dunaliella salina teodoresco is a green alga capable of producing high concentrations of carotenoids , i.e. more than 8% of dry weight ( ben - amotz et al .
good hplc - based methods already exist for the separation , detection and quantification of a wide variety of carotenoids ( fraser et al .
. however , these methods do not give information about the intracellular localisation of the carotenoids .
to gain more insight into , for example , the milking process to extract carotenoids ( hejazi and wijffels 2004 ) , it would be advantageous to have an imaging technique at our disposal that can visualise these carotenoid - containing globules .
electron micrographs of dunaliella cells show small globules in the chloroplast , labelled as carotenoid - containing lipid droplets ( ben - amotz et al .
2004 ; hejazi and wijffels 2004 ; borowitzka and siva 2007 ) . however , a disadvantage of electron microscopy is that it can not be used on living cells .
fluorescence microscopy is a noninvasive imaging technique and can be used to study cellular processes in vivo .
the detection of endogenous fluorescence from cell components or exogenous fluorescence from added dyes can be used to show and localise cellular components in vivo .
an example of an endogenous fluorophore is chlorophyll which is widely known for its autofluorescence ( papageorgiou and govindjee 2004 ) . also , fluorescence of carotenoids in solution has been demonstrated ( gillbro and cogdell 1989 ; bondarev 1997 ) .
however , intracellular carotenoids as part of the photosystem are known to mainly quench fluorescence emitted by other cell components ( lichtenthaler 1987 ; chappelle et al . 1991 ) . in this paper , we show that the carotenoid - containing globules produced by d. salina emit green fluorescence that can be detected with confocal laser scanning microscopy ( clsm ) .
absorbance and fluorescence measurements show that the fluorescence most likely originates from the -carotene in these globules .
dunaliella salina ccap 19/18 was obtained from ccap ( culture collection of algae and protozoa , oban , uk ) .
cells were grown and stressed in a flat - panel photobioreactor with a working volume of 2,500  ml and a light path of 3  cm .
the cell suspension was mixed by gassing ( 0.6 n2 l.min ) , and ph was controlled at ph  7.5 by adding co2 pulses .
the temperature was kept at 30c by circulating temperature - controlled water through the reactor water jacket . for the growth period ,
the reactor was illuminated from one side with an average irradiance of 206  mol photonsm s ( high - pressure sodium lamp ) . for the stress period ,
the irradiance was raised to 1,672  mol photonsm s by moving the lamp closer to the reactor .
small amounts ( 15  ml ) of green cells harvested during the growth period and orange cells harvested during the stress period were used for microscopy purposes . at the end of the cultivation
8  40  ml cell suspension was centrifuged at 1,000g for 15  min .
each pellet was resuspended in 40  ml of 30  mm nacl and centrifuged at 12,000g for 10  min . to break the cells ,
each pellet was resuspended in 4  ml demineralised water and again centrifuged at 12,000g for 10  min .
the supernatant was mixed with a solution of 2  ml of 50% sucrose in 10  mm tris 
hcl ( ph  8.0 ) . on top of this mixture , 0.5  ml 10  mm tris 
this mixture was centrifuged for 2  h at 48,000g to separate the globules from the chloroplast membranes .
the absorbance and autofluorescence of the globules were measured using a spectrophotometer ( avaspec-2048-usb , avantes , the netherlands ) and a spectrofluorimeter ( jobin yvon fluorolog fl3 - 22 , horiba ) , respectively . as reference sample , 10  mm tris 
the excitation wavelengths for the emission spectra of the autofluorescence were 450 , 488 and 510  nm .
for emission , the bandwidth was 0.6  nm and the step size was 0.2  nm .
bright field microscopy pictures from cells and globule suspensions were made with a microphot fluorescence microscope ( nikon ) or a ck 40 bright field microscope ( olympus ) equipped with an olympus ax 70 camera .
fluorescence microscopy pictures of cells and globule suspensions were made with a clsm ( zeiss lsm 510-meta 18 ) .
the clsm was connected to an inverted microscope ( axiovert 200  m ) with differential interference contrast ( dic ) . for excitation ,
the argon diode laser ( 30  mw , 488  nm ) was used . for the detection of emitted fluorescence
, we applied the bp505 - 530/lp650 filtre combination . a zeiss 639 -plan fluar oil objective ( na 1.45 )
the obtained clsm pictures were a combination picture of images from three channels , namely a dic microscopy image from the cell , an image from the green fluorescence between 505 and 530  nm and an image from the red fluorescence of 650  nm and higher .
8  40  ml cell suspension was centrifuged at 1,000g for 15  min .
each pellet was resuspended in 40  ml of 30  mm nacl and centrifuged at 12,000g for 10  min . to break the cells , each pellet was resuspended in 4  ml demineralised water and again centrifuged at 12,000g for 10  min .
the supernatant was mixed with a solution of 2  ml of 50% sucrose in 10  mm tris 
hcl ( ph  8.0 ) . on top of this mixture , 0.5  ml 10  mm tris 
this mixture was centrifuged for 2  h at 48,000g to separate the globules from the chloroplast membranes .
the absorbance and autofluorescence of the globules were measured using a spectrophotometer ( avaspec-2048-usb , avantes , the netherlands ) and a spectrofluorimeter ( jobin yvon fluorolog fl3 - 22 , horiba ) , respectively . as reference sample ,
the excitation wavelengths for the emission spectra of the autofluorescence were 450 , 488 and 510  nm .
for emission , the bandwidth was 0.6  nm and the step size was 0.2  nm .
bright field microscopy pictures from cells and globule suspensions were made with a microphot fluorescence microscope ( nikon ) or a ck 40 bright field microscope ( olympus ) equipped with an olympus ax 70 camera .
fluorescence microscopy pictures of cells and globule suspensions were made with a clsm ( zeiss lsm 510-meta 18 ) .
the clsm was connected to an inverted microscope ( axiovert 200  m ) with differential interference contrast ( dic ) . for excitation ,
the argon diode laser ( 30  mw , 488  nm ) was used . for the detection of emitted fluorescence
a zeiss 639 -plan fluar oil objective ( na 1.45 ) was used for all imaging experiments .
the obtained clsm pictures were a combination picture of images from three channels , namely a dic microscopy image from the cell , an image from the green fluorescence between 505 and 530  nm and an image from the red fluorescence of 650  nm and higher .
when dunaliella salina cells are stressed , they start to produce carotenoids . the green cell which is dominated by the chloroplast starts to turn orange .
the chloroplast shrinks , chloroplast membranes decrease in size and carotenoid - containing lipid globules are formed .
light microscopy clearly showed this difference between non - stressed and stressed cells ( fig . 
fluorescence microscopy also showed that a distinct difference in fluorescence pattern arose after stress ( fig . 
1a , b bright field microscopy picture of non - stressed ( a ) and stressed ( b ) d. salina cells .
c , d fluorescence microscopy pictures ( clsm ) of non - stressed ( c ) and stressed ( d ) d. salina cells .
scale bars=10  m . the clsm pictures are combination images of three channels : dic microscopy , green fluorescence ( 505530  nm ) and red fluorescence ( > 650  nm ) .
green fluorescence comes from the globules , red fluorescence from chlorophyll remains a , b bright field microscopy picture of non - stressed ( a ) and stressed ( b ) d. salina cells .
c , d fluorescence microscopy pictures ( clsm ) of non - stressed ( c ) and stressed ( d ) d. salina cells .
scale bars=10  m . the clsm pictures are combination images of three channels : dic microscopy , green fluorescence ( 505530  nm ) and red fluorescence ( > 650  nm ) .
green fluorescence comes from the globules , red fluorescence from chlorophyll remains to prove that this green fluorescence was emitted by the carotenoid - containing globules , we isolated the globules ( fig . 
light microscopy showed clear orange globules , and with the clsm , the green fluorescence again was detected .
this fluorescence was emitted by the globules and not by the solution as can be seen in fig . 
the absorbance and emitted fluorescence from the globule suspension were measured with a spectrophotometer and spectrofluorimeter , respectively , and are shown in figs .  2 and 3 . 

3emission spectra of carotenoid - containing globules for three different excitation wavelengths ( 450 , 488 and 510  nm ) absorbance spectrum of carotenoid - containing globules .
the peak at 670  nm was absorbance from chlorophyll emission spectra of carotenoid - containing globules for three different excitation wavelengths ( 450 , 488 and 510  nm ) in the absorbance curve , the carotenoid peak is clearly visible ( 350550  nm ) .
next to this peak , a very small peak at 670  nm was visible , originating from some chlorophyll contamination still left in the solution . in the fluorescence emission graph ( fig . 
3 ) , this chlorophyll contamination showed a very high emission peak at 670  nm . at 560  nm , a second ,
much smaller peak was visible , which must have originated from another component of the suspension , most likely the carotenoid - containing globules .
with different excitation wavelengths , this emission peak did not shift its position with respect to the wavelength . to determine whether the fluorescence peak at 560  nm was emitted by the -carotene in the carotenoid - rich globules
, we compared the relative values for the quantum yields of the chlorophyll peak and the 560-nm peak to see whether these compare to values found in literature for chlorophyll and carotenoid fluorescence .
dunaliella salina stores secondary carotenoids ( predominantly -carotene ) in lipid globules in the chloroplast when cultivated under stress conditions .
non - stressed cells are dominated by the chloroplast , emitting red fluorescence originating from the chlorophyll in the thylakoid membranes .
when the cells are stressed , the red fluorescence from chlorophyll partly disappears as the thylakoid membranes are broken down . at the same time , the cells start to produce carotenoid globules and green fluorescence appears simultaneously .
dunaliella salina cells show this distinct difference in red and green fluorescence pattern between non - stressed and stressed cells .
we determined the relation between the carotenoid - containing globules and the green fluorescence and studied the possibility to visualise and localise these lipid globules inside the cells in vivo using this autofluorescence .
( 1982 ) , are composed almost entirely of lipids . over 90% of the lipids
are neutral lipids , with -carotene as the dominant fraction ( 65% of dry weight ) .
the carotenoid peak in the absorbance curve ( 350550  nm ) showed great similarity to the absorption spectrum of an 80% acetone extract of the purified -carotene globules from d. bardawil , found by ben - amotz et al .
( 1982 ) , and resembles the absorbance peak of pure -carotene in solution ( kandori et al .
the peak originated mainly from -carotene , but other carotenoids might have contributed as well , just as some chlorophyll contamination .
this originated probably from some chloroplast membrane remainders that were connected to the lipid globules .
chlorophyll has absorbance peaks between 400500 and > 600  nm ( lichtenthaler 1987 ) .
the small peak visible at 670  nm in the absorbance graph therefore must originate from chlorophyll still left in the solution .
the fact that with different excitation wavelengths the emission peak did not shift its position with respect to the wavelength proves that the peak is indeed fluorescence and can not be ascribed as a raman peak ( shanker and bane 2008 ) .
the high relative intensity of the emitted fluorescence from the relatively small chlorophyll contamination coheres with its high quantum yield .
the ratio between the absorbance peaks and the emission peaks can be used to determine the ratios in quantum yields ( shanker and bane 2008 ) .
the quantum yields for chlorophyll vary from 0.02 to 0.33 depending on whether measured in various solutions or in the cell ( forster and livingston 1952 ; latimer et al .
( 1994 ) , the quantum yield for -carotene is 1.7    10 .
this results in a ratio between the quantum yields of -carotene and chlorophyll varying from approximately 100 to 2000 .
the ratio in fluorescence between both peaks lies between 0.07 and 0.11 when we divide the maximum height of both peaks ( e.g. 3.0    10 : 2.7    10 for 488-nm excitation wavelength ) .
the ratio between the absorbance peaks is around 14 when we divide the maximum height of both peaks ( 0.64:0.05 ) .
consequently , the ratio between the relative values for the quantum yields for both peaks in this research is somewhere between 100 and 200 .
comparing these to literature shows that the emission peak at 560  nm most likely originates from the -carotene in the lipid globules . in conclusion ,
d. salina stores secondary carotenoids in lipid globules when cultivated under stress conditions . to visualise these carotenoid - containing globules , cslm can be used in combination with the autofluorescence of cell components .
this autofluorescence of the cells is emitted by chlorophyll in the red part of the spectrum and by the carotenoid globules in the green part of the spectrum . when comparing the relative quantum yields obtained from the absorbance and fluorescence emission spectra with data from literature
, we can conclude that the green fluorescence most likely is emitted by the -carotene that comprises the main part of the carotenoid - rich globules .
 dunaliella salina is a halotolerant green alga that is well known for its carotenoid producing capacity . 
 the produced carotenoids are mainly stored in lipid globules . for various research purposes , such as production and extraction kinetics 
 , we would like to determine and/or localise the carotenoid globules in vivo . in this study , we show that the carotenoid - rich globules emit clear green fluorescence , which can be used in , for example , fluorescence microscopy ( e.g. clsm ) to obtain pictures of the cells and their carotenoid content . 