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Paper text:
the dna sequence encoding avi peptide was inserted between 139140 amino acid positions of vp1 in aav2 helper plasmid , named as avi - aav2 .
a quatral plasmid cotransfection method was used to produce the raav2 vectors used in this study . briefly , one vector plasmid pssaav - cmv - lacz , one aav helper plasmid avi - aav2 , one bira - egfp plasmid , and one mini adenovirus function helper plasmid pf6 , were cotransfected into hek293 cells cultured in roller bottles at a ratio of 1:1:1:2 .
after extensive buffer exchange against phosphate - buffered saline with 5% d - sorbitol , the peak fractions of purified virus were pooled and stored at 80 c before administration .
alexa fluor 647-labeled streptavidin was agitated with aav - biotin for 1 hour in 4  degree before experiment .
a mixture of full / empty capsid ratio of approximately 10:1 was used for bulk and single - molecule experiments .
a hela cell line stably expressing the gfp - conjugate of pom121 was used , and freshly split cells were grown overnight on coverslips in dulbecco s modified eagle medium supplemented with 10% fetal bovine serum .
for living cell detection , alexa fluor 647-labeled raav2 were added to the gfp - pom121 hela cell line with virus : cell ratio as 10,000:1 .
the speed microscope includes an olympus ix81 equipped with a 1.4 na 100 oil - immersion apochromatic objective ( uplsapo 100x , olympus , center valley , pa ) , a 35 mw 633  nm he - ne laser ( melles griot , carlsbad , ca ) , a 120 mw arkr tunable ion laser ( melles griot ) , an on - chip multiplication gain charge - coupled device camera ( cascade 128 + , roper scientific , tucson , az ) and the slidebook software package ( intelligent imaging innovations , denver , co ) for data acquisition and processing .
a 17-nm focal length lens was added to set an epi - fluorescent setup where the diameter of the efficient excitation region was about 12 m . gfp and alexa fluor 647 fluorescence were excited by 488 and 633  nm lasers , respectively .
the two lasers were combined by an optical filter ( fff555/646 di01 , semrock , rochester , ny ) , collimated and focused into an overlapped illumination volume in the focal plane .
the green and red fluorescence emissions were collected by the same objective , filtered by a dichroic filter ( di01- r405/488/561/635 - 25x36 , semrock ) and an emission filter ( nf01- 405/488/561/635 - 25x5.0 , semrock ) and imaged by an identical ccd camera .
the position of the ne was determined at super - accuracy by fitting the fluorescence of gfp - pom121 as follows .
the pixel intensities within a row or a column approximately perpendicular to the ne were fit with a gaussian .
the peak position of the gaussian for a particular set of pixel intensities was considered the ne position for that row and column .
the peak positions of a series of such gaussians were then fit with a second - degree polynomial , yielding the orientation of the ne within the entire image .
the following rules were then used to select a single npc and determine its orientation , which needs to be perpendicular to the ne on the equator of the nucleus and to the y direction of the cartesian coordinates ( x , y ) in the ccd camera : ( i ) to focus on a gfp - npc with eight copies of gfp - pom121 , individual gfp - npcs on the ne were selected when their fluorescence intensity was ~8-fold that of a single gfp ; ( ii ) we chose a fluorescent npc on the equator of the nucleus such that the tangent of the ne at the location of this npc was parallel to the y - direction of the cartesian coordinates ( x , y ) in the ccd camera ; and ( iii ) we examined the ratio of gaussian widths in the long and short axes of the chosen gfp - npc fluorescence spot , which needed to fall between 1.74 and 1.82 . within this range
, an illuminated npc only has a free angle of 1.4 to the perpendicular direction to the ne .
the localization precision for fluorescent npcs , immobile fluorescence molecules , and moving fluorescence molecules was defined as how precisely the central point of each detected fluorescent diffraction - limited spot was determined .
for immobile molecules or fluorescent npcs , the fluorescent spot was fitted to a 2d symmetrical or an elliptical gaussian function , respectively , and the localization precision was determined by the standard deviation of multiple measurements of the central point . however , for moving molecules , the influence of particle motion during image acquisition should be considered in the determination of localization precision . in detail , the localization precision for moving substrates (  ) was determined by an algorithm of =f(16(s2+a2/12)/9n+8b2(s2+a2/12)2/a2n2 ) , where f is equal to 2 , n is the number of collected photons , a is the effective pixel size of the detector , b is the standard deviation of the background in photons per pixel , and s = s02 + 1/3dt , s0 is the standard deviation of the point spread function in the focal plane , d is the diffusion coefficient of substrate in the npc and t is the image acquisition time . in our measurements ,
the localization precision was 69  nm for moving raav2s according to the above equations .
due to the inevitable vibration of npcs in the ne of living cells , the localization precision of the npc centroid was ~6  nm .
additionally , based on the detection of 230 immobile alexa fluor 647-labeled gfp molecules adsorbed on a coverslip , the system error of aligned red and green fluorescence channels was determined to 3.0    0.1  nm .
therefore , the overall tracking precision for raav2s import through the gfp - labeled npc in living cells was estimated to ~912  nm .
experimental measurements are reported as mean  standard error of the mean , unless otherwise noted .
the dna sequence encoding avi peptide was inserted between 139140 amino acid positions of vp1 in aav2 helper plasmid , named as avi - aav2 .
a quatral plasmid cotransfection method was used to produce the raav2 vectors used in this study . briefly , one vector plasmid pssaav - cmv - lacz , one aav helper plasmid avi - aav2 , one bira - egfp plasmid , and one mini adenovirus function helper plasmid pf6 , were cotransfected into hek293 cells cultured in roller bottles at a ratio of 1:1:1:2 .
after extensive buffer exchange against phosphate - buffered saline with 5% d - sorbitol , the peak fractions of purified virus were pooled and stored at 80 c before administration .
alexa fluor 647-labeled streptavidin was agitated with aav - biotin for 1 hour in 4  degree before experiment .
a mixture of full / empty capsid ratio of approximately 10:1 was used for bulk and single - molecule experiments .
a hela cell line stably expressing the gfp - conjugate of pom121 was used , and freshly split cells were grown overnight on coverslips in dulbecco s modified eagle medium supplemented with 10% fetal bovine serum .
for living cell detection , alexa fluor 647-labeled raav2 were added to the gfp - pom121 hela cell line with virus : cell ratio as 10,000:1 .
the speed microscope includes an olympus ix81 equipped with a 1.4 na 100 oil - immersion apochromatic objective ( uplsapo 100x , olympus , center valley , pa ) , a 35 mw 633  nm he - ne laser ( melles griot , carlsbad , ca ) , a 120 mw arkr tunable ion laser ( melles griot ) , an on - chip multiplication gain charge - coupled device camera ( cascade 128 + , roper scientific , tucson , az ) and the slidebook software package ( intelligent imaging innovations , denver , co ) for data acquisition and processing .
a 17-nm focal length lens was added to set an epi - fluorescent setup where the diameter of the efficient excitation region was about 12 m . gfp and alexa fluor 647 fluorescence were excited by 488 and 633  nm lasers , respectively .
the two lasers were combined by an optical filter ( fff555/646 di01 , semrock , rochester , ny ) , collimated and focused into an overlapped illumination volume in the focal plane .
the green and red fluorescence emissions were collected by the same objective , filtered by a dichroic filter ( di01- r405/488/561/635 - 25x36 , semrock ) and an emission filter ( nf01- 405/488/561/635 - 25x5.0 , semrock ) and imaged by an identical ccd camera .
the position of the ne was determined at super - accuracy by fitting the fluorescence of gfp - pom121 as follows .
the pixel intensities within a row or a column approximately perpendicular to the ne were fit with a gaussian .
the peak position of the gaussian for a particular set of pixel intensities was considered the ne position for that row and column .
the peak positions of a series of such gaussians were then fit with a second - degree polynomial , yielding the orientation of the ne within the entire image .
the following rules were then used to select a single npc and determine its orientation , which needs to be perpendicular to the ne on the equator of the nucleus and to the y direction of the cartesian coordinates ( x , y ) in the ccd camera : ( i ) to focus on a gfp - npc with eight copies of gfp - pom121 , individual gfp - npcs on the ne were selected when their fluorescence intensity was ~8-fold that of a single gfp ; ( ii ) we chose a fluorescent npc on the equator of the nucleus such that the tangent of the ne at the location of this npc was parallel to the y - direction of the cartesian coordinates ( x , y ) in the ccd camera ; and ( iii ) we examined the ratio of gaussian widths in the long and short axes of the chosen gfp - npc fluorescence spot , which needed to fall between 1.74 and 1.82 . within this range
, an illuminated npc only has a free angle of 1.4 to the perpendicular direction to the ne .
the localization precision for fluorescent npcs , immobile fluorescence molecules , and moving fluorescence molecules was defined as how precisely the central point of each detected fluorescent diffraction - limited spot was determined . for immobile molecules or fluorescent npcs , the fluorescent spot was fitted to a 2d symmetrical or an elliptical gaussian function , respectively , and the localization precision was determined by the standard deviation of multiple measurements of the central point . however , for moving molecules , the influence of particle motion during image acquisition should be considered in the determination of localization precision . in detail , the localization precision for moving substrates (  ) was determined by an algorithm of =f(16(s2+a2/12)/9n+8b2(s2+a2/12)2/a2n2 ) , where f is equal to 2 , n is the number of collected photons , a is the effective pixel size of the detector , b is the standard deviation of the background in photons per pixel , and s = s02 + 1/3dt , s0 is the standard deviation of the point spread function in the focal plane , d is the diffusion coefficient of substrate in the npc and t is the image acquisition time . in our measurements ,
the localization precision was 69  nm for moving raav2s according to the above equations . due to the inevitable vibration of npcs in the ne of living cells , the localization precision of the npc centroid was ~6  nm .
additionally , based on the detection of 230 immobile alexa fluor 647-labeled gfp molecules adsorbed on a coverslip , the system error of aligned red and green fluorescence channels was determined to 3.0    0.1  nm .
therefore , the overall tracking precision for raav2s import through the gfp - labeled npc in living cells was estimated to ~912  nm .
experimental measurements are reported as mean  standard error of the mean , unless otherwise noted .
adeno - associated virus ( aav ) has been developed as a promising human gene therapy vector . 
 particularly , recombinant aav vector ( raav ) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane , endosomal membrane , and nuclear envelope ( ne ) . 
 so far , the aav transduction mechanism has not been explored thoroughly at the single viral particle level . in this study 
 , we employed high - speed super - resolution single - point edge - excitation sub - diffraction ( speed ) microscopy to map the events of single raav2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 912  nm and 220  ms . 
 data reveal that raav2 particles are imported through nuclear pore complexes ( npcs ) rather than nuclear membrane budding into the nucleus . 
 moreover , approximately 17% of the raav2 molecules starting from the cytoplasm successfully transverse the npcs to reach the nucleoplasm , revealing that the npcs act as a strict selective step for aav delivery . 
 this study lastly suggests a new pathway to improve aav vectors for human gene therapy .