two of the mainstay techniques in single - molecule research are optical trapping and single - molecule fluorescence . previous attempts to combine these techniques in a single experiment  and on a single macromolecule of interest  have met with little success , because the light intensity within an optical trap is more than ten orders of magnitude greater than the light emitted by a single fluorophore . instead , the two techniques have been employed sequentially , or spatially separated by distances of several micrometers within the sample , imposing experimental restrictions that limit the utility of the combined method . here , we report the development of an instrument capable of true , simultaneous , spatially coincident optical trapping and single - molecule fluorescence . we demonstrate the capability of the apparatus by studying force - induced strand separation of a rhodamine - labeled , 15 base - pair segment of double - stranded dna , with force applied perpendicular to the axis of the dna molecule . as expected , we observed abrupt mechanical transitions corresponding to the unzipping of dna at a critical force . transitions occurred concomitant with changes in the fluorescence of dyes attached at the duplex ends , which became unquenched upon strand separation . through careful optical design , the use of high - performance spectral notch filters , a judicious choice of fluorophores , and the rapid acquisition of data gained by computer - automating the experiment , it is possible to perform combined optical trapping and single - molecule fluorescence . this opens the door to many types of experiment that employ optical traps to supply controlled external loads while fluorescent molecules report concurrent information about macromolecular structure . among the most useful single - molecule approaches are optical traps ( ' optical tweezers ' ) and fluorescence . the combination of these two biophysical techniques in a single assay offers a powerful tool for studying molecular systems , by allowing direct correlations to be made between nanoscale structural changes , reported by single - molecule fluorescence , and biomechanical transitions , probed by piconewton forces generated with optical traps . here , we demonstrate the feasibility of this combination by simultaneously observing single - molecule fluorescence changes and force - induced strand separations in dye - labeled double - stranded dna duplexes . prior attempts to marry optical trapping with single - molecule fluorescence failed to achieve spatially coincident fluorescence and trapping within the sample . in all cases reported , the optical trap was physically separated from the region of fluorescence excitation by a distance of up to several micrometers , or the techniques were applied sequentially . the comparatively large separation avoids problems caused by the high flux of infrared light within the optical trap , which can reduce fluorophore lifetimes ( through unwanted two - photon excitation of dyes or other destructive photochemical mechanisms ) and contributes to high levels of background light , which can readily obscure the weak fluorescence signal . a large physical separation imparts its own drawbacks , however , imposing severe restrictions on the experimental geometries that can be achieved . moreover , it requires that optical forces be applied remotely , generally through a suspended filament geometry  that is , through a tether , rather than directly to the macromolecule of interest . the elastic compliance of a tether increases with its length , and thermal measurement noise ( brownian noise ) increases in proportion to this compliance . our design strategy circumvents many earlier problems and achieves spatially coincident optical trapping and single - molecule fluorescence , making this a true combination of the two technologies . the optical layout of our instrument   ( see also the additional diagram ) was designed from the outset to permit simultaneous single - molecule fluorescence and optical trapping . the choices of dyes and excitation wavelengths were optimized to minimize two - photon excitation and achieve acceptably long fluorescence lifetimes in the trap . the laser wavelengths used for trapping , nanoscale position detection , and fluorescence were all broadly separated . attention was paid to the rejection of stray light and the selection of all optics , including the use of high - efficiency holographic notch filters ( kaiser optical , ann arbor , usa ) to reject laser excitation and detection wavelengths before the photodetectors . finally , the apparatus is computer - automated , greatly reducing the set - up time once a candidate fluorophore is identified and permitting rapid measurements prior to photobleaching . the instrument incorporates a microscope plus three lasers : one for trapping ( 1064 nm ) , a second for position detection ( 827 nm ) , and a third for fluorescence excitation ( 514 nm ; see the additional diagram ) . laser excitation light is coupled through the microscope objective into the specimen as an evanescent wave ( single - sided total internal reflection fluorescence , tirf ) . in our experimental geometry ( figure 1a ) , a 1,010 base - pair dna duplex with an overhanging segment was attached at one end ( via a biotin - avidin linkage ) to a polystyrene bead 500 nm in diameter and annealed at its distal end to a 15 base - pair oligonucleotide , anchored directly to the cover - glass surface ( via a digoxygenin - antidigoxygenin antibody linkage ) . tetramethylrhodamine ( tamra ) dyes conjugated to nucleotides were placed on complementary bases , one on the 3 ' end of the shorter 15-mer and the other on the 5 ' end of the long strand ; in these positions , the dye proximity causes fluorescence to be quenched . a recent study has shown that rhodamine self - quenching arises from the formation of noncovalent dimers based on stacked xanthine rings , leading to an approximately 30-fold decrease in fluorescence signal . the distance range probed by dye self - quenching is therefore exceedingly short ( about 12 nm ) , and smaller than most distances probed by fluorescence resonance energy transfer ( fret ) . the dna - bead complex was trapped and the microscope stage was moved at constant velocity , pulling the bead from the center of the trap and thereby increasing the load until rupture ( ' unzipping ' ) of the 15 base - pair duplex occurred . experiments were conducted in an assay medium consisting of 100 mm na - phosphate buffer ph 7.5 , 0.1% tween detergent and 0.1% beta mercaptoethanol , with 3.0 mg / ml bovine serum albumin added as a blocking protein ; buffers were degassed lightly under house vacuum before use in flow chambers . bead position , accurate to the nanometer level , was recorded with a quadrant photodiode   while fluorescence was monitored by counts on an avalanche photodiode ( apd ; eg&g optoelectronics , gaithersburg , usa ) , collected through a confocal pinhole . the area of regard of the apd was limited to a region of approximately 950 nm diameter in the specimen plane . fluorescence and position traces were both collected at 200 hz and filtered at 100 hz ; fluorescence count rates were further smoothed with a three - point boxcar filter . records of bead position were converted to force based on the measured stiffness of the trap . prior to rupture , force increased monotonically , and the low light levels indicated the quenched dye state ( figure 1b ) . at rupture , the force dropped from around 9 pn to 0 pn and the light level increased , indicating that the two fluorophores separated , unquenching the dye bound to the 15-mer remaining on the surface . after several seconds , this dye photobleached in a single step and the light signal returned to background levels . we also conducted similar experiments with single dyes bound to either the long strand or to the 15-mer ( data not shown ) . as expected , dyes bound to the long strand fluoresced normally and light levels decreased abruptly upon rupture as the bead carrying the dna strand was pulled beyond the excitation zone . records of light levels with dyes bound to the 15-mer show no such decrease , but instead bleached at some variable time later . the latter experiment supplies a control showing that rupture corresponds to strand dissociation , as expected , and not to the breakage of the digoxygenin - based link holding the dna to the surface . using the methods described here , it is possible to build up histograms of the forces required to unzip ( or to shear ) duplex dna molecules of various lengths under a variety of conditions and rates of loading ( m.j.l . when suitably combined , optical trapping and single - molecule fluorescence supply information beyond what can be learned with either technique alone , permitting structure and mechanics to be probed within a single macromolecule . traces corresponding to force - induced mechanical transitions may be challenging to interpret , because of difficulties in determining the precise location at which an applied force produces a conformational change . simultaneous fluorescence measurements can resolve this ambiguity by pinpointing the location(s ) of structural transitions . such assignments are especially important in the study of complex systems : for example , the folding / unfolding studies of nucleic acids and polypeptides , or conformational rearrangements associated with the translocation of processive enzymes . moreover , simultaneous optical trapping and single - molecule fluorescence measurements supply information about the sequence of molecular events . relative timing information can be used , for example , to establish the relationship between substrate binding and stepping motion in motor proteins , or to probe molecular mechanisms underlying conformational changes . we anticipate that this technique will have broad applicability to the study of fundamental biological questions , by providing a direct means to study coupling between biochemical and biomechanical reaction cycles . m.j.l . was a recipient of a postdoctoral fellowship from the jane coffin childs foundation . a combined optical trapping and fluorescence experiment to unzip dna . ( a ) a cartoon of the simplified experimental geometry ( not to scale ) . a bead was tethered by a digoxygenin - based linkage ( blue and yellow ) to the coverglass surface through a dna molecule , consisting of a long segment ( black ) joined to a shorter 15 base - pair strand that forms a duplex region ( red ) . the bead ( blue ) was captured by the optical trap and force was applied to unzip the short duplex . tetramethylrhodamine ( tamra ) dyes attached at the ends of the dna strands provide a fluorescence signal ( red dots ) . ( b ) simultaneous records of force ( red trace ) and fluorescence , measured as the photon count rate ( blue trace ) . the dye unquenched at the point of rupture , and later bleached at t  9 sec . see text for further details . a simplified diagram of the optical layout of our instrument ( see also ) . the inset is a cartoon showing a detail of the specimen in the region illuminated by the three lasers ( not to scale ) . fluorescence excitation of the sample is supplied by evanescent wave illumination through the microscope objective over a region of several square micrometers ( green ) . overlapping within this region , the optical - trapping laser ( pink ) and position - detection laser ( yellow ) beams are coaxial and brought to diffraction - limited spots near the coverglass , as shown . an optically trapped bead tethered to the coverglass surface the area of regard of the fluorescence photodetectors through a confocal pinhole is indicated ( dark gray ) . the main diagram shows the instrument itself , which is based on an inverted microscope with a nano - positionable three - dimensional piezo stage and equipped with a mercury arc lamp ; the key components are shown in the center of the diagram . also shown is the quadrant photodiode ( qpd ) subsystem used to detect changes in the position of the trapped bead . the input optics , including all three lasers , are shown to the right of the microscope inside the box labeled in red . the position - detection pathway is shown in orange , the trapping - laser pathway in red , the fluorescence - excitation pathway in blue and the fluorescence - emission pathway in dark green . the trapping laser beam can be moved electronically by means of acousto - optic deflectors ( aods ) placed at optical planes conjugate to the back focal plane of the objective . the output optics , including a cooled , intensified charge - coupled device ( ccd ) camera , a conventional black - and - white ccd camera , and two silicon avalanche photodiodes ( sapds ) , are shown to the left of the microscope , inside the box labeled in green . the identities of other optical elements are : b , beam ; d , dichroic ; f , filter ; l , lens ; p , polarizer ; s , shutter ; fm , flipper mirror .
backgroundtwo of the mainstay techniques in single - molecule research are optical trapping and single - molecule fluorescence . 
 previous attempts to combine these techniques in a single experiment  and on a single macromolecule of interest  have met with little success , because the light intensity within an optical trap is more than ten orders of magnitude greater than the light emitted by a single fluorophore . instead , the two techniques have been employed sequentially , or spatially separated by distances of several micrometers within the sample , imposing experimental restrictions that limit the utility of the combined method . here , we report the development of an instrument capable of true , simultaneous , spatially coincident optical trapping and single - molecule fluorescence.resultswe demonstrate the capability of the apparatus by studying force - induced strand separation of a rhodamine - labeled , 15 base - pair segment of double - stranded dna , with force applied perpendicular to the axis of the dna molecule . as expected , we observed abrupt mechanical transitions corresponding to the unzipping of dna at a critical force . 
 transitions occurred concomitant with changes in the fluorescence of dyes attached at the duplex ends , which became unquenched upon strand separation.conclusionsthrough careful optical design , the use of high - performance spectral notch filters , a judicious choice of fluorophores , and the rapid acquisition of data gained by computer - automating the experiment , it is possible to perform combined optical trapping and single - molecule fluorescence . 
 this opens the door to many types of experiment that employ optical traps to supply controlled external loads while fluorescent molecules report concurrent information about macromolecular structure .