benzo[a]pyrene ( bap ) , an extensively researched 
 carcinogen and mutagen , belongs to the high - molecular - weight polycyclic 
 aromatic hydrocarbon ( pah ) group of chemicals .
bap and other pahs 
 are derived as a result of incomplete combustion and are released 
 into the environment by both natural and anthropogenic activities
. 
 some of the proven emission routes are domestic emission and wastes , 
 automobile exhaust , industrial emission , agricultural activities , 
 and also some natural sources .
metabolism 
 of bap activates several mixed - function enzymes in the cell and results in the progression of different 
 types of cancer in human and animals .
being hydrophobic , bap has been 
 found to accumulate in several organisms , including microalgae , mussels , and mice .
microalgae are the primary producers in 
 the food chain and can 
 grow in three diverse ecosystems : fresh water , marine environments , 
 and soil .
they support many different life forms that are exclusively 
 dependent on them for food and play a vital role in the environment .
microalgae growing in these environments are 
 exposed to different pollutants , which may have natural or anthropogenic 
 sources .
accumulation of pollutants by microalgae has an immediate 
 effect on the higher organisms in the food chain that feed on them .
the use of microalgae for the removal of different 
 organic pollutants is also gaining importance , apart from their use as pollution indicators .
studies related to the catabolic ability of microalgae 
 to remove bap were started recently .
it has been shown that the green 
 alga metabolizes bap primarily to cis - dihydrodiols by a dioxygenase enzyme system similar to that 
 in bacteria and also forms bap sulfate 
 ester and glucose conjugates .
most mammals 
 and terrestrial plants follow the monooxygenase enzymatic pathway 
 to metabolize bap .
most of the 
 biological effects exerted by bap are mediated through 
 aryl hydrocarbon receptors on the cell membrane . in mammalian cells
, bap is enzymatically converted into 
 bap - diol epoxide , which can complex with nucleotides to form nucleotide 
when these adducts are incorporated into 
 dna during dna synthesis , the dna polymerase is prevented from moving 
 further as a result of interference by the adduct , which results in immature termination of dna synthesis . 

thus , defective metabolism or defective regulation of cellular metabolism 
 occurs , which leads to apoptosis or proliferation of cells .
localization of bap in murine macrophages 
 and human fibroblasts 
 has been demonstrated using fluorescence microscopy .
fluorescence imaging of the bap distribution in embryonic 
 and larval medaka tissues has also been shown .
it is essential to study the uptake and accumulation mechanisms 
 of bap in microalgae , as they are one of the primary producers in 
 the food chain .
even though there have been previous reports showing 
 accumulation of bap in this system , intracellular 
 localization and aggregation have not yet been studied using the most 
 recent fluorescence microscopy tools .
determination of the bioaccumulation 
 of organic compounds using standard analytical techniques such as 
 gc and hplc is invasive , as it requires killing of the organisms used 
 for the study and the use of solvents to extract the organics .
fluorescence 
 microscopy is a noninvasive technique that does not require harmful 
 solvents for the extraction of organics .
it also provides information 
 about the accumulation pattern of hydrophobic pollutants inside single 
 cells . indeed , further studies are needed to develop quantitative 
 microscopy tools to quantify the uptake of bap and other fluorescent 
 pollutants by microalgal cells .
a microalgal cell contains a wide 
 spectrum of autofluorecent molecules , and thus , it is difficult to 
 distinguish fluorescence from additional molecules such as bap accumulating 
 in the cell unless a specific fluorescence signal that can be unequivocally 
 attributed to bap can be found . in an endeavor to study the accumulation 
 of bap and to visualize the intracellular localization , we used fluorescence 
 lifetime imaging microscopy ( flim ) with the phasor approach and confocal 
 microscopy with number and brightness ( n&b ) analysis .
the 
 use of fluorescence microscopy and flim in algal studies was 
 started recently , and these techniques have also 
 been used extensively to study biological structures such as photosynthetic 
 structures of plants .
one advantage of 
 flim is that the lifetime information can be represented as a phasor 
 plot , thereby allowing better separation of fluorescent components 
 and the identification of a specific lifetime signal from bap .
as 
 the background fluorescence of plant cells has a very short lifetime , 
 any fluorescent molecule that is different from the background can 
 be well - separated in the phasor plot .
the difference used could be 
 either wavelength , which can be separated by filters , or lifetime , 
 which can be resolved by a phasor plot .
hence , we used flim as the 
 technique to detect and image the interaction of the fluorescent pollutant 
 bap with the microalga , chlorella sp . 

we show that flim can be used to follow the accumulation of bap in chlorella cells .
our results suggest that bap accumulates 
 in lipid bodies and/or vacuoles of chlorella for a long time ( imaged up to 3 weeks ) and that there is frster 
 resonance energy transfer ( fret ) between bap and photosystems of the 
 cells .
the localization of bap in chlorella cells could be visualized while avoiding the background fluorescence 
 due to fluorescing molecules in the algal photosystems and the quenching 
 of bap fluorescence due to fret .
our n&b analysis suggests that 
 bap predominantly exists as monomers rather than as aggregates inside 
 the chlorella cells .
, originally 
 isolated from a soil sample and maintained in the phycology laboratory 
 at the centre for environmental risk assessment and remediation of 
 the environment ( cerar ) , university of south australia , was used in 
 this study .
the culture was maintained in bold s basal medium 
 ( bbm ) as described earlier .
the culture 
 was grown in 100 ml erlenmeyer flasks containing 25 ml of bbm in an 
 orbital shaker set at 150 rpm under 3  36 w cool white fluorescent 
 light ( 100 mol of photosynthetic photon flux density ) 
 at 24  2 c .
( 3 
  10 cells ml ) in 25 ml of bbm 
 placed in 100 ml sterile erlenmeyer flasks .
the cultures were examined 
 in an olympus bx41 epifluorescence microscope for the presence of 
 any contaminating fluorescent materials or debris in the medium other 
 than the autofluorescent microalgal cells .
the culture was exposed 
 to 100 m bap in culture medium added from a concentrated stock 
 solution prepared in n , n - dimethylformamide 
 ( dmf ) and incubated in an orbital shaker under constant illumination 
 at 24  2 c as described above .
the samples 
 were drawn for confocal microscopy analysis after 1 , 5 , 12 , and 24 
 h of incubation with bap .
bap and 
 nile red images were 
 obtained with a leica sp5 inverted microscope using a 63 , 1.2 
 na water immersion objective .
bap was prepared in dmf , resuspended 
 in bbm , incubated with chlorella cells , 
 and used for imaging .
bap was excited with a 405 nm laser , and emission 
 was set at 450550 and 600750 nm simultaneously in 
 two channels . for nile red staining ,
nile red was prepared in dmf 
 ( 10 mm ) and diluted into bbm to a final concentration of 100 
 m .
nile red ( 100 m ) was incubated with chlorella cells in 100 ml of bbm for 1 or 2 h and 
 imaged in the confocal microscope .
the fluorophore was excited using 
 a 488 nm laser , and the emission window was set to 500550 
 nm .
flim was carried 
 out using a leica sp5 microscope with a 405 nm pulsed laser at a frequency 
 of 40 mhz .
the emission window was set at 450550 nm to collect 
 the emission from bap and 600750 nm to collect the autofluorescence 
 ( predominantly photosystems i and ii ) of algal cells .
the image sizes 
 were set at 256  256 pixels , and the signals were collected 
 for 120 s. the flim data were analyzed using simfcs software .
leica sp5 time - correlated 
 single - photon 
 counting ( tcspc ) lifetime image files ( * .sdt ) were opened in the simfcs 
 flim analysis module , and the phasor plot was generated using the 
 phasor explorer page of simfcs .
the sample files were opened , and the different 
 phasor plots for each file were generated and analyzed either to obtain 
 lifetimes or to see quenching processes indicative of fret .
the histogram of the time 
 delays at each pixel is transformed into a phasor plot , which is like 
 a vector .
the phasor plot is a two - dimensional histogram where the 
 values of the sine  cosine transforms are represented in a polar 
 plot .
each pixel of the image has a point in the phasor plot . in a 
 reciprocal manner ,
each point of the phasor plot can be mapped to 
 a pixel of the image .
hence , each molecular species has a specific 
 phasor ; molecules can be identified by their position in the phasor 
 plot .
n&b analysis was 
 done according to the procedure of dalal et al . and digman et al .
a time series 
 of 100 frames with a resolution of 256  256 pixels , a pixel 
 size of 49.7 nm , and a pixel dwell time of 8 , 12 , or 20 s was 
 collected using a nikon c1-z confocal imaging system ( nikon eclipse 
 te2000-e with a 63 , 1.2 na water immersion objective , hanson 
 institute , adelaide ) .
background counts were collected with 
 the laser off and the detectors on using the same gain and offset 
 settings as used for collecting the time series .
the * .ids files of 
 the nikon confocal microscope were converted to * .bin files and imported 
 into simfcs , and n&b analysis was performed . from the background files ,
the s factor and  
 factor were estimated and used to analyze the data .
total organic carbon and 
 inorganic carbon were measured using a total organic carbon analyzer 
 ( 1010 toc analyzer , oi analytical , college station , tx , usa ) .
phosphorus 
 and potassium were measured from the aqua regia extract of sediments 
 using inductively coupled plasma mass spectrometry ( icp - ms ) ( agilent 
 7500 series , agilent technologies , tokyo , japan ) .
both ph and electrical 
 conductivity were measured using the smart chem - lab laboratory analyzer 
 ( tps pty ltd . , brisbane , australia ) and are reported in table s2 in 
 the supporting information .
sediments were spiked with bap 
 at two different concentrations ( 5 and 20 mg l ) , and 20 g samples of spiked and unspiked sediments were weighed 
 into sterile petri dishes .
then the chlorella cells were added to the sediments at a concentration of 5  
 10 cells ( g of sediment ) and incubated 
 under continuous 3  36 w cool white fluorescent light at 24 
  2 c .
samples were taken for fluorescence microscopy analysis 
 after 1 , 5 , 12 , and 24 h of incubation .
cells that had bap ( blue fluorescence ) 
 and cells without bap ( red fluorescence ) were counted separately . 

the effects of incubation time , bap concentration , and sediment type 
 and their interaction on the bap uptake by chlorella sp . were analyzed by three - way anova using minitab 16 statistical 
 software .
bap dissolves in organic solvents 
 because of its hydrophobic nature , and molecules are monodispersed 
 in these solvents . however , to understand the uptake , accumulation , 
 and degradation using biological systems , bap must be suspended in 
 an aqueous medium .
bap was dissolved in dmf and subsequently resuspended 
 in bbm to the required concentration and used in this study .
hence , 
 it is appropriate to image bap in dmf and an aqueous medium and measure 
 the lifetimes in these media .
the image of bap fluorescence in dmf 
 is very uniform ( figure 1a ) because the molecules 
 are dissolved .
however , at the resolution of our microscope it is 
 not possible to see smaller molecular aggregates .
the image of bap 
 in the aqueous medium shows aggregates of large size ( figure 1b ) . comparing these two images and taking into account 
 the hydrophobic nature of bap
, we concluded that bap forms aggregates 
 in bbm but is monodispersed in dmf .
these observations were confirmed 
 later , as discussed in number and brightness analysis below .
( a , 
 b ) laser confocal microscopy images of bap in ( a ) dmf and ( b ) 
 bbm .
( c , d ) phasor plots of lifetime images of bap in ( c ) dmf and 
 ( d ) bbm .
fluorescein 
 was used as a lifetime standard ( 4.1 ns ) to reference the phasor plot 
 ( the phasor plot with the fluorescein lifetime is not shown ) .
the 
 bap in dmf lifetime image was imported into simfcs and plotted in 
 the phasor plot ( figure 1c ) .
the lifetime was 
 found to be a relatively single exponential at 14 ns , as shown by 
 the phasor cluster located very close to the universal circle ( where 
 all of the single - exponential decays are found ) .
for all of 
 the solution and suspension samples , the fluorescence 
 decay data were extracted from the images and analyzed using globals 
 for spectroscopy software .
the fluorescein 
 data were fit to a single component at 4.1 ns ( figure s1a in the supporting information ) ; the deviations were 
 very small .
similarly , bap in dmf was analyzed by the same algorithm 
 ( figure s1b ) .
there was a misfit of unknown 
 origin at the very beginning of the data .
however , the fit was much 
 better when performed avoiding first part of the curve ( figure s1c ) .
the recovered value of the lifetime 
 using the least - squares analysis algorithm in globals for spectroscopy 
 was 14.5 ns .
one potential issue is that the intensity for this sample 
 was very weak and there was a very large background .
also , the range 
 that can be used for the b&h card was small , and there were artifacts 
 at the beginning and the end of the time trace .
the 
 flim data of bap in bbm in the phasor plot are scattered compared 
 with those for bap in dmf ( figure 1d ) , probably 
 because the sample had very weak fluorescence and was aggregated . 

the average lifetime lies inside the semicircle with a broader distribution 
 compared with that for bap in dmf .
this is expected because of the 
 nature of bap in the aqueous medium , which forms aggregates that are 
 affected by self - quenching .
hence , the lifetime is shortened , shifting 
 the phasor cluster toward smaller phase angles .
the average lifetime 
 for this sample was 7 ns , and the lifetimes were distributed between 
 12 and 4 ns .
the lifetime of bap in dmf was measured 
 earlier by iwata and co - workers using 
 the pulsed excitation method and further 
 verified by them using the frequency domain method , and the lifetime was found to be 14.7 ns .
this value is 
 close to the lifetime that we obtained using lifetime imaging ( 14 
 ns ) .
furthermore , we obtained the lifetime of bap in an aqueous medium 
 ( bbm ) .
used a tcspc instrument 
 to measure the lifetime of bap in methanol and found the lifetime 
 to be 45 ns , which confirms an earlier report by imasaka et al .
these measurements were carried out in cuvettes , 
 and the data were fitted to obtain the lifetimes .
the advantage with 
 the phasor plot is that no fitting of the data is required .
furthermore , the lifetime distribution in an 
 image can be better visualized using the phasor plot . the broader 
 lifetime distribution of bap in bbm ( figure 1d ) compared with bap in dmf ( figure 1c ) and 
 the chlorella sp .
background lifetime 
 ( figure 2d , e ) are examples where the phasor 
 plot gives a better understanding of the data , as there are no calculations 
 or nonlinear fitting involved .
however , 
 when the data do not show a distribution , as in the case of bap in 
 dmf , fitting routines could be used as well .
( a  c ) lifetime 
 images of a chlorella cell : ( a ) intensity 
 image ; ( b ) reciprocal plot selected by the cursor 
 in the phasor plot in ( d ) ; ( c ) reciprocal plot selected by the cursor 
 in the phasor plot in ( e ) .
( d , e ) phasor plots of fluorescence lifetimes 
 of a chlorella cell .
has several fluorescent molecules , 
 among which chlorophyll a 
 and b are the major ones .
flim of chlorella sp . was 
 done as a control to understand the lifetime present in the background . 
 as a result of energy transfer from photosystem ii to i and further 
 down in the electron transport system ,
the lifetime measured from the phasor plot ( figure 2d , e ) varied between 1.5 and 0.2 ns .
the photosystems 
 could not be distinguished , as the photosystem signals were collected 
 in one channel .
however , it is possible to separate regions of the 
 cell with different lifetimes using the phasor plot .
the majority 
 of the pixels lifetimes were very short , around 200 ps in regions 
 that can be associated with membranes , confirming that the background 
 lifetime could arise from photosystems .
the lifetime image of 
 a chlorella cell is shown in figure 2a . from this image ,
the phasor plot was generated 
 ( figure 2d , e ) . in a reciprocal selection ( from 
 figure 2d ) , a large cluster of phasor values 
 was selected by the red cursor ( red circle ) , and the pixels corresponding 
 to that cluster are represented in red in figure 2b .
similarly , another cluster was selected in figure 2e by the green cursor ( green circle ) , and the corresponding 
 pixels are painted in green in figure 2c .
however , it is 
 difficult to interpret these results unequivocally as arising from 
 a specific photosystem .
additional information could be obtained , 
 for example , by using different quenchers to find out the distribution 
 of certain fluorescent molecules in the photosystems . the phasor plot representation 
 of the background 
 lifetime of chlorella cells , which 
 is due to photosystems i and ii , is shown in figure 2d , e .
similarly , the bap lifetimes in dmf and bbm are plotted 
 in phasor form in figure 1c , d . when we combine 
 these two components ( bap and background ) ,
the lifetime of bap is 
 reduced from 14 ns to about 8 ns ( figure 3a ) . 

we considered two possibilities : ( 1 ) that the reduction in the lifetime 
 is due to the linear combination of the background lifetime and the 
 bap lifetime in the same pixel , and ( 2 ) that there could be quenching 
 of the bap fluorescence , making the bap lifetime become shorter .
it 
 is a property of the phasor plot that the linear combination of lifetimes 
 would fall somewhere on line b drawn in figure 3a , depending on the contributions from the two components ( f1 and 
 f2 ) .
as the phasor plot shows , no phasor points 
 are seen on that line .
hence , the bap lifetime could be quenched by 
 either photosystem i or ii , or both .
if quenching of the donor bap 
 is due to fret in which the photosystem acts as an acceptor , the phasor 
 would fall anywhere in the quenching trajectory marked a in figure 3a .
the red cursor in 
 the phasor plot in figure 3a selects the major 
 phasor cluster , which has been reciprocally shown in red in figure 3b .
( a ) phasor plot showing the bap lifetime distribution 
 in chlorella sp . the green line labeled 
 as b represents 
 linear combinations of the lifetimes of bap and chlorella background fluorescence , and the green curved trajectory labeled 
 as a represents fret between bap and the photosystems .
( b ) reciprocal 
 selection from the phasor plot by the red cursor c in ( a ) , showing 
 the bap fluorescence lifetime distribution in chlorella sp .
however , the overall trend was 
 that bap was being quenched by the 
 photosystems of chlorella cells .
the 
 streaking curved nature of the phasor plot ( figure 3a ) suggests varying quenching efficiencies of bap fluorescence . 
 finally , another possibility is that bap lifetime is shortened as 
 a result of self - quenching .
the 
 flim data suggest 
 that there could be self - quenching of the bap fluorescence and/or 
 energy transfer from bap to the photosystems .
n&b analysis is 
 an appropriate method to see whether there are aggregates of bap in chlorella cells .
time series ( 100 frames each ) of 
 bap in dmf , bap in bbm , and bap in chlorella cells were taken and analyzed .
, molecules have 
 an average brightness ( bav ) of about 6.937 , 
 which after calibration corresponds to about 142  000 counts 
 molecule s. the brightness 
 distribution is relatively narrow ( figure 4i ) .
there is no evidence of dimers , which would have b = 13 , or of larger aggregates .
( a  d ) maps of brightness vs fluorescence 
 intensity for bap in ( a ) dmf , ( b ) bbm , and ( c , d ) chlorella sp . cells . ( e )
image of bap in dmf showing corresponding pixels 
 selected by the cursor ( red circle ) in ( a ) .
( f ) image of bap in bbm 
 showing corresponding pixels selected by the cursor in ( b ) , corresponding 
 to high - brightness aggregates . (
cells showing corresponding pixels 
 selected by the cursor in ( c ) , corresponding to average - brightness 
 particles .
cells showing corresponding pixels selected by the cursor in 
 ( d ) , corresponding to high - brightness aggregates above average .
( i  k ) 
 histograms showing brightness distributions of bap in ( i ) dmf , ( j ) 
 bbm , and ( k ) chlorella sp . cells . in bbm ,
bap forms aggregates . 
 figure 4b 
 shows the distribution of intensity and brightness of the aggregates . 

a wide range of distribution of particles with varying brightness 
 is shown in figure 4j . in figure 4b ,
the cursor ( red circle ) selects high - brightness pixels , 
 and the corresponding bigger aggregates are highlighted in the image 
 shown in figure 4f .
if we compare bap in dmf 
 and bap in bbm , bap exists as monodispersed molecules in dmf whereas 
 it clearly aggregates in bbm . in chlorella cells ,
apart from background , 
 there are varying intensity distributions ( figure 4c , d ) . a majority of pixels corresponding to the bap fluorescence 
 are selected by the red circle in figure 4c 
 and are mapped in red in figure 4 g .
the average 
 brightness is bav = 5.33 ( figure 4k ) , which is slightly less than that for bap in 
 dmf ( figure 4i ) , indicating low aggregation 
 and some quenching .
( b > 6 ) , which are selected by the red circle in figure 4d and painted in red in figure 4h .
figure 4 g shows the reciprocal selection 
 of pixels selected by the cursor ( red circle ) in figure 4c , and similarly , figure 4h shows the 
 reciprocal selection by the cursor in figure 4d .
spectroscopic studies of bap monomer , dimer , and trimer had been 
 done by fioressi et al .
bap forms aggregates 
 in aqueous media and is monodispersed in organic solvents . in a cell , 

as bap prefers to partition into lipid droplets and the membrane portion , 
 it is in a monomeric form .
when bap is taken up by a cell , it has 
 to pass through the lipid bilayer , where it attaches as aggregates 
 to the surface and then might dissociate
it is a general hypothesis 
 that organic compounds pass passively through plant and animal cells .
however , experimental studies of individual 
 organic pollutants and their interactions with different organisms 
 are decisive if there is to be a clear depiction about the interaction 
 properties of the pollutant with diverse organisms and cell types .
chlorella cells were incubated with bap at a final 
 concentration of 1 , 40 , or 100 m prepared in bbm and imaged .
there was no detectable difference in the accumulation 
 patterns seen for the different incubation regimes of 5 min and 48 
 h using confocal microscopy .
the cells showed accumulation of bap 
 in specific pockets ( figure 5b  e ) .
these 
 pockets could be lipid bodies and/or vacuoles considering the localization 
 pattern of bap .
( a ) confocal 
 microscopy image of chlorella cells 
 showing background photosystem fluorescence . ( b ) confocal 
 microscopy image showing bap fluorescence in chlorella cells .
( c ) overlap of chlorella sp . 
 background fluorescence and bap fluorescence ( scale bar = 20 m ) . 

( d ) higher - magnification image showing fluorescence of bap inside chlorella sp . cells ( scale bar = 5 m ) .
( e ) 
 confocal microscopy image of a chlorella cell showing pictate staining of bap fluorescence .
( f ) confocal 
 microscopy image of a chlorella cell 
 showing the lipid - specific fluorescent stain nile red .
nile red , a lipid - specific dye , was 
 used to determine whether bap accumulation occurs in lipid bodies 
 of chlorella cells .
nile red is excited 
 at 488 nm and its emission maximum is at 525 nm , whereas bap is excited 
 at 405 nm and its emission is collected between 450 and 550 nm . as 
 the emissions of these two dyes partially overlap , it is difficult 
 to do simultaneous imaging and show colocalization .
hence , bap and 
 nile red images were measured separately using different chlorella cells , and the patterns are shown in figure 5e , f , respectively .
compared to nile red staining , bap staining is more punctuated . in order to determine the bap uptake assay by microalgae , 
 fluorescence microscopy analysis was carried out on chlorella cells to detect the bap uptake from sediments . 

between the two sediments ( denoted 
 as s1 and s2 ) , even though their nutrient contents differ greatly 
 ( table s2 in the supporting information ) .
bap uptake could not be detected in the algae during the first 
 and fifth hours of incubation , and all of the cells showed only background 
 fluorescence .
however , the number of cells with bap fluorescence started 
 to increase after 12 h of incubation ( figure 6 ) . during the first few hours of incubation , the quantity of bap 
 taken up by microalgae may not be sufficient to detect using fluorescence 
 microscopy .
moreover , no clear morphological differences between the chlorella cells exposed to the bap - spiked sediments 
 and unspiked sediments were observed .
earlier , wu et al . observed morphological changes in aspergillus fungus using fluorescence microscopy . 

this shows the differential response between microalgae and fungi 
 to bap at the microscopic level .
fluorescence microscopy analysis of bap 
 uptake by chlorella sp . in two different 
 sediments ( 5 and 20 
 are bap concentrations ; s1 and s2 are sediment types ; and 1 , 5 , 12 , 
 and 24 are incubation periods ) .
analysis by three - way anova indicates that bap concentration , 
 incubation 
 time , and sediment type all have significant effects ( p > 0.01 ) on the bap uptake by chlorella sp .
a significant influence of bap concentration and incubation 
 time was seen . a significant influence of incubation time and sediment 
 type ( p > 0.01 ) on bap uptake was also observed . 

however , there was no significant interaction between bap concentration 
 and sediment type on the bap uptake by chlorella ( p > 0.01 ) .
flim , confocal microscopy , and 
 n&b techniques can be used to 
 study the uptake , accumulation , and aggregation of bap in living cells . 

the limitations of imaging of bap localization in chlorella cells is that there is background fluorescence from photosystems 
 and other molecules and there is fret between bap and other molecules 
 in the cell .
however , flim can be used to distinguish the background 
 fluorescence from bap fluorescence .
the advantages of 
 using the flim  phasor and n&b analyses are that bap fluorescence 
 can be distinguished from the background fluorescence and aggregation 
 of bap molecules can be visualized inside the cells . in the case of chlorella sp . , bap accumulates at high concentrations 
 inside the cells , preferentially in lipid bodies .
bap forms aggregates 
 of various sizes in an aqueous medium ( bbm ) , whereas it exists as 
 monomers or small aggregates inside the cell .
this study lays the 
 foundation for studying the interaction of microalgae and fluorescent 
 xenobiotics such as bap using confocal microscopy .
indeed , there is 
 more potential for utilizing and refining this technique to study 
 the interaction of fluorescent pollutants in other diverse microorganisms .
 benzo[a]pyrene ( bap ) , a polycyclic aromatic hydrocarbon 
 ( pah ) , is one of the major environmental pollutants that causes mutagenesis 
 and cancer . bap has been shown to accumulate in phytoplankton and 
 zooplankton . 
 we have studied the localization and aggregation of bap 
 in chlorella sp . 
 , a microalga that 
 is one of the primary producers in the food chain , using fluorescence 
 confocal microscopy and fluorescence lifetime imaging microscopy with 
 the phasor approach to characterize the location and the aggregation 
 of bap in the cell . 
 our results show that bap accumulates in the lipid 
 bodies of chlorella sp . and that there 
 is frster resonance energy transfer between bap and photosystems 
 of chlorella sp . 
 , indicating the close 
 proximity of the two molecular systems . 
 the lifetime of bap fluorescence 
 was measured to be 14 ns in n , n - dimethylformamide , 
 an average of 7 ns in bold s basal medium , and 8 ns in chlorella cells . 
 number and brightness analysis suggests 
 that bap does not aggregate inside chlorella sp . 
 ( average brightness = 5.330 ) , while it aggregates in the supernatant . 
 in chlorella grown in sediments spiked 
 with bap , in 12 h the bap uptake could be visualized using fluorescence 
 microscopy . 
Introduction
Experimental
Design
Results and Discussion