in order to recapitulate the lytic is in an alignment suitable for super - resolution imaging , we utilized glass coated with antibodies directed against the nk cell activating receptor nkp30 and adhesion receptor cd18 , as described previously . the human nk cell line , nk92 , was prepared in single cell suspension and adhered to antibody - coated glass for 20 min then fixed . after fixation , cells were permeabilized and stained for f - actin using phalloidin alexa fluor 488 and for the lytic granule component perforin using pacific orange - conjugated anti - perforin antibody . using sequential scanning , we evaluated actin via phalloidin alexa fluor 488 in sted and anti - perforin via the pacific orange secondary antibody in both sted and confocal imaging modes . images were acquired using leica asaf software then exported to volocity software ( perkin elmer ) and thresholded using the same settings in all cases to allow for quantitative comparison of the images . as we had previously identified , both f - actin and lytic granules were present throughout the synapse . there was a qualitative improvement in the resolution of the lytic granules imaged using the sted modality ( fig .  1a , red ) , when compared with confocal ( fig .  1b , red ) . to quantitatively compare resolution , we measured a single granule in both sted and confocal and determined the full width at half maximum ( fwhm ) using leica asaf software ( fig . fwhm measures the width of the fluorescence intensity peak and thus reflects the ability to separate or resolve objects . as suggested by our observations , analysis confirmed greater resolution in sted , with a fwhm value of 90 nm , whereas fwhm in confocal was 210 nm . visualization of lytic granules imaged by cw - sted and confocal on f - actin . nk92 cells were adhered to glass coated with antibody to activating ( nkp30 ) and adhesion ( cd18 ) receptor then fixed , permeabilized and stained for perforin and actin . cells were imaged using cw - sted ( actin , green ) and either cw - sted or confocal ( perforin , red ) . shown is the same cell with granules detected by sted ( a ) or confocal ( b ) . a region of interest is enlarged to show greater resolution of granules ( center panel ) . ( c ) full width half maximum ( fwhm ) measurements of confocal ( green line ) and sted images ( red line ) . horizontal dashed lines show half maxima , vertical dashed lines show width at half maxima . ( d ) representative line profile of pixel intensities of actin ( green line ) and perforin ( red line ) taken from a line bisecting a single granule ( shown in white in sted image enlargement ) . the increased resolution we were able to identify using sted resulted in an ability to define lytic granules of an apparent smaller size . thus , with this improved ability to distinguish lytic granules , we sought to confirm our earlier finding that lytic granules , while located in areas of f - actin hypodensity , were either in minimally sized clearances in contact with or atop f - actin filaments . in order to accomplish this , we measured line profiles of fluorescence intensity for perforin and f - actin staining . consistent with our earlier findings , we found an intersection of line profiles ( fig . this indicates that lytic granules are closely associated with f - actin and thus secreted through minimally sized clearances . actin reorganization at the is is a critical prerequisite for cytotoxicity in both adaptive and innate immune effector cells . it is required for cell surface receptor rearrangements , cell activation signaling and for the subsequent polarization of lytic granules to the is . previous studies performed using 3d reconstruction of confocal images , however , have resulted in a model for secretion in which lytic granules are expelled through a central clearance of actin in both nk cells and their adaptive counterpart , the cytotoxic t lymphocyte . detailed super - resolution analysis of the nk cell lytic synapse by two independent laboratories suggests a new paradigm for cytotoxicity in which a pervasive actin network is present and acts not as a barrier but a facilitator for secretion . with this new understanding it will be interesting to see if this model extends to other immune cells undergoing directed secretion of both specialized secretory lysosomes and cytokines . alternatively , it may represent an additional checkpoint utilized by cells of the innate immune system as they access pre - armed functions . using dual color sted nanoscopy of lytic granules on actin filaments in nk cells we have shown in unprecedented resolution details of this new paradigm .
natural killer ( nk ) cells are innate immune effectors that eliminate diseased and tumorigenic targets through the directed secretion of specialized secretory lysosomes , termed lytic granules . 
 this directed secretion is triggered following the formation of an immunological synapse ( is ) , which is characterized by actin re - modeling and receptor organization at the interface between the nk cell and its susceptible target . 
 actin at the is has been described to be permissive to secretion by forming a large central clearance through which lytic granules are released . 
 however we , and others , have recently shown that the actin network in nk cells at the is is dynamic yet pervasive . 
 these efforts used multiple high resolution imaging techniques to demonstrate that the actin network does not act as a barrier to secretion , but instead enables the secretion of lytic granules through minimally sized clearances . in our recent publication 
 we visualized actin using continuous wave stimulated emission depletion ( cw - sted ) and lytic granules using the confocal modality . here 
 we report for the first time dual channel sted nanoscopy of nk cell lytic granules on actin filaments .