transmission electron 
 microscopy ( tem ) is one of the main tools 
 to investigate the morphology of materials , from subnanometer to micrometer 
 length scales .
however , the interaction of electrons with materials , 
 and especially beam sensitive structures such as polymers , biological materials , or 
 zeolites , causes different types of radiation 
 damage , e.g. , atomic displacement , electrostatic charging , sputtering , 
 radiolysis , and knock - on damage .
these 
 mechanisms operate at different length scales : at subnanometer length 
 scales , knock - on damage and atomic displacement can result in distorted 
 crystal lattices , while morphology changes due to heating , electrostatic 
 charging and sputtering are visible at nanometer and micrometer length 
 scales . hence , for beam sensitive materials , beam damage constitutes 
 a physical limit and determines the resolution that can be achieved 
 in imaging , diffraction , and electron tomography .
therefore , it is important to analyze and understand beam 
 damage to facilitate the study of these ( beam sensitive ) materials 
 with a minimum amount of artifacts .
the degradation of crystal 
 lattices , at subnanometer length scales , 
 can be studied by electron diffraction .
it is well established that 
 electron beam damage causes the fading of diffraction spots and rings 
 in protein crystallography , and it has 
 been shown that it is affected by the temperature of the material , 
 the electron flux through the material , and the accumulated amount 
 of electrons transmitting the material .
furthermore , this effect is easily quantified by following the intensity 
 of diffraction rings or spots as a function of accumulated dose .
the effects of beam damage at nanometer and micrometer length 
 scales 
 are dependent on and specific to the utilized imaging mode but mainly 
 relate to materials loss by sputtering , and shrinkage .
the degradation of materials causes artifacts in the form of changes 
 in contrast or local intensity , while mass loss will result in an 
 increase in overall intensity . in the 
 literature , this phenomenon is mostly studied by using the difference 
 image of two images with different accumulated electron doses .
these techniques , however , is not commonly used to follow beam damage 
 over a multitude of images , which is 
 essential for e.g. electron tomography .
the 
 analysis of organic photovoltaics ( opvs ) is a good example 
 where beam sensitivity plays an important part .
since the morphology of this photoactive 
 layer , i.e. , the distribution of molecules , including their nanoscale 
 phase separation , has a large impact on the overall efficiency of 
 opvs , tem has become a standard characterization tool .
common opv 
 materials are e.g. poly(3-hexylthiophene ) ( p3ht ) and phenyl - c61-butyric acid methyl ester ( pcbm ) .
these materials show diffraction 
 rings instead of diffraction spots , resulting from the random orientation 
 of crystallites or the presence of amorphous phases .
therefore , radial 
 averaging of diffraction patterns is a suitable way to record and 
 analyze the intensity change of p3ht and pcbm diffraction signals upon electron beam damage . in the analysis 
 of opvs ,
tem samples are usually created by floating 
 the photoactive layer on water from a water - soluble polymer film .
the resulting water that is present in the sample 
 might play a role in beam damage mechanisms as well , for example , 
 by creating oxygen radicals .
an oxygen- and water - free sample preparation 
 method is therefore introduced to specifically study the influence 
 of sample preparation . in this
paper , beam damage is studied 
 in both diffraction and bright 
 field imaging , investigating the effect of electron dose rate , temperature , 
 and sample preparation .
more specifically , ( 1 ) beam damage of the 
 crystallinity is quantified via radially averaged diffraction patterns , 
 ( 2 ) mass loss is assessed via changes in intensity and standard deviation 
 of the mean intensity , and ( 3 ) shrinkage is determined by monitoring 
 the movement of markers , while a method is introduced to determine 
 shrinkage or expansion via the use of the normalized cross - correlation .
poly(3-hexylthiophene ) ( p3ht , plexcore os2100 ) 
 was supplied by plextronics , while pcbm ( ( 6,6)-phenyl - c61-butyric acid methyl ester , purity 99% ) was purchased from solenne 
 b.v . and the solvent o - dichlorobenzene ( odcb ) 
 from sigma - aldrich .
pedot : pss ( clevios p vpal 4083 ) was purchased 
 from heraues gmbh .
tem grids ( quantifoil , r2\2 200-mesh cu ) was purchased 
 form quantifoil micro tools gmbh .
p3ht and pcbm were dissolved in 
 odcb , resulting in a solution with 1 wt % p3ht and 1 wt % pcbm .
the 
 solution was stirred at 70 c for 24 h to ensure complete dissolving 
 of the materials .
a custom - made specimen holder was used to 
 facilitate direct spin - coating on tem grids .
four recesses were laser 
 ablated ( 20 m deep with a diameter of 3 mm ) on 5 , 10 , 15 , and 
 20 mm from the center of a 50  50 mm glass plate .
a custom spring 
 was designed to lock the tem grids in the recesses , with minimal altering 
 of flow behavior of the solution during spin - coating .
glass plates 
 were cleaned by ( 1 ) ultrasonication in acetone for 30 min , ( 2 ) rubbing 
 with soap and rinsing with demineralized water , ( 3 ) ultrasonication 
 in isopropanol for 30 min , and ( 4 ) uv - ozone for 30 min .
first , pedot : pss was spin - coated on 25  25 mm glass plates , 
 at 3000 rpm for 60 s , resulting in a 50 nm thick water - soluble layer . 

the p3ht : pcbm solution was subsequently spin - coated at 2000 rpm for 
 120 s , which results in a 25 nm thick layer . the opv thin films prepared 
 on the pedot : pss spin - coated glass plates were submerged in water 
 to dissolve the pedot : pss layer , resulting in an opv thin film floating 
 on a water surface .
this film was picked up on a glow - discharged tem 
 grid , dried at room temperature , and subsequently annealed in air 
 at 120 c for 10 min . after floating of the opv layer on water 
 and pickup by the tem grid
, there is a small chance that some pedot : pss 
 will remain on the sample surface .
however , due to the small surface - to - volume 
 ratio , the diffraction and imaging experiments are dominated by the 
 bulk information , including oxygen and water which can penetrate into 
 the bulk .
furthermore , this adds a significant advantage to the new 
 direct spin - coating method , since there certainly will be no pedot : pss 
 present during this method . in one of the conventional prepared samples , 
 silver particles ( 10 nm diameter , dissolved in toluene with 
 oleylamine as capping agent )
were added to the p3ht : pcbm solution 
 before spin - coating . to exclude water and oxygen being present 
 in the opv thin film , 
 another approach was followed . here
, the opv thin film was directly 
 spin - coated on a tem grid inside a glovebox ( m - braun labmaster glove 
 box system ) at 500 rpm for 10 min .
the directly spin - coated samples 
 were annealed at 120 c for 10 min inside a glovebox .
note that 
 the direct spin - coating method is not used as an alternative to opv 
 device fabrication but as an alternative tem sample preparation method . 

it is not possible to achieve water- and oxygen - free tem analysis 
 by preparing a sample using the common floating method , hence the 
 need for a direct spin - coating method . for clarity , the sample code 
 used in the results section
the samples were loaded into the tu / e 
 cryo titan ( fei company ; now thermo fisher scientific ) , which was 
 operated at 300 kv and is equipped with a field emission gun .
diffraction 
 patterns were acquired at dose rates of 0.1 , 1 , 10 e/( s ) , with exposure times of 5 , 0.5 , and 0.05 s ( keeping the total 
 dose per pattern constant ) , at a camera length of 1.15 m. for the 
 bright field imaging experiments , the dose rates were set at 1 , 10 , 
 and 100 e/( s ) , with exposure times of 5 , 0.5 , and 
 0.05 s ( again , keeping the total dose per image constant ) , respectively , 
 at a magnification of 18k . for high accumulated dose experiments , 
 the screen was flipped down to realize constant irradiation and flipped 
 up to acquire intermediate images .
diffraction patterns were radially averaged 
 using an in - house matlab script ( details in the supporting information , section 1 ) and fitted by a nonlinear 
 least - squares method using the lsqnonlin command in matlab ( details 
 in results section and figure 1b ) .
the signal intensities of p3ht ( at 0.256 
  ) and pcbm ( at 0.217  ) were normalized to their initial intensity and followed as a function 
 of accumulated electron dose .
( a ) fading of diffraction rings of a p3ht : pcbm 
 bulk heterojunction 
 at room temperature , before and after exposure to 50 e /  .
( b ) radial average of diffraction pattern as shown in ( a ) 
 decomposed in its different components by least - squares fitting .
prior to further analysis , bright 
 field images were aligned via 
 cross - correlation to avoid processing artifacts from sample drift . 

the aligned data stack was cropped to the overlap area present in 
 all images for analysis . from the resulting aligned stack of images , 

the average intensity , standard deviation , and the normalized cross - correlation 
 coefficient with the first image of the series were measured as a 
 function of accumulated electron dose .
finally , to assess local 
 deformations , the aligned stack was cut 
 into 16 subareas of equal size .
these subarea stacks were individually 
 aligned via cross - correlation . from this alignment , the shift of each 
 individual subarea with respect to the first image in the x and y direction was used to calculate 
 the displacement of each subarea .
furthermore , the relative displacement 
 from the center of the total image was calculated by measuring the 
 distance between the center of the subarea and the center of the total 
 image .
more details can be found in the results section and supporting information section 
 2 .
figure 1a shows 
 a diffraction pattern obtained from a typical p3ht : pcbm bulk heterojunction 
 prepared via the conventional sample preparation ( cf / rt/10 ) , before 
 and after exposure to 50 e /  at room temperature . 

it is evident that the sharp outer ring , depicting the ( 020 ) lattice 
 spacing in p3ht , is decreasing in intensity faster than the broad 
 inner ring , characterizing pcbm nanocrystals , which is in agreement 
 with previous studies . since the pcbm 
 is inherently broader than the p3ht ring , indicating a large part 
 of pcbm being amorphous , a slower decrease in diffraction intensity 
 for pcbm
the 
 radially averaged diffraction pattern of the p3ht : pcbm bulk heterojunction 
 is presented . to quantitatively analyze the decrease in intensity 
 as a function of accumulated electron dose , it is necessary to separate 
 neighboring peaks .
therefore , to independently address the intensity 
 of the pcbm peak at 0.217  and the p3ht 
 peak at 0.256  , the spectrum was fitted 
 to a model function by least - squares fitting .
this function contains 
 ( in the same order as in eq 1 ) a gaussian describing the pcbm peak , a gaussian describing 
 the p3ht peak , a gaussian located between 0.285 and 0.323  describing ( inseparable ) secondary components of 
 both p3ht and pcbm , a power law to estimate the inelastic 
 background , and a constant representing residual dark current in the 
 diffraction patterns .
the variables a1 and a2 describe the intensity of the 
 pcbm and p3ht signal , respectively.1 to study the 
 effect of electron dose rate on the decrease of intensity of p3ht 
 and pcbm diffraction rings , dose series were acquired at various dose 
 rates , i.e. , 0.1 , 1 , and 10 e/( s ) .
the dose per 
 image was kept constant at 0.5 e /  by adjusting the 
 exposure time accordingly .
because of differences in absolute peak 
 height for pcbm and p3ht , and changes in pcbm / p3ht ratio in different 
 regions of the sample , all peaks were normalized to the peak intensity 
 of pcbm ( a10 ) and p3ht ( a20 ) of the initial 
 diffraction pattern ( a1,20 ) .
figure 2 shows the decrease in relative intensity 
 ( a1,2i / a1,20 ) for p3ht ( a ) and pcbm ( b ) acquired 
 at three different dose rates ( cf / rt / x ) , in the presence 
 of water and oxygen .
the results confirm that the relative intensity 
 of the p3ht diffraction ring decreases faster than the relative intensity 
 of the pcbm diffraction ring , as seen in figure 1a . in all three cases the critical dose for 
 p3ht , which we define by the accumulated electron dose at which the 
 relative intensity decreases to 1/e ( 37% ) , 

is about 1619 e /  ( table 2 ) , as calculated from an exponential decay 
 fit .
most notably , changing the electron dose rate from 0.1 to 10 
 e/( s ) has no significant effect on the fading of 
 the diffraction rings , i.e. , the critical dose .
fading of relative diffraction 
 intensity ( a / a ) as a 
 function of accumulated dose for p3ht 
 ( a ) and pcbm ( b ) at different dose rates . for visibility reasons
the critical dose of pcbm was obtained by an exponential decay 
 extrapolation of the curves in figure 2b ( details are presented in the supporting information , section 3 ) .
therefore , the critical 
 dose for pcbm is less well - defined than the critical dose for p3ht .
table 2 shows that this critical 
 dose is between 265 and 412 e /  . despite the large 
 error due to extrapolations
, it is clearly shown that the critical 
 dose of pcbm is more than 10 times larger than the critical dose of 
 p3ht .
therefore , in this system , the stability of p3ht is the limiting 
 factor .
the results show that , using a dose of 0.1 e /  per image , at least 160 diffraction patterns can be acquired , opening 
 up the possibility of using high electron dose techniques such as 
 electron diffraction tomography for organic crystals . to complement the presented results , studies on beam damage effects 
 for each component individually and nanocomposites of different ratios 
 should be considered .
, dose rates between 0.1 and 10 e/( s ) do not have an effect on the rate of relative intensity loss 
 of both p3ht and pcbm .
this is in agreement with previous studies 
 on the decrease of features in the eels spectrum of other organic 
 materials , but in contrast with observations of karuppasamy 
 et al . in single - particle cryo - electron microscopy .
note that karuppasamy et al . used an electron microscope 
 operating at 120 kv , significantly decreasing the inelastic mean free 
 path . at 300 kv , as in our experiments
, changing the sample thickness 
 or increasing the electron dose rate to values above 10 e/( s ) might therefore cause an electron dose rate effect on 
 sample damage .
nonetheless , this falls beyond the scope of this paper 
 and is therefore not investigated . as the decrease in diffraction 
 intensity
is related to the breaking 
 of chemical bonds , beam damage will change the homo and lumo levels 
 of opv materials , which can be measured with electron energy loss 
 spectroscopy ( eels ) . based on the 
 low - loss eels results presented in ref ( 17 ) , a critical dose can be estimated which is similar 
 to our diffraction results .
this clearly illustrates the close relationship 
 between diffraction intensities and energy levels in opv materials 
 with low - dose diffraction being somewhat simpler to carry out and 
 more broadly available for critical dose measurements .
the above results were obtained with a standard opv 
 bulk heterojunction sample imaged at room temperature .
additional 
 dose series were acquired in cryogenic conditions , as this has been 
 shown to reduce beam damage in beam sensitive materials .
we furthermore studied the effect 
 of sample preparation using either the conventional sample preparation 
 ( cf ) or by direct spin - coating ( ds ) on a tem grid in a water and oxygen - free 
 environment ( glovebox ) , facilitated by a custom - made tem specimen 
 holder as described in the methods section .
figure 3 shows the 
 influence of temperature and sample preparation on the decrease of 
 relative intensity of the p3ht ( a ) and the pcbm ( b ) signal , with a 
 dose per image of 0.5 e /  acquired with a dose rate 
 of 10 e/( s ) .
it is clearly visible that the sample 
 cf / cryo/10 shows a slower fading of the diffraction signal for both 
 the pcbm and the p3ht signal .
the critical dose increases from 16 
 e /  at room temperature to 108 e /  at 80 k ( table 3 ) . hence , cryo - preservation is shown again to be essential in the 
 analysis of beam sensitive materials . more importantly , having a quantitative criterion 
 or threshold for determining exposure limits
as presented here will 
 provide a framework to maximize useful information before beam damage 
 influences the validity of acquired data .
fading of relative diffraction 
 intensity ( a / a ) for 
 p3ht ( a ) and pcbm ( b ) as a function of 
 accumulated dose , showing the effect of sample preparation at room 
 temperature ( rt ) and at 80 k ( cryo ) .
sample preparation effects , i.e. , the presence or absence of oxygen 
 and water , are only minor at room temperature .
however , at cryogenic 
 conditions , the rate of relative intensity loss for p3ht ( figure 3a , purple triangles ) 
 is significantly decreased by a water- and oxygen - free sample preparation 
 method ( ds / cryo/10 ) , increasing the critical dose from 108 
 to 188 e /  ( table 3 ) .
a detailed analysis of the significance 
 of these results is presented in the supporting information , section 4 . the observed difference in critical 
 dose for p3ht and pcbm with 
 or without oxygen and water being present at low temperatures
these molecules 
 will form radicals upon electron beam irradiation , thus decreasing 
 the crystalline order at lower accumulated electron doses .
because 
 of the fact that the samples have been in high vacuum for at least 
 30 min before imaging , it is expected that water and oxygen will diffuse 
 out of the material at rt conditions and thus only be present in small 
 amounts in the imaged thin film
. this explains why sample preparation 
 seems to have little influence when measuring at room temperature . 
 for cryogenic conditions
the exclusion of water and oxygen significantly 
 improves the stability of p3ht exposed to an electron beam .
furthermore , 
 for the room temperature experiments , annealing in air might cause 
 minor damage .
this also could explain the observed critical dose , 
 causing the critical dose for the cf / rt/10 to be lower than the critical 
 dose for ds / rt/10 . the values in table 3 also suggest that a water- and oxygen - free 
 method increases the 
 critical dose of pcbm in cryogenic conditions . in this case , a linear 
 extrapolation was used , since the adjusted r value for the exponential decay fits were too low ( 0.85 ( exponential 
 fit ) compared to 0.95 ( linear fit ) ) . since the pcbm intensity decay 
 will probably not behave in a linear fashion
, one could suggest that 
 the difference between the pcbm critical doses at cryogenic conditions 
 is therefore not significant .
since pcbm is more hydrophobic than 
 p3ht , and it is known that p3ht forms a charge - transfer complex with 
 oxygen , it can indeed be expected that 
 the presence of water and oxygen has less influence on the decay of 
 the pcbm diffraction ring . in imaging mode , 

beam damage can present itself as among others ( 1 ) shrinkage or expansion 
 of an organic thin film and ( 2 ) sample 
 thinning due to mass loss .
both effects 
 were measured by acquiring dose series of samples prepared by cf and 
 ds methods .
these series of images were acquired at different dose 
 rates and at either room temperature or cryogenic conditions .
to study 
 sample deformations , the normalized cross - correlation coefficient 
 ( nccc ) of an image with respect to the first image within the series 
 was calculated .
sample thinning due to mass loss was assessed by the 
 relative decrease in average pixel intensity and the accompanying 
 standard deviation .
figure 4a shows the 
 decrease of the 
 nccc with respect to the original image as a function of accumulated 
 dose at dose rates of 1 , 10 , and 100 e/( s ) ( cf / rt / x ) .
local differences in thickness , small deviations in 
 intensity due to microscope instabilities , and high contrast contaminations 
 cause the nccc to start at different values for each series . therefore , 
 the trend of the curves is studies instead of the absolute nccc values . 

it is clearly visible that the nccc drops quickly before a more stable 
 regime is reached after approximately 150180 e /  , as calculated from the intersect of two linear fits : one 
 fitted to the first 50 e /  and one fitted between 
 750 and 1000 e /  ( for details , see supporting information , section 5 ) .
identical fit regimes 
 were chosen in every experiment to ensure a fair comparison between 
 data sets , at room temperature and at cryogenic conditions .
( a ) cross - correlation 
 coefficient with respect to the first image 
 of three dose series ( 1 , 10 , and 100 e/( s ) ) as 
 a function of accumulated dose . for visibility reasons ,
( b ) cross - correlation coefficient with the first 
 image of a dose series acquired at 10 e /  at room 
 temperature , starting at 750 e /  .
an exponential 
 fit is added to calculate the critical dose . for cf / rt/100 ,
the quick initial drop is less evident ( a 
 slope 
 of ( 6.73  0.04 )  10 as compared 
 to ( 1.32  0.04 )  10 and ( 1.67 
  0.05 )  10 for cf / rt/1 and cf / rt/10 , 
 respectively ) , which is likely caused by pre - exposure with a higher 
 dose before starting the dose series .
an exposure of 0.25 s to check 
 that there was no carbon film in the entire field of view caused a 
 pre - exposure of 0.25 and 2.5 e /  for the cf / rt/1 
 and cf / rt/10 samples , respectively , which is small compared to the 
 dose limit .
however , for the cf / rt/100 , this pre - exposure will be 
 25 e /  .
this will cause significant damage , and therefore 
 the nccc for the last image will be higher , as the first image is 
 already more damaged .
after the cutoff dose is reached , a more stable 
 second regime starts , as indicated by a much smaller slope (  
 1  10 ) .
since the cutoff dose and the slope 
 of the second regime are fairly similar between dose rates of 1 to 
 100 e/( s ) , it is concluded that the dose rate effect 
 is negligible , similar to the diffraction data .
although the 
 slope of the second damage regime is much smaller , 
 beam damage still occurs . in a similar way as introduced for the diffraction 
 data , a quantitative criterion can be set to calculate the critical 
 dose , at which the initial nccc decreases with a factor of e. to estimate this critical dose for imaging , a dose series 
 is acquired at 10 e/( s ) , reaching an accumulated 
 dose of 30  000 e /  .
an exponential decay curve 
 is fitted to the nccc decay of this data set , starting at 750 e /  to exclude effects from the first damage regime . calculating 
 the critical dose imaging results in a value of ( 2.6  0.2 )  
 10 e /  ( for details ,
since 
 the nccc is a measure of similarity , it will entail among 
 others sample deformation and contrast loss or intermixing . to show 
 that the initial drop in cross - correlation is caused by sample deformations , 
 the stack of aligned images acquired at 10 e/( s ) 

was cut into 16 equal subimages ( figure 5a ) , and these different subimages were aligned 
 by the normalized cross - correlation to the next image of the dose 
 series .
this approach facilitates the measurement of displacement 
 of each subimage , i.e. , expansion or contraction of the thin film 
 as a function of accumulated dose .
it is clearly visible that the displacement of the subimages at 
 the corners and edges of the stack is larger than the displacement 
 of the subimages at the center of the stack ( figure 5b ) , indicating movement to or from the center 
 of the stack . calculating the relative increase in distance between 
 the center of each subimage and the center of the total image results 
 in a shrinkage of 0.70  0.14% at an accumulated dose of 1000 
 e /  .
( a ) schematic representation of cutting a stack of images 
 in 16 
 parts ( left to middle ) and using the cross - correlation to find the 
 shift in 1 part of the image compared to the original image ( right ) , 
 as a representation of shrinkage or expansion .
( b ) the average shift 
 of the 4 corner subareas , the 8 edge subareas , and the 4 center subareas , 
 acquired at 10 e/( s ) .
( c ) relative decrease in 
 standard deviation of the mean intensity as a function of accumulated 
 dose for samples prepared by the common floating approach at room 
 temperature , at three different dose rates . for visibility reasons , 

the validity and accuracy of the method above has been proven 
 against 
 an approach that uses the movement of high contrast silver makers 
 to determine shift , as presented in the supporting information , section 2 . as a different measure to assess 
 beam damage
, we quantify the change 
 in average intensity that can be interpreted as a change in mass 
thickness 
 due to either mass loss or deformations of the thin film . to minimize 
 the effects of microscope instabilities , two low - magnification overview 
 images were taken , before and after the dose series . from these two 
 images
the intensity ratio of the nonilluminated and illuminated region 
 before and after the series was calculated ( table 4 ) .
because of the occurrence of lateral shrinkage , 
 one would expect the sample to become thicker ( assuming volume preservation ) , 
 since material is moving toward the center .
however , table 4 shows an insignificant change in intensity , 
 which can be explained by the fact that the relative shrinkage is 
 small .
furthermore , thinning caused by mass loss will increase the 
 average intensity , compensating for the thickening effect of shrinkage . 
 before and after images are shown in the supporting information , section 7 .
last , figure 5c 
 shows the relative decrease in standard deviation of the mean intensity 
 of the aligned images .
shrinkage would result in a higher standard 
 deviation , since more data will move into the field of view .
it is , 
 however , clear that the standard deviation decreases , again pointing 
 out that mass loss plays a significant role in beam damage processes . 

the fast drop in relative standard deviation at the onset of beam 
 damage might be caused by quick mass loss of material at the surface 
 of the sample removing sample roughness .
after this initial drop , 
 a more stable regime is reached where mass loss plays a less significant 
 role since the sample has reached a more equilibrated state . from this , we can again conclude that in imaging mode a dose rate 
 between 1 and 100 e/( s ) has a negligible effect 
 on beam damage .
furthermore , we can conclude that the minimal pre - exposure 
 of polymer thin films or microtome sections , often applied prior to 
 acquiring electron tomography data sets , can now be quantified to limit artifacts in imaging . during this 
 regime , mass loss and shrinkage
occur , while after this regime , a 
 second regime will set in , significantly reducing the rate of electron 
 beam damage at higher doses .
furthermore , a critical dose in imaging 
 is calculated , which results in a critical dose of more than 2500 
 e /  . even when the cutoff dose of the first damage 
 regime is subtracted , this leaves more than enough dose available 
 for acquiring tomography data sets ( e.g. , tilting from 70 
 to + 70 in 1 steps , with 10 e /  per image 
 results in a total dose of 1410 e /  ) .
figure 6a shows 
 the decline of the nccc with respect to the first image as a function 
 of accumulated dose , for series acquired at room temperature and cryogenic 
 conditions , and prepared by either the cf or ds methods .
comparing 
 the room temperature measurements , one can see that the decrease in 
 nccc in the first regime does not significantly change for ds method , 
 as the calculated slope is ( 1.57  0.12 )  10 compared to ( 1.67  0.05 )  10 for the cf method .
however , the cutoff between the 
 two damage regimes decreases from 156  9 to 96  12 e /  .
this indicates that the rate of damage is similar for both 
 the cf and the ds method but that the total damage in the first regime 
 will be smaller for the ds method ( the relative decay of nccc at the 
 cutoff dose decreases from 24% for the cf method to 15% for the ds 
 method ) , indicating a more stable system .
( a ) decrease in nccc 
 with respect to the first image for dose series 
 acquired at 10 e/( s ) , at room temperature and at 
 cryogenic conditions , from samples prepared by either the conventional 
 floating method or the oxygen- and water - free method . ( b ) cross - correlation 
 with the first image of a dose series acquired at 10 e /  at room temperature of a sample prepared by the oxygen- and 
 water - free method .
( c ) mean - squared displacement of the 12 edge subareas and the 
 4 center subareas calculated by alignment via cross - correlation .
the 
 images were acquired at 10 e /  at room temperature 
 from a sample prepared by either the common floating approach or the 
 oxygen- and water - free method .
( d ) see part c , but for images acquired 
 at cryogenic conditions . for visibility reasons , every fifth data 
 point is shown .
the second , more stable 
 regime shows a relatively constant nccc 
 for the ds method , since the slope of this regime decreases from ( 12.7 
  0.3 )  10 for the cf method to ( 6.9 
  0.2 )  10 without water and oxygen 
 present .
the critical dose therefore increases from ( 2.6  0.2 ) 
  10 e /  for the cf method to ( 5.0 
  0.4 )  10 e /  for the ds 
 method ( for details , see supporting information , sections 5 and 6 ) .
this again indicates that the ds method will 
 create a more stable system during electron beam radiation .
figure 6c shows 
 the displacement of parts of the images acquired at room temperature 
 by using the alignment method as described before .
it is clearly visible 
 that by using the ds method , the shrinkage of the thin film decreases 
 significantly , from 0.70  0.18% to 0.40  0.14% .
the sample prepared 
 by the ds method is more stable , since it is clear that the absolute 
 decrease in relative standard deviation is smaller for this method . 
 just as the nccc behavior , a second more stable regime sets in .
the 
 smaller slope in this regime for the ds method ( figure 6b , black squares vs red circles ) indicates 
 a larger stability .
all in all , this leads to the conclusion that 
 at room temperature an oxygen- and water - free sample preparation method 
 significantly enhances sample stability . after subtracting the initial 
 pre - exposure regime , an electron dose of 5
 10 e /  is still available before reaching the critical dose imaging , 
 which is about twice as much as for a sample prepared by the cf method . 

furthermore , since the relative decay of nccc at the cutoff dose is 
 smaller for the ds method , the acquired data set in the second regime 
 will show the most resemblance to the sample in the initial , nondamaged 
 state .
figure 6a also 
 shows the evolution of the nccc as a function of accumulated dose 
 in cryogenic conditions .
clearly , the two damage regimes remain visible . 
 the slope of the first regime decreases from ( 1.57  
 0.12 )  10 for ds / rt/10 to ( 9.25 
  0.76 )  10 for ds / cryo/10 and to a 
 minimum of ( 9.53  0.57 )  10 for cf / cryo/10 .
this indicates that cryo - preservation could be useful 
 when acquiring small data sets with a low total dose , since the nccc 
 changes more slowly , and one can therefore acquire images as close 
 as possible to the original , nondamaged state .
the cutoff between the 
 two damage regimes increases from 156  
 9 e /  for cf / rt/10 to 219  19 and 271  
 28 e /  for cf / cryo/10 and ds / cryo/10 , respectively . 
 with a relative decay of 19% at the cutoff dose for cf / cryo/10 , in 
 comparison to 37% for ds / cryo/10
, it can be concluded that for small 
 data sets with relatively low doses a conventional floating method 
 at cryogenic conditions is best applicable , since the nccc decreases 
 slowly over a larger amount of dose . in cryogenic conditions
, 
 the second regime behaves differently 
 in comparison to room temperature conditions , since the nccc is increasing 
 again .
therefore , it was impossible to determine a useful critical 
 dose imaging , based on an nccc decay by a factor of e. the increase in nccc can be explained by an inversion of the displacement , 
 i.e. , initial shrinkage followed by expansion or vice versa .
this 
 is confirmed by the displacement of subareas , shown in figure 6d . at an accumulated dose of 
 400 e /  ,
the relative shrinkage is calculated to 
 be 0.39  0.16% and 0.79  0.42% for cf / cryo/10 
 and ds / cryo/10 , respectively .
this means that the sample will expand 
 first , before it starts to shrink , in contrast to room temperature 
 conditions where shrinkage occurs directly from the onset of beam 
 damage . whether a stable regime will appear after shrinkage in cryogenic 
 conditions , just as in room temperature conditions , is not investigated .
because of the initial expansion , one would expect an initial decrease 
 in standard deviation , since the information on less material will 
 be spread over the same amount of pixels .
this effect is seen in figure 6b . however , for the 
 cf / cryo/10 sample , this effect is minimal .
local mass loss , which 
 is expected since oxygen and water can be removed easily , will increase 
 the standard deviation , explaining the smaller decrease in relative 
 standard deviation . in cryogenic conditions , in contrast with 
 room temperature conditions , 
 the sample seems to be destabilized by an oxygen- and water - free sample 
 preparation method .
furthermore , even without oxygen and water being 
 present , the stability at high doses is less in cryogenic conditions 
 than at room temperature .
further details on intensity changes of 
 areas before and after exposure can be found in supporting information section 7 .
by 
 combining diffraction and imaging data , we have investigated 
 electron beam damage effects in p3ht - pcbm thin films over multiple 
 length scales .
analysis of the fading of electron diffraction rings 
 allows us to quantify the beam sensitivity of each component of the 
 nanocomposite individually .
this shows that pcbm is 15 times more 
 stable based on the criterion of the critical dose diffraction , i.e. , 
 the accumulated electron dose at which the intensity of the corresponding 
 diffraction ring decreases with a factor of e. for 
 electron dose rates between 0.1 and 10 e/( s ) , no 
 dose rate effects are observed .
cryo - preservation increases beam stability 
 of both materials with the critical dose for p3ht significantly increasing 
 from 16 to 108 e /  , while the critical dose for pcbm 
 increases from 392 to 596 e /  .
most importantly , 
 it is shown that excluding water and oxygen during sample preparation 
 further improves the beam stability of these materials , reaching a 
 critical dose of 188 e /  for p3ht , which corresponds 
 to a 10-fold increase over conventional sample preparation and room 
 temperature diffraction .
beam damage in imaging mode was analyzed 
 in terms of mass loss 
 and thin film deformations .
the normalized cross - correlation coefficient 
 ( nccc ) between images contains contributions from among others contrast 
 loss and intermixing of phases . here
, the nccc is used to assess sample 
 deformation , as the nccc is followed as a function of accumulated 
 dose .
first , it is shown that the dose rate effect is negligible for 
 dose rates between 1 and 100 e/( s ) .
second , experiments 
 at room temperature show a quick initial decrease in nccc before a 
 stable second regime is reached . in this first regime , shrinkage and 
 mass loss occurs , as shown via the movement of subimages and changes 
 in relative standard deviation .
the second , more stable regime is 
 used to calculate a critical dose imaging , defined by the dose at 
 which the initial nccc is decreased with a factor of e. an oxygen- and water - free sample preparation method increases this 
 critical dose imaging from 2600 to 5000 e /  . furthermore , 
 since the relative decay of the nccc at the cutoff dose is lowest 
 in oxygen and water free conditions , data acquired in this second 
 regime will be closest to the sample in a nondamaged state . in cryogenic 
 conditions ,
the first regime shows a slower decrease of the nccc , 
 indicating that the first regime is more stable than in room temperature 
 conditions . in the second regime , however , the nccc increases again . 

this is due to the fact that after an initial expansion of the thin 
 film the sample starts to shrink . in summary , we suggest to 
 use an oxygen- and water - free sample 
 preparation method combined with cryogenic conditions in diffraction 
 mode , while for imaging an oxygen- and water - free sample preparation 
 method is best suited combined with data acquisition at room temperature 
 ( high total doses , large data sets ) and a conventional floating method 
 is best suited for cryogenic condition ( small total doses , small data 
 sets ) .
we believe that the quantitative analysis of tem beam damage 
 as illustrated here will become a standard tool to optimize tem imaging 
 conditions and sample preparation protocols in the future .
 in 
 transmission electron microscopy ( tem ) the interaction of an 
 electron beam with polymers such as p3ht : pcbm photovoltaic nanocomposites 
 results in electron beam damage , which is the most important factor 
 limiting acquisition of structural or chemical data at high spatial 
 resolution . 
 beam effects can vary depending on parameters such as 
 electron dose rate , temperature during imaging , and the presence of 
 water and oxygen in the sample . 
 furthermore , beam damage will occur 
 at different length scales . to assess beam damage at the angstrom 
 scale 
 , we followed the intensity of p3ht and pcbm diffraction rings 
 as a function of accumulated electron dose by acquiring dose series 
 and varying the electron dose rate , sample preparation , and the temperature 
 during acquisition . from this 
 , we calculated a critical dose for diffraction 
 experiments . in imaging mode , 
 thin film deformation was assessed using 
 the normalized cross - correlation coefficient , while mass loss was 
 determined via changes in average intensity and standard deviation , 
 also varying electron dose rate , sample preparation , and temperature 
 during acquisition . 
 the understanding of beam damage and the determination 
 of critical electron doses provides a framework for future experiments 
 to maximize the information content during the acquisition of images 
 and diffraction patterns with ( cryogenic ) transmission electron microscopy . 