briefly , the output of a 445 nm diode laser is spatially 
 filtered and adjusted to 2 mm beam diameter before passing through 
 two acousto - optic deflectors ( aod , gooch and housego ) . the beam deflections 
 generated by the aods are imaged with telecentric lenses into the 
 back focal plane of an oil immersion objective ( olympus plapon 1.42 
 na , 60 ) after passing through a polarizing beam splitter ( pbs ) . the small beam diameter underfills the back aperture of the objective 
 to generate a focal spot of 1 m full width at half - maximum 
 ( fwhm ) . a quarter wave plate before the objective causes any reflected 
 and scattered light by the sample to get reflected by the pbs before 
 being imaged onto a cmos camera ( photonfocus mv - d1024 - 160-cl-8 ) at 
 either 167 or 333 magnification by choosing the appropriate 
 focal length imaging lens . the incident power is adjusted to achieve 
 near - saturation of the cmos camera amounting to 2.5 and 10 kw / cm at the sample at 1.0 ms exposure time for the two magnifications . the two aod channels are scanned in a sawtooth fashion by separate , 
 phase - locked function generators at 84 and 83 khz , respectively . both 
 the absolute and relative frequencies are chosen to induce the smallest 
 detectable fluctuations in the background light intensity on the time 
 scale of the camera exposure time . even though the frequency difference 
 ( 1 khz ) nominally suggests a minimum exposure time of 1 ms , this requirement 
 is relaxed by the large spot size . for a fwhm of 1 m , few tens 
 of scans over an area of 10  10 m are sufficient 
 to generate a highly uniform illumination . at the given scan speeds , 
 this process only takes 100 s , much faster than the 
 shortest exposure time . any spot broadening induced by the limited 
 speed of the acoustic wave in the deflector only serves to further 
 smoothen the illumination . rapid scanning and illumination of an area 
 four times larger than what is imaged by the camera avoids the introduction 
 of diffraction fringes from the edges of the sawtooth pattern and 
 eventually allows for shot noise limited sensitivity toward the 10 level . to produce 
 images such 
 as figure 1c it is necessary to remove any 
 constant background caused by residual reflections and illumination 
 inhomogeneities . to do so we record 100 images while manually moving 
 the sample stage and then replace each pixel by the temporal median 
 value of the frame sequence to generate an optimal flat field image 
 that is independent of the sample . after division by the flat field 
 image , we obtain sample - specific images with shot noise limited sensitivity , 
 which simplifies the initial alignment and choosing an appropriate 
 region . finding the correct focal point is critical and can be estimated 
 by maximizing the contrast from individual actin filaments , although 
 fine adjustment during the recording is necessary . the latter is achieved 
 by recording a single image and subtracting it from the live preview 
 that only reveals changes in the sample scattering compared to the 
 original background image . in the presence of myosin 5a hmm , such 
 a subtraction leads to differential images such as those shown in supporting information movie s3 . to reduce the 
 effects of sample drift along the optical axis , we stabilize the focus 
 by monitoring the back - reflection of a totally internal reflected 
 beam at 633 nm . to generate an image containing all the static 
 iscat features we performed a temporal median filter over the range 
 of images in which the myosin 5a hmm molecule was processive , typically 
 greater than 1000 frames . alternatively , consecutive frames that lacked 
 myosin 5a hmm signals were averaged together to produce the static 
 iscat background image . contrast values for single myosin 5a hmm were 
 determined by the pixel value corresponding to the center of mass 
 of the point spread function . for myosin 5a detection , we initially 
 used 166 magnification , 
 104  104 pixel field of view at 63.6 nm / pixel , a 
 frame rate of 1.7 khz and time averaged the differential images to 
 10 hz . for tracking , we increased the magnification to 333 with 
 a 128  128 pixel field of view and a frame rate 
 of 1.0 khz to improve the electron count and therefore the signal - to - noise 
 ratio . nanometric tracking was performed by time averaging the differential 
 images to 25 hz and then fitting the point spread function to a two - dimensional 
 gaussian . rabbit skeletal muscle actin and mouse 
 myosin 5a hmm with a c - terminal gfp were 
 prepared as described and stored in liquid nitrogen until used . a 
 20 m actin stock solution was prepared in polymerization buffer 
 ( 10 mm imidazole , 50 mm kcl , 1 mm mgcl2 , 1 mm egta , ph 
 7.3 containing 1.7 mm dtt , 3 mm atp ) . actin was diluted to 200 nm 
 in motility buffer ( mb ; 20 mm mops ph 7.3 , 5 mm mgcl2 , 
 0.1 mm egta ) . 1.5 , 24  
 50 mm , vwr ) were cleaned by sequential rinsing with milli - q water , 
 ethanol , and water . they were then dried under a stream of dry nitrogen 
 and exposed to uv / ozone for 8 min at 50 w power using a plasma cleaner 
 ( diener electronic , plasma system femto ) . a single flow cell was then assembled 
 using double - sided transparent tape ( scotch ) and a second cover glass 
 ( no . 1 , 24  40 mm , vwr ) . the flow cell was rinsed with 
 1 mg / ml solution of poly(ethylene 
 glycol)-poly l - lysine ( peg - pll ) branch copolymer ( surface 
 solutions susos , switzerland ) in phosphate buffered saline and incubated 
 for 30 min . after 5 min of incubation , the chamber was washed with mb and 
 the surface was blocked by adding 1 mg / ml bsa in mb and subsequent 
 incubation for 5 min . a solution of 210 nm myosin ( mb containing 
 40 mm kcl , 5 mm dtt , 0.1 mg / ml bsa , and 5 m calmodulin ) was 
 added , incubated for 5 min , and then washed . myosin 5a hmm 
 was diluted to 50 nm in buffer containing 10 mm mops ( ph 7.0 ) , 2 mm 
 mgcl2 , 0.1 mm egta , and 40 mm kcl . a 5 l drop of 
 sample was applied to a carbon - coated copper grid ( pretreated with 
 uv light ) and stained with 1% uranyl acetate .
optical detection of individual proteins 
 requires fluorescent labeling . 
 cavity and plasmonic methodologies enhance single molecule signatures 
 in the absence of any labels but have struggled to demonstrate routine 
 and quantitative single protein detection . here , we used interferometric 
 scattering microscopy not only to detect but also to image and nanometrically 
 track the motion of single myosin 5a heavy meromyosin molecules without 
 the use of labels or any nanoscopic amplification . 
 together with the 
 simple experimental arrangement , an intrinsic independence from strong 
 electronic transition dipoles and a detection limit of < 60 kda , 
 our approach paves the way toward nonresonant , label - free sensing 
 and imaging of nanoscopic objects down to the single protein level .