the ultrastructural localization of a molecule requires that a certain degree of electron - density , resulting in adequate electron scattering , be present in sufficient amount .
binding of heavy atoms can be accomplished easily with regards to proteins , nucleic acids and other cellular components , in some cases with a high degree of specificity . however , the sub - cellular localization of ions is a challenge , since in many cases ions are diffusible and not easily kept in place after the procedures of sample preparation for electron microscopy ( em ) .
many ions can offer enough scattering in spite of not being heavy metals , such as manganese , while others necessitate of blocking or precipitating techniques to be visualized .
in particular , calcium ions are easily diffusible inside the cell , and even when protein - bound are not easily detectable . the pioneering paper by tandler et al .
demonstrated the presence of calcium ions by em via pyroantimonate precipitation , with a good yield of the final product . the end product of the reaction , however , is probably overrated , since the technique is capable of stabilizing and precipitating calcium and other bivalent cations , such as magnesium , as proved by electron spectroscopic imaging ( esi ) analysis . since their introduction by tsien and coworkers ,
calcium studies have used specific ca - sensitive fluorochromes , such as fura-2 , indo-1 and fluo-4 , coupled to wide - field digitized video fluorescence microfluorimetry and confocal microscopy to image acute elevations in intracellular ca concentration ( [ ca]i ) with high spatio - temporal resolution .
cytosolic and nuclear ca signals regulate a multitude of vital cellular processes , such as gene transcription , cell cycle progression , cytoskeletal and chromatin remodelling , and migration . however , proper ca handling within the endoplasmic reticulum ( er ) , the most abundant intracellular ca reservoir , is also crucial for cell - fate decisions . the steady - state er ca level ( [ ca]er ) does not only control synthesis , folding , export and trafficking of proteins , but it also fuels mitochondrial bioenergetics thereby suppressing autophagy .
low affinity ca indicators , such as mag - fura 2 , and genetically - encoded probes , such as the bioluminescent protein aequorin and the gfp - based fluorophores camgaroos , pericams , and cameleons , have been devised to assess er ca levels .. nevertheless , these sophisticated tools do not provide any reliable information on ca distribution within the heterogeneous structure of the er . we propose in this paper a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques .
maranto and sandell and masland have proposed the possibility of photoconverting fluorochromes into an electrondense end product .
the technique utilizes the photo - oxidation of diaminobenzidine ( dab ) in presence of the emission of an excited fluorochrome , and it has been shown to be efficient on different dyes and conditions .
this method combines the specificity of available fluorophores to the high spatial resolution offered by transmission em to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles .
the present manuscript sought to exploit dab photo - oxidation of mag - fura 2 for visualizing er ca distribution in hela cells , which are among the most widespread model cell lines used for studying intracellular ca signaling .
hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence ,
cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) .
the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition .
the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 .
all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) .
intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m .
cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy .
an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software .
instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st .
louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs .
thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel
, some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above .
moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above . fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature .
once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) .
ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv .
hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence ,
cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) .
the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition .
the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 .
all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) .
intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m .
cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy .
an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software .
instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st .
louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs .
thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel
, some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above .
moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above .
fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature .
once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) .
ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv .
calcium labeling with mag - fura 2 results in highly fluorescent staining mainly localized in the cytoplasm .
after incubation of the cells with 20 mm ca for 1 h , the signal is present in large vesicles in the cytoplasm and around the cell nucleus , as well as in elongated structures probably related to mitochondria ( figure 1a ) .
the faint signal visible in the nucleus is very probably due to the superposition of the cytoplasmic layer surrounding it .
when the cells are incubated in pss , the signal is lower ( figure 1b ) and mostly present in the perinuclear vesicles , while the cytoplasmic signal far from the nucleus somehow disappears . in the last condition , without calcium and in the presence of egta , some labeling is still visible , although to a much reduced level ( figure 1c ) .
this continuous presence of ca represents very probably the last ions the cell can not allow to lose , a sort of surviving threshold concentrations .
supplementary figure 1 confirms that altering extracellular ca concentrations results in a consequent change in er ca homeostasis , which may have important consequences also on cytosolic ca dynamics . at em level
, the controls did not show any electron dense precipitates , both in the absence of the fluorochrome or after aldehyde blockade ( not shown ) ; consequently , the end product obtained in the mag - fura 2 stained cells is due to the fluorochrome photoconversion .
the dab photoconversion is an efficient way to stabilize the ions and to visualize them .
it must be underlined here that we observe a thin section , 60 - 80 nm thick , corresponding , consequently , only to a fraction of the fluorescent signal .
after pss incubation , the cells show a distinctive positivity ( figure 2a ) . in some cases it is possible to see the presence of precipitates without the direct association with smooth or rough er ( ser or rer , respectively ) membranes , likely related to unbound ca ions
( figure 2b ) , and electron dense material inside the vesicles is also positive ( figure 2c ) .
a possible suggestion of ca internalization or extrusion is shown in figure 2 d , e , where the deposits are present on the outer membrane .
when the cells are incubated without calcium and in the presence of egta , the deposits are extremely reduced , although still present on the outer cell membrane , suggesting the presence of an extruding mechanism ( figure 2f ) . on the contrary ,
the diffuse , low density end product is almost absent from the cytoplasm . at higher ca concentration in the medium ( figure 3a ) , the dark precipitate is detectable on the inner membrane of cytoplasmic vesicles possibly pertaining to ser , and in discrete foci with no apparent association with vesicles or membranes .
the deposits are present within electron dense vesicles ( figure 3b ) and electron lucent structure , in some cases associated , and suggesting the idea of passage of ions from one compartment to another .
in other cases , dab - positive material is present on the membranes of complex systems such as multivesicular bodies ( figure 3c ) or on the outer membranes of cytosolic vesicles ( figure 3d ) .
interestingly , calcium deposits are detectable on the outer cell membrane ( figure 3e ) in spots , localized and separated , suggesting the presence of calcium channels . in figure 3f ,
the end product of the dab photoconversion is a speckled , dense precipitate of varying size .
however , another abundant end product is present as small sized dots , showing a markedly lower electrondensity ( figure 3f , inset ) and a widespread distribution .
we show in this paper that mag - fura 2 photoconversion via dab oxidation is an efficient way for localizing ca ions at em level . moreover ,
this could be considered also as a means of stabilizing their presence , thus limiting the loss of ions . as for the method ,
photoconversion is easily carried out and reproducible , can be obtained on a good amount of cells , since the exposition in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp .
the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary . in these conditions
, the blackness of the product stands out from the background . in our conditions , in fact , ca ions are immediately detectable within the vesicles and the tubules of the ser , while it is much more difficult to see their presence when in the cytosol , due to their low electrondensity .
this is a clear advantage over the conventional imaging of mag - fura 2 fluorescence that must be removed by the cytosol in order to study truly er signals .
this is achieved by permeabilizing the plasma membrane with brief applications of antibiotics or detergents , but this procedure may destroy intracellular signaling pathways , dilute cytosolic modulators of er ca release / sequestration , and disassemble the cytoskeletal elements that maintain the contact between cell membrane and er . alternatively , the whole - cell configuration of the patch - clamp technique could be exploited to remove the dye , but this experimental approach is rather expensive and challenging , requires highly skilled electrophysiologists and has a quite low throughput .
the final yield of photoconverted mag - fura 2 is in direct relation with the amount of ca present in the sample considered . in our condition
, the ca - enriched medium gave a higher end product staining than the normal physiological conditions or than ca deprivation .
it is interesting that in several cases , dense precipitates could be found in areas suggesting the local accumulation of ca nearby the mouth of plasmalemmal ca channels .
accordingly , such precipitates are evident when extracellular ca concentration is increased , which is consistent with the notion that constitutive ca inflow from the extracellular milieu is enhanced .
this unexpected outcome of dab photoxidation is extremely relevant in light of the master role served by local ca microdomains generated by extracellular ca influx , which regulate cell functions as diverse as cycle progression , proliferation , migration , nitric oxide production , and adenylyl cyclase activity .
one thing that should be underlined here is that we could not visualize the ser tubule filled with the end product .
this could be due to three different causes : i ) ca can be dispersed ( lost ) in the conditions used here ; ii ) local intraluminal ca concentration is less than expected ( which is less probable as it is unlikely to be lower than 100 m ) ; or iii ) dab photoconversion is not efficient enough for mag - fura . by comparing fluorescence images with em micrographs ,
it is clear the difference in the signal coming from vesicles and the elongated filamentous structures , the latter being much less fluorescent .
the relative low yield when comparing fluorescence images is definitely related to the extremely lower amount of end product which can be found within a 60 - 80 nm thin section in relation to the fluorescence originating from the whole cell . in addition , albeit [ ca]er fluctuates between 100 and 500 m , ca accumulates in specific regions of the er that may be missed in our sections . moreover , releasable ca is actually trapped by oligomers of the ca - binding proteins calnexin and calreticulin : these form a concealed ca source that is not in rapid equilibrium with the [ ca]er , but is easily accessible to ca - releasing channels upon extracellular stimulation .
thus , the lack of ser tubules or cisternae filled with the end product of the photo - conversion , i.e. , ca , is not surprising .
finally , the photoconversion process seems to be efficient , and gives an end product more reasonable than the precipitation technique utilized , for instance , by tandler et al . in the latter case ,
an important amount of precipitates were found in the cell and especially in the nucleus ; our method gives a much finer end product , and this allows a superior resolution and localization . in conclusion , this method is a reliable and efficient technique for stabilizing and visualizing ca ions at em .
it gives a high resolution end product thus allowing the fine structural ca localization . in principle
, this approach could provide important qualitative information about the amount and sub - cellular distribution of calcium ions both in cultured cells and , more importantly , in ex vivo samples , which are far less amenable to high resolution ca imaging of er ca levels .
 we propose a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques . we have applied a new approach by photo - oxidation of diaminobenzidine in presence of the emission of an excited fluorochrome specific for calcium detection . 
 this method combines the selectivity of available fluorophores to the high spatial resolution offered by transmission electron microscopy to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles . 
 we show in this paper that mag - fura 2 photoconversion via diaminobenzidine oxidation is an efficient way for localizing ca2 + ions at electron microscopy level , is easily carried out and reproducible , and can be obtained on a good amount of cells , since the exposure in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp . 
 the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary . 
Introduction
Materials and Methods
Cell culture
Intracellular Ca2
Transmission electron microscopy
Results
Discussion