then phosphate release Dantzig et al. Similar studies on cardiac fibers showed this model also is applicable to cardiac myosin Araujo and Walker, More recent studies of the rate of the working stroke in skeletal Muretta et al.

The data from previous experiments on fast-skeletal myosin with the UFFC system support that the model of Figure 6 also applies to this myosin isoform Capitanio et al. The present data provide the most direct evidence for this ordering of events after actin attachment in cardiac muscle myosin. High resolution crystal structures of myosin have been used to suggest that phosphate must be released before the stroke occurs Llinas et al.

In addition, some models of contraction developed to explain a wide variety of muscle fiber data have suggested that phosphate release and the working stroke are not tightly coupled Caremani et al. The possibility remains that other myosin isoforms which exhibit different kinetic properties than muscle myosin, such as non-muscle myosins V and VI, may proceed through actin binding, the working stroke, and phosphate release with different dynamics or even different sequences Llinas et al.

Specific experiments on those systems are needed to clarify the mechanisms. Experiments were conducted in flow cells constructed and prepared as previously described Woody et al. The calculated ionic strength of the experimental solutions was held constant across ATP and P i conditions. The values of the components and total ionic strength of the solutions for the various P i and MgATP conditions are given in Supplementary file 1 Table 3. Myosin was added to nitrocellulose-coated chambers and allowed to attach nonspecifically to 2. Experiments were performed with trap stiffness 0.

When a myosin molecule was found on a pedestal bead, a stage stabilization system was engaged Woody et al. The ultra-fast force clamp was then engaged, and 5—10 min of data were recorded for each applied force. Occasional filament breakage prevented collection at all forces 1. The optical trapping setup used in these experiments was described previously in detail, Woody et al.

Briefly, polarization-split nm beams are steered by two 1D electro-optical deflectors EODs, Conoptics, Inc into a 60x water immersion objective Nikon. The laser light collected from the chamber of an oil immersion condenser is projected onto two quadrant photodiodes conjugate to the back focal plane of the condenser for direct force detection Gittes and Schmidt, Experiments were conducted otherwise as previously described Capitanio et al. The trap moved under the applied force for nm before the applied force was switched to the opposite direction and the cycle of motions back and forth repeated.

To eliminate effects that tension on the actin filament might have on myosin interaction, the forces were distributed between the two beads so that the pretension on the filament was constant even as the applied force changed magnitude Figure 5—figure supplement 3. The force signals and feedback output signals to the EODs were digitized at kHz through a kHz anti-aliasing filter in the sum and difference amplifiers for the x - and y -direction difference signals from the photodiode currents.

The positions of the traps were determined from the EOD driving signals and optical calibration measurements of corresponding bead displacements. We added a novel real-time drift and slope correction DSC system to our UFFC instrument to improve stability and accuracy of the force signal as a result of thermal changes and other variations. This system is described in the below and Figure 5—figure supplement 3. Relative to acousto-optic deflectors AODs more commonly used in dynamic optical trap instruments, the electro-optic deflectors EODs used here provide a much smoother angular deflection of the IR beams constant slope of deflection vs.

Even with regular alignment, thermal drifts and or air currents produced instrumental 0. While the feedback system is briefly paused, the trap positions are not updated and when myosin is not interacting with actin the only force on the beads is from the pretension applied to the filament. The force signals observed during each pause can be used to reset the zero-force voltage and calculate the setpoint in the feedback loop to the recorded F 0 level plus the applied force.

We implemented this system on the FPGA feedback controller such that every 10 ms the feedback loop pauses twice, first when the trap position reaches the top of the set excursion distance, and again when the trap position is at the bottom of the excursion Figure 5—figure supplement 3c. Each pause lasts 2 ms, and the force signals from the last 1.

These individual values are averaged over 20 pauses to calculate the setpoint during the experiment. Either the top or bottom pause allows correction for the slow thermal drifts. Additionally, by using both the top and bottom F 0 values and the known excursion distance, the value of the EOD slope is be calculated Figure 5—figure supplement 3.

This slope value can then be used in the FPGA to update the setpoint value as the trap positions change in order to account for the slope in the force signals. Examples of the drift offset and the calculated slope values over a 30 s trace are shown in Figure 5—figure supplement 3d. When the data are analyzed, the recorded force signals can be transformed into the actual force signals by using a similar algorithm in the analysis software that was used in real time on the FPGA Figure 5—figure supplement 3e. When detecting and characterizing binding events, the forces and positions of the traps when the feedback was paused are ignored.

This DSC system markedly improved reliability and consistency within and across experiments, reduced the width of the velocity distributions, lowered effective deadtimes, and improved data reliability at lower forces. The smoothing varies with the applied force but is constant across experimental conditions.

Because higher loads cause faster motion of the actin, less smoothing is required and shorter events can be detected at higher loads. Small corrections are applied to the start- and end-times of the events to account for delay based on the Gaussian filter, as previously described Capitanio et al. From each data trace, a theoretical minimum detectable event length deadtime was calculated based on the velocity distributions and the threshold value as previously described Capitanio et al.

Events were designated as occurring either during hindering or assisting load based on the direction of actin motion when binding occurred and the polarity of the actin as determined by initial non-feedback experiments. For a given force, all events which were shorter than the largest calculated deadtime for traces recorded at that force were excluded from the analysis. The deadtimes, set to be constant across different biochemical conditions, were 2. Events from molecules at the same magnitudes and directions of force were pooled for each given condition e.

Each molecule contributed equally to the fitted values as described previously Woody et al. The observed amplitudes of each phase are reported as well as the deadtime corrected amplitudes Figure 2—figure supplement 1 , which consider how many events are likely missed due to the experimental deadtime and assuming exponential duration components Woody et al. Ensemble averages were carried out as have been described previously Woody et al.

The beginnings of the events were aligned based on their detected time of binding from the crossing of the smoothed velocity trace through the detection threshold as previously described Capitanio et al. The trap position for the leading bead the bead with the higher magnitude of force applied was taken as the position of the actin filament, as there is less influence of non-linear end compliance on this more highly loaded bead. Ensembles were weighted such that each molecule contributed equally to the final average. For the 10 mM P i data and for analysis of the 0 P i data which was directly compared to that with 10 mM P i Figures 4 and 5 , events longer than 25 ms were included because the total working stroke size continued to increase as events between 15 ms and 25 ms were excluded Figure 3—figure supplement 1b , which could be due to reversals and detachments occurring later in an interaction when P i is present.

Quantification of the ensemble average was done programmatically, using the methods and parameters reported below.

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The minimum position of the ensemble average within the first millisecond of detected actin binding was set to be zero displacement. The size of the initial displacement was calculated from the raw unfiltered ensemble averages by finding the time t init when the ensemble average reached its maximum within the first 1. The minimum and maximum positions were used to normalize the displacements to range from 0 to 1, as plotted in Figure 4a,b. The normalized displacement data between the minimum position and t init was used to fit a single exponential rise with an amplitude of 1 to determine the rate of the working stroke as reported in Figure 4c.

The location and position of the dip were determined by taking a 1 ms moving average of the ensemble and finding the minimum point between t init and 15 ms after the detected binding time. The size and timing of the dip are reported as the difference between the time and displacement of initial stroke and the minimum dip respectively.

Simulations of individual traces were performed using a Monte-Carlo based method Gillespie, using the rates and transitions given in Figure 5—figure supplement 1. Once all states were assigned for a given simulated interaction the last state is always a detached state , a position value was assigned to each state based on the mechanical properties of the states.

The simulated position values were drawn for each interaction from a Gaussian distribution with a 0. The position trace before the initial interaction was a constant slope with a velocity similar to what is observed in the data for the simulated applied force. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included. Thank you for submitting your article "Single molecule mechanics resolves the earliest events in force generation by cardiac myosin" for consideration by eLife.

Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Olga Boudker as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Michael Geeves Reviewer 2 ; Arne Gennerich Reviewer 3.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The reviewers and I felt that this is a significant piece of work that needs to be widely read and understood. It is a major technical achievement — to have such high time resolution optical trap data and the authors have chosen an ideal protein to use. This allows fine detail of myosin attachment and the subsequent events to be mapped in great detail. Here for the first time the authors appear to be seeing myosin heads weakly attached to actin and the transition through the power stroke to a strongly attached form, significantly with phosphate P i still present in the motor.

Each of these is a significant observation and if the interpretations are correct then the results have a big impact on some of the major questions about how myosin generates force. The interpretation of the data will be argued over for some time.

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Arguments have swung back and forth about the order of the power stroke and P i release steps, which comes first? The inability to measure these events directly has led to intense debate. Here they come down very firmly on power stroke followed by P i release. Another important question is what is the nature of the weakly-attached pre power-stroke state and how much load can it bear? Importantly the transition forward is less likely when attachment occurs at high load not because of load inhibition of the forwards step but because of acceleration of the reverse step.

Overall it was agreed that this work is important and that your conclusions are well supported by the data. In places the reviewers felt that the presentation needs to be improved by changes to the text. The details are important and should not be left in the Materials and methods alone. For a general reader the possibility of isoform differences between myosins needs to be addressed in relation to the major conclusions.

This is not mentioned in the paper but will add a complication to interpretations of the data. The authors should address why HMM is used and how this affects their interpretation. This requires some comment and may be one of the very interesting features of the data that requires further explanation.

In contrast Figure 2—figure supplement 2 is instead as showing symmetry, a single d value for assisting and hindering loads. There has been a lot of debate about asymmetry in the crossbridge stiffness and its consequences. This is worth commenting on. For example:. There is a substantial literature on the human protein including from some of the authors. The work on human form should surely be a better reference. Bagshaw and Trentham, , is all work in the absence of actin.

We also have added to the Discussion about the isoform in response to questions 2 and 3 as mentioned below. We expounded upon the last paragraph to further emphasize that there may be differences between the various myosin isoforms. The distance parameters d values obtained from the Bell fit have been used as an estimate of the force dependence of the kinetic transitions being observed. This asymmetry in force sensitivity may be related to myosin stiffness, but it does not have to be.

We interpreted the difference in the d values for k f for assisting vs. For k int , we have interpreted the single d value for assisting and resisting loads to indicate that a single transition is affected for both directions of force which slows under assisting load.

This directionality is consistent with reversals of the working stroke. We thank the reviewer for pointing this out and have updated the references to cite the most relevant work done on the human protein, namely Deacon, et al. We thank the reviewer for pointing this out and have updated the references to include Stein, Chock, and Eisenberg, and Brenner The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Single molecule mechanics resolves the earliest events in force generation by cardiac myosin

This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited. Article citation count generated by polling the highest count across the following sources: Crossref , PubMed Central , Scopus.

Cited 0 Views Annotations Open annotations. The current annotation count on this page is being calculated. Cite this article as: eLife ;8:e doi: Figure 1. Download asset Open asset. Figure 2 with 3 supplements see all. Figure 3 with 1 supplement see all. Figure 4 with 1 supplement see all. Figure 5 with 3 supplements see all. Figure 6. Phosphate release and force generation in cardiac myocytes investigated with caged phosphate and caged calcium A Araujo JW Walker Biophysical Journal 70 — The characterization of myosin—product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction CR Bagshaw DR Trentham Biochemical Journal — Rapid dissociation and reassociation of actomyosin cross-bridges during force generation: a newly observed facet of cross-bridge action in muscle B Brenner PNAS 88 — Sliding Filament Mechanism in Muscle Contraction, Single-Channel Recording.

Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state R Dominguez Y Freyzon KM Trybus C Cohen Cell 94 — Clifton NJ — To the extent no commercially available, they can be operatively integrated as indicated herein. Some examples are illustrative. Housing 12 can be made of any number of materials and can include appropriate structure and components to reduce noise into system As can be appreciated by those of skill in the art, the software would be adapted to speak to the AFM sub-system and understand and use the language or protocols of whatever commercial embodiment of AFM is utilized.

In particular, in could instruct operation of xyz scanner 18 and other AFM operation including the AFM laser not shown and photodiode quadrant detectors not shown. And, additionally, it would control any piezo stages 16 , lasers 30 , and other control features. And, furthermore, it would communicate with the AFM sub-system and with photodiodes 20 and camera 24 to allow recordation, manipulation, and evaluation of the data they measure or capture.

See website at ni. Such programming is frequently used for automating the usage of processing and measuring equipment, including but not limited to data acquisition, instrument control, test automation, analysis and signal processing, and industrial automation on a variety of platforms e. It can be interface with many libraries with a large number of functions e. The number of advanced mathematic blocks for functions such as integration, filters, and other specialized capabilities usually associated with data capture from hardware sensors is immense.

In addition, LabVIEW includes a text-based programming component called MathScript with additional functionality for signal processing, analysis and mathematics. Another known software component is PicoView see wwww. A scripting language can be used to control the several applications in system As can be appreciated by those skilled in the art, variants of the components in 10 may include piezo stages and programmable controllers. For further discussion of system 10 and its possible modes of operation see Examples infra. This can allow easy interchangeability or substitution to and from housing 12 FIG.

One example would be interchangeable modules for laser s One module could include a single laser and ancillary operational components in its own housing, on its own substrate or board, or otherwise a sub-assembly that could easily added or removed including connection and orientation to other components. A different module could contain a laser of different characteristics. A still further module could include a plurality of lasers in a form that could be hooked up to system 10 and selectively operated in system Another example would be regarding the detecting components of system Different photodiodes 20 could be in modular form and inserted or removed according to selection by the user.

The modules, boards, or sub-assemblies would be of a size that occupies a sub-set of the space inside housing By methods obvious to those skilled in the art, they can be configured or come packaged with the necessary hardware to operationally connect them into system This could include instructions or different accessories or alternatives to accomplish these ends.

As can be appreciated by those skilled in the art, piezo stage 16 presents a sample surface 52 see FIG. As will be indicated later, samples 50 can take many different forms. One example of particular emphasis here is a sample with one or more fluorescence molecules e. The FM laser s and optics would be selected according to conventional principles regarding the particular FM functionality desired for an application.

As indicated in FIG. The components would be operatively connected, computer system 40 initialized, and the GUI could guide the operator through start up, set up, any calibration procedures, control and manipulation, and recording of information, including simultaneous smAFM-FM. Additional understanding of operation of system 10 can be derived from specific applications described below.


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The present system 10 successfully combines single molecule AFM with FRET to apply forces on individual biological molecules and simultaneously monitor their conformational dynamics. The integrated smAFM-FM can be applied to biomedical research, drug discovery, disease diagnosis, ultra-sensitive bio-sensing, nanotechnology, and material science applications, to name a few.

The system can be used to apply forces on individual molecules or nanoscale objects and simultaneously monitor the structure, dynamics, and optical properties. Stand-alone AFM has limitations regarding structural changes in molecules as they interact. These can, however, be identified by attaching florescent probes to the molecules and monitoring the structure and dynamics using FRET.

This was mounted on a home built sample scanning confocal microscope The AFM sub-system is mounted on the optical microscope 14 with a homebuilt low-noise stage An intuitive GUI to control optomechanical and electronic components of system 10 in a synchronized fashion and to display data and real time is included. The former conditions important for studying nanoscale materials and polymers, while the latter is important for studying biological molecules and living cells. For simultaneous AFM-FRET measurements, macromolecules labeled with donor fluorophores and their binding partners labeled with exceptor dyes are immobilized on the substrate and AFM-tip respectively, and allowed to interact, the tip and substrate are separated to rupture the molecular complex and measure its bond strength with pN resolution.

Simultaneously, donor and exceptor fluorescence are collected by objective 61 , spectrally separated and detected by the two APDs to determine FRET in real time. The FRET time traces indicate changes in the structure of the bound molecules with nanometer resolution and the dynamics of them binding with millisecond time resolution. For simultaneous single molecule AFM-fluorescence spectral measurements, florescent nanoparticles like semiconductor nanocrystals or biomolecules labeled with fluorophores are immobilized in a substrate, a calibrated force is exerted on the molecules using the AFM tip while simultaneously the fluorescence is collected by objective 60 and dispersed by high resolution grating 22 on to back-thinned EMCCD camera By referring to FIGS.

In FIG. Dark state is also illustrated. As the AFM tip applies a force on the tetrapod by pressing it, tetrapod fluorescence emission intensity increases simultaneous with increasing force the fluorescence emission shifts to longer wavelengths. Single molecule fluorescence and single molecule force measurements with AFM are two powerful techniques, however, each suffers from limitations overcome by system The set up for these examples is shown in FIGS. Tip localization is illustrated in FIGS.

The AFM is located over the focused excitation laser beam 30 by recording Raleigh scattered light as the AFM tip is scanned over the laser beam. To confirm that the apex for the AFM tip corresponds to the maxima in its Raleigh scattered image, we used the tip to move a 20 nm fluorescence bead located over the excitation FM laser. Fluorescence from the bead FIG. The AFM tip was scanned over the FM laser beam, as the tip displaced the bead, an abrupt decrease in fluorescence was observed. Scale bar in FIGS. As can be appreciated by those skilled in the art, the foregoing example illustrates benefits and capabilities of system Other beneficial applications are, of course, possible.

Several others are discussed in this description. These examples are neither inclusive nor exclusive of possible uses of system Reference should also be taken to FIGS. To confirm that the apex of the AFM tip corresponds to the maxima in its Raleigh scattered image, we use the tip to move a 20 nm fluorescence bead located over the excitation FM laser. Fluorescence from the beam FIG. Scale bar: nm. A critical obstacle in using a sharp AFM probe for combined single molecule AFM-FM measurements is the low probability of interaction between fluorescent molecules on the substrate and the sharp AFM tip.

Commercially available AFM tips have a radius of curvature of approximately 10 nm. Similarly a single molecule has a size of approximately 1 nm to 10 nm. Since the dimensions of both the fluorescent molecules and the radius of curvature of a sharp AFM tip are significantly smaller than these images, the tip cannot be positioned exactly over the fluorescent molecule using diffraction limited optics.

This hurdle can be overcome by accurately positioning the AFM tip over a fluorescent molecule. This exemplary embodiment allows the immobilized fluorescent molecule and the AFM tip to be precisely aligned in registry at the center of the confocal microscope laser beam—the FM laser beam. The FM laser beam has a known, fixed location in space, and a known width and other characteristics.

It can be similar or the same to a typical excitation laser used in FM. The FM laser is thus used both for FM fluorescence microscopy functions and information about a sample e. To locate the AFM tip over a fluorescent molecule we use the following steps see also the flow chart and illustrations at FIG. We repeat these steps with every fluorescent molecule. See FIG. See also, Yildiz, et al.

We accomplish this by following the six steps listed above to locate the AFM tip and single molecule at the center of the laser beam. In the sixth step, we also send the trigger signal indicating start of AFM tip movement to the EMCCD camera to synchronize fluorescence spectra acquisition. The AFM tip is then used to press or stretch the molecule while recording corresponding changes in fluorescence spectra. See, e. See also Choi, C. As can be appreciated by those skilled in the art, the apparatus and methods discussed above can be applied in a variety of ways. Some have been mentioned. The Figures and Examples below, provide a compilation of some of those applications.

See also, Li, H. Below is some additional discussion of either those examples or others. We disclose the invention of an integrated single molecule Atomic Force Microscope-Fluorescence Microscope smAFM-FM for biomedical research, drug discovery, disease diagnosis, ultra-sensitive bio-sensing, nanotechnology and materials science applications. Single molecule fluorescence can report on the structure and dynamics of molecules while AFM can be used to load and manipulate molecules and measure their interactions.

However each of these techniques suffers from limitations that can be overcome using a combined smAFM-FM approach. For example, with a stand-alone AFM it is difficult to determine the structural changes in molecules as they interact. These can however be identified by attaching fluorescent probes to the molecules and monitoring their structure and dynamics using FRET.

Similarly using smAFM-FM it is possible to apply forces on materials and monitor changes in their optical properties, an approach which is critical for the design of nanoscale optical force sensors. Here we describe a smAFM-FM instrument using which we can apply forces on individual molecules or nanoscale objects and simultaneously monitor their structure, dynamics and optical properties.

A schematic of our instrument is shown in FIG. Fluorescence is collected by the objective, spectrally separated and detected either by two single photon counting avalanche photodiodes APD or dispersed by a high resolution grating onto a back-thinned EMCCD camera. The AFM is mounted on the optical microscope using a homebuilt low-noise stage.

We have also developed an intuitive graphical user interface to control all the optomechanical and electronic components of the microscope in a synchronized fashion and to display data in real time. The former condition is important for studying nanoscale materials and polymers while the latter is important for studying biological molecules and living cells. For simultaneous single molecule AFM-FRET measurements, macromolecules labeled with donor fluorophores and their binding partners labeled with acceptor dyes are immobilized on the substrate and AFM-tip respectively and allowed to interact.

The tip and substrate are separated to rupture the molecular complex and measure its bond strength with pN resolution. Simultaneously, donor and acceptor fluorescence are collected by the objective, spectrally separated and detected by the two APDs to determine FRET in real time. The FRET time traces indicate changes in the structure of the bound molecules with nm resolution and the dynamics of unbinding with ms time resolution.

For simultaneous single molecule AFM-Fluorescence spectral measurements, fluorescent nanoparticles like semiconductor nanocrystals or biomolecules labeled with fluorophores are immobilized on a substrate. A calibrated force is exerted on the molecules using the AFM tip while simultaneously the fluorescence is collected by the objective and dispersed by a high resolution grating onto a back-thinned EMCCD camera. When the AFM tip exerts force on a tetrapod by pressing it red trace , tetrapod fluorescence emission intensity increases black trace. As the force exerted on the tetrapod increases, its fluorescence spectra shifts to longer wavelengths brown trace is spectra at low force and blue trace is spectra at high force.

We were able to demonstrate, for the first time in the world, that a single tetrapod changes its optical properties when subjected to an external force FIGS. Simultaneously with increasing force, the fluorescence emission shifts to longer wavelengths FIG. Life science research: Determining the structure and dynamics of biological molecules such as DNA and proteins. These measurements can be conducted both in vitro as well as in living cells.

Determining the interaction of biomolecules with receptors and toxins. Drug discovery: Direct observation of targeted drug delivery and drug binding to target molecules in vitro, in live cells and tissue. This will enable rational design of better pharmaceutical products. Nanoscience and nanotechnology: Characterization of the optical properties of nanoscale materials like semiconductor nanocrystals, nanotubes and nanowires.

Measuring the force dependent optical properties of these materials will enable development of new technologies like optical force sensors, touch screen displays, and nanoscale biomedical diagnostic devices. Material science: Characterization of materials with novel force dependent optical properties. Developing more efficient organic semiconducting polymers by determining the relationship between the optical and electronic properties and polymer structure.

Environment research: Detection of toxins and pollutants at the ultimate single molecule resolution. Identifying chemical properties of the toxin at the single molecule level using its spectral signature. Optical MEMS industry: Design of Optical MEMS devices that rely on external stress to change optical properties, for example pressure sensors, disk-drive heads, biosensors, and optical switches. The focus of this project is to develop an integrated single molecule Atomic Force Microscope-Fluorescence Microscope smAFM-FM for biomedical research, drug discovery, disease diagnosis, and ultra-sensitive bio-sensing applications.

Single molecule fluorescence, particularly Fluorescence Resonance Energy Transfer FRET , and single molecule force measurements with the Atomic Force Microscope AFM are two powerful techniques that are widely used to study single molecules and their interaction with receptors, toxins, and pharmaceuticals. Single molecule FRET can report on the structure and dynamics of biomolecules while AFM can be used to manipulate molecules and measure their interactions.

For example, with a standalone AFM it is difficult to determine the structural changes in biological molecules as they interact. The smAFM-FM instrument applies forces on individual biological molecules and simultaneously monitor their structure and dynamics. This microscope has increased capabilities, efficiency, bandwidth, and ease of use. A tip scanning AFM is mounted on a sample scanning confocal microscope. Macromolecules for e. The substrate is raster scanned to image individual donor labeled receptors and locate them under the AFM tip.

The tip and substrate are bought into contact so that receptor and ligand bind; and then withdrawn to rupture the receptor-ligand complex and measure its bond strength with 1 pN resolution. Simultaneously, donor and acceptor fluorescence is measured with single photon counting avalanche photodiodes to determine FRET in real time. The FRET time traces indicate changes in the structure of the bound biomolecules with 1 nm resolution and the dynamics of unbinding with 1 ms time resolution. This experiment will showcase smAFM-FM's capabilities to simultaneously measure the interaction of single biomolecules with pN force resolution, their structure with nm distance resolution, and dynamics with ms time resolution.

The instrument will have a similar optical scheme as FIG. The instrument can be, for example, integrated with a commercially-available AFM platform e. A pulsed laser will be used to measure single molecule fluorescence and FRET lifetimes and perform anti bunching measurements. Additionally, the fluorescence will be dispersed using a grating onto a CCD camera to obtain single molecule fluorescence spectra. Examples of the components of FIG.

Single molecule FRET measures energy transfer between fluorescent dyes tagged to biomolecules; and can report on the conformational state and the dynamics of the biomolecules. AFM can measure the interaction between molecules immobilized on a cantilever and surface and can also be used to mechanically manipulate molecules. However each of these techniques suffers from limitations that can be overcome by the use of a combined single molecule AFM-FRET approach. For example, with a stand-alone AFM it is difficult to determine precisely where force exerts its effect within a macromolecular complex.

This location can however be identified by attaching fluorescent probes to molecular sub-domains and monitoring their motion using single molecule FRET. The components. The microscope setup is shown schematically in FIG. In the instrument, a tip scanning AFM will be mounted on a sample scanning confocal microscope. The substrate will be raster scanned to image individual donor labeled receptors and locate them under the AFM tip. The tip and substrate will be bought into contact so that receptor and ligand bind; and then withdrawn to rupture the receptor-ligand complex and measure its bond strength with 1 pN resolution.

Simultaneously, donor and acceptor fluorescence will be measured with single photon counting avalanche photodiodes to determine FRET in real time. The FRET time traces will indicate changes in the structure of the bound biomolecules with 1 nm resolution and the dynamics of unbinding with 1 ms time resolution.

Alternatively, a pulsed laser system will be used to detect fluorescence and FRET lifetimes. This laser will also be used for fluorescence antibunching measurements to detect the number of independent emitters in conjugated molecules as the conformation of the biomolecule is altered with the AFM.

In addition, the instrument can be used to measure the force dependent optical properties of semiconductor nanocrystals and conjugated molecules such as organic semiconductors and photosynthetic proteins. For these experiments the fluorescence will be dispersed using a grating onto a CCD camera to obtain single molecule fluorescence spectra. This technology satisfies critical academic and industrial needs to decipher fundamental biological processes, such as molecular recognition, drug discovery, and structure-function relationships in biomolecules.

The instrument can also be used to determine the conformation dependent optical properties of organic semiconductors used in light emitting devices; where molecular structure plays an important role in device function. With reference to FIG. When the streptavidin was cut, an abrupt loss of fluorescence signal was measured. The fluorescence reappeared when the streptavidin was re-pasted on the surface. See also Example 1 which discusses details about such tetrapods and other concepts regarding use of the system To validate this technique, we will measure the force induced shearing of dye-labeled, double stranded DNA.

With stand-alone AFM it is difficult to determine precisely where force exerts its effect within a macromolecular complex. The present system 10 and alignment methods can improve on these tasks. Bond strength can be measured, e. For further information refer to attached Examples above. Using a set up similar to FIG. Macromolecules e. The tip in some substrate will be brought into context so that receptor and ligand bind, and then withdrawn to rupture the receptor-ligand complex and measure its bond strength with one pN resolution.

Simultaneously, donor and exceptor of fluorescence will be measured with single photon counting avalanche photodiodes to determine FRET in real time. The FRET time traces will indicate some changes in the structure of the bound biomolecules with 1 nm resolution in the dynamics of them binding with one ms time resolution. Alternatively, a pulsed laser system will be used to detect fluorescence in FRET lifetimes.

This laser will also be used for fluorescence antibunching measurements to detect a number of independent emitters in conjugated molecules as the conformation of the biomolecule is altered with the AFM. In addition, the instrument can be used to measure the force dependent optical properties of semiconductor nanocrystals and conjugated molecules, such as organic semiconductors and photosynthetic proteins. For these experiments, the fluorescence will be dispersed using a grating on to a CCD camera to obtain single molecule fluorescence spectra.

Applications include academic and industrial needs to decipher fundamental biological processes such a molecular recognition, drug discovery, and structure-function relations in biomolecules. The instrument can also be used to determine the conformation dependent optical properties of organic semiconductors and used in light emitting devices, where molecular structure plays an important role in device function.

It can therefore be appreciated that system 10 and its methods are a combined single molecule AFM-confocal fluorescence microscope capable of simultaneously measuring fluorescence time traces, spectrum, and forces of single molecules. As will be appreciated by those skilled in the art, the invention can take many forms and embodiments.

Variations obvious to those skilled in the art will be included within the invention, which is not limited by the specific exemplary embodiments presented herein. For example, the specific commercial components utilized to form system 50 can vary according to need or desire. Additionally, the specific samples under investigation can vary.

Similar to other commercial analogs e. Regarding the modules, preassembled small plug in modules for different ones of these functions can be prepared. The operator can simply change as needed. Examples of generic components for system 10 can include the following. As is appreciated by those of skill in the art, obvious variations are possible. Examples of equipment and components that can be used in one set up include but are not limited the following:. Year of fee payment : 4. An apparatus, system, and method of integrating atomic force microscopy AFM and fluorescence microscopy FM.

One particular application is to simultaneous single molecular fluorescence with AFM force spectroscopy.

Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements
Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements
Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements
Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements
Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements
Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements
Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements
Correlation Force Spectroscopy for Single Molecule Measurements Correlation Force Spectroscopy for Single Molecule Measurements

Related Correlation Force Spectroscopy for Single Molecule Measurements



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