melanins are biological pigments that 
 are found in human skin in two forms : black / brown eumelanin and red / brown 
 pheomelanin . pump
this 
 technology has shown that increased eumelanin content correlates with 
 increasing severity of a melanoma diagnosis .
recently , we found that pump  probe microscopy is also sensitive 
 to the iron content in eumelanin ; however , 
 under the conditions previously used to differentiate eumelanin from 
 pheomelanin , iron - loaded eumelanin shares a similar pump 
probe 
 response with pheomelanin , making them difficult to discriminate . characterizing the factors that influence melanin s
the histological 
 diagnosis of melanoma is fraught with uncertainty ; new indicators could potentially increase the certainty 
 of diagnosis , which could not only help catch melanomas that might 
 otherwise be missed but also decrease wasted healthcare costs associated 
 with false positives .
melanoma metastases have elevated ferritin levels , which may result in increased iron content in 
 cutaneous melanin that would be detectable using pump  probe 
 microscopy .
oxidative stress helps melanomas progress toward metastasis ; oxidative damage to melanins could be indicative 
 of malignancy .
additionally , melanin aggregate size may be relevant 
 to melanoma diagnosis because small melanin aggregates are less efficient 
 at converting absorbed light to heat .
they 
 may , instead , produce free - radicals , which could lead to malignancy .
this work presents the pump  probe response of iron - loaded 
 eumelanin , eumelanin with metals removed , and pheomelanin , at a wider 
 range of wavelengths than previous work . in general
, we find that at shorter pump wavelengths pu , the response crosses over from a predominantly ground - state 
 bleaching signal to an excited state absorption signal .
the crossover 
 wavelength pu , x is different for each type of melanin , 
 and we find that iron shifts eumelanin s pu , x to shorter wavelengths . to identify the mechanism by which iron 
 shifts pu , x , we characterize the effects of chemical 
 oxidation , photodamage , and aggregate size .
we find that iron s 
 modification of the eumelanin pump  probe response can not be 
 attributed to differences in aggregation or raman resonances .
after 
 considering recent theoretical models of eumelanin s optical 
 absorption , we conclude that iron shifts pu , x as 
 a consequence of broadening the near - infrared absorption bands of 
 individual chromophores in the heterogeneous melanin aggregate .
another challenge to differentiating types of melanin stems from 
 the changes in pump  probe response exhibited with increasing 
 optical intensity .
probe 
 response on optical intensity , finding that eumelanin likely exhibits 
 a pump- and probe - dependent saturation , whereas pheomelanin and iron - loaded 
 eumelanin exhibit a nonlinear excited state absorption . at very high 
 power levels , the iron - loaded eumelanin , in particular , undergoes 
 irreversible changes to the pump  probe response .
the laser 
 exposure necessary to cause these changes is similar to the previously 
 reported levels necessary to enhance eumelanin fluorescence .
this suggests that the previously reported  activation  
 of eumelanin fluorescence may be caused in part by the dissociation 
 of metal ions or the selective degradation of iron - containing melanin , 
 thus reducing fluorescence quenching .
eumelanin from sepia officinalis was purchased from sigma - aldrich 
 and washed with edta to remove metals , as previously reported .
a portion of the edta washed eumelanin was then 
 saturated with iron(iii ) chloride according to the previously reported 
 method .
iron content was measured with 
 inductively coupled plasma mass spectrometry ( icp - ms ) , as previously 
 reported : the iron - saturated eumelanin 
 contained 27  494 ppm iron , and the edta - washed eumelanin contained 
 only 30 ppm iron .
sepia melanin was 
 separated by aggregate size , as previously reported . to prepare for imaging ,
the eumelanin samples were suspended 
 in a drop of ultrapure water and evaporated on a slide to produce 
 a thin layer of melanin particles .
the synthetic pheomelanin , a fine 
 powder , was embedded in agarose , thinly sliced , and allowed to dry , 
 as previously reported .
photodamage 
 and oxidative damage studies in human eumelanin were conducted on 
 black human hair cross sections .
these were prepared by first embedding 
 the hair in paraffin wax and then slicing the paraffin block into 
 5 m slices .
a solution of 
 5% hydrogen peroxide continuously flowed over the black hair cross 
 section while images were acquired .
the laser system used for 
 the majority of pump  probe experiments ( illustrated in figure 1 ) was a mode - locked , titanium - sapphire laser ( chameleon , 
 coherent , inc . ) pumping a tunable , intracavity frequency - doubled optical 
 parametric oscillator ( mira - opo , coherent , inc . ) , both producing optical 
 pulses with 5 nm bandwidth .
the pulses from each source were compressed 
 with prism pairs down to approximately 250 fs fwhm , as measured by 
 two - photon absorption cross - correlation in rhodamine-6 g .
the pump 
 beam was intensity - modulated using an acousto - optic modulator ( aom ) 
 at 2 mhz .
the pump and probe beams were combined collinearly and sent 
 into a home - built scanning microscope fitted with a 40 , 0.75 
 na objective ( olympus ) .
both beams transmitted through the sample 
 were collected with a 1.1 na condenser ( olympus ) .
the pump was blocked 
 using a stack of appropriate chromatic filters , and then the probe 
 was detected by an amplified photodiode . a lock - in amplifier ( stanford 
 research systems 844 ) detected the modulation passed from the pump 
 beam to the probe beam .
normal imaging conditions were around 0.1 
 mw of each beam measured at the sample .
scan speeds were 
 normally 25 ms / line with 512 pixels per line across a 400 m 
 field of view .
backscattered fluorescence was collected using a 600 
 nm short - pass dichroic filter ( thorlabs fes0600 ) and a photomultiplier 
 tube ( hamamatsu , r3896 ) .
a separate system was used to 
 simultaneously measure the pump  probe response and fluorescence 
 for hair samples .
the system is similar to the one described above 
 with minor alterations , primarily consisting of equipment .
the probe 
 beam is generated with a tsunami ti : sapphire oscillator ( newport ) 
 tuned to 810 nm , which also pumps an opal - opo ( newport ) tuned to 1430 
 nm ( chosen to optimize output power and stability ) .
the output is 
 then doubled with a bbo crystal to produce a 730 nm pump beam .
the 
 two beams are then coupled into a commercial zeiss lsm 510 microscope . 

custom detection instrumentation , including lock - in - amplifier , integrate 
 pump  probe microscopy with other detection ports of the lsm 
 microscope ( e.g. , confocal and multiphoton detection ) .
the cross - correlation 
 of the two beams measured in rhodamine-6 g is 190 fs . pump 
probe response curves were acquired by varying the time 
 delay between the pump and probe pulses using a motorized stage , as 
 a stack of images was acquired .
probe response curves 
 shown here are averages of the entire field of view . by convention
, 
 we set the lock - in amplifier phase so that a positive signal indicates 
 an increase in absorption of the probe .
figure 2 shows the pump  probe response of edta - washed sepia eumelanin , iron - loaded sepia eumelanin , and synthetic pheomelanin , at pr = 
 815 nm probe , scanning the pump from pu = 700 to 
 pu = 725 nm in 5 nm increments .
the data are normalized 
 to either positive or negative peaks ( whichever is greater ) to facilitate 
 comparison of the shape of the pump  probe response curves . 
 we refer to signals that require the pulses to be overlapped in time 
 as  instantaneous  , and signals that persist beyond 
 the 250 fs cross - correlation of the pulses as  time - delayed  . 

instantaneous signal contributions may include two - photon absorption 
 ( positive signal ) , stimulated raman gain ( negative , only if pu < pr ) , stimulated raman loss ( positive , 
 only if pu > pr ) , and optical 
 kerr lensing ( which is minimized by using a high - na condenser optic ) .
time - delayed signal contributions may include 
 excited state absorption ( positive ) , stimulated emission ( negative , 
 only if pu < pr ) , and ground 
 state bleaching ( negative ) . though we 
 can not rule out stimulated emission , ground state bleaching is a more 
 likely explanation for the negative signals we report here ( see discussion ) .
edta - washed eumelanin has an 
 excited state absorption for all pump wavelength shown here ( 700 nm 
  pu  725 nm ) and has a small instantaneous 
 negative signal only for the lowest energy pump , pu = 725 nm .
iron - loaded eumelanin has an excited state absorption 
 at pu  710 nm with a longer lifetime , and 
 a negative signal with both instantaneous and time - delayed components 
 at pu  715 nm .
pheomelanin has a negative 
 instantaneous at all pump wavelengths ( 700 nm  pu  725 nm ) ; its time - delayed signal shows ground state 
 bleaching at pu  715 nm and an increasingly 
 dominant excited state absorption with pu < 715 
 nm , confirming the trend established by our previous measurements .
data 
 are normalized to the largest peak and smoothed using a 5 point , second - degree 
 polynomial savitzky  golay filter to facilitate comparison of 
 response curves with variable snr levels .
though the addition of iron to 
 eumelanin only causes a slight change to the optical absorption spectrum , it causes a dramatic change to the pump 
the negative response of iron - loaded eumelanin 
 at pu  715 nm is short - lived and may , in 
 principle , be attributable to either stimulated raman loss or ground 
 state bleach with a short lifetime . given the 5 nm bandwidth of our 
 pulses , the wavelengths scanned in figure 2 can stimulate raman loss in the probe for transitions in the range 
 of 12802120 cm . in this range ,
eumelanin 
 has resonances at 1400 cm and 1600 cm , with a weak iron - specific raman line at 1470 cm . to test whether stimulated raman scattering 

is a significant contribution to the pump  probe response , we 
 conducted two measurements -- one with the pu and 
 pr swapped , the other with a wider range of pu . swapping the pump  probe wavelengths will confirm 
 the presence of a significant raman contribution if the instantaneous 
 signal changes sign : in one configuration , pu < 
 pr , stimulated raman scattering induces a loss in 
 the probe ( negative signal ) . in the other configuration , pu > pr , stimulated raman scattering induces 
 a gain in the probe ( positive signal ) . in figure 2 ,
pu < pr , and the 
 negative instantaneous signals at pu = 715 , 720 , 
 and 725 nm are possibly stimulated raman loss .
but in figure 3 , where we swapped pu and pr , the response of iron - loaded eumelanin at pr = 715 , 720 , and 725 nm does not change sign , indicating that raman 
 scattering is not a major contribution .
data are normalized 
 to the largest peak . in this arrangement ( pu > 
 pr ) , stimulated raman gain is expected to produce 
 a positive instantaneous signal . scanning a wider range of pump wavelengths will confirm a 
 raman contribution to the signal if the magnitude of the instantaneous 
 pump  probe response varies proportionally to the raman spectrum . 
 to span all three raman lines associated with eumelanin and with iron - loaded 
 eumelanin ( 13501650 cm including associated 
 raman line bandwidths ) , we measured the response of iron - loaded eumelanin 
 at pr = 805 nm , scanning the pump from pu = 712 to pu = 728 nm in 4 nm increments . 

( the opo was not stable at 815/745 nm , so we shifted the scan to start 
 at 805/728 nm .
although this may change the time - delayed response , 
 the raman response remains unaffected , as it depends only on the difference 
 in pump and probe wavelength . ) to compare the amplitude of signals 
 across different wavelengths , we normalize the pump  probe data 
 by the magnitude of the 2-photon absorption cross - correlation measured 
 in rhodamine 6 g .
figure 4 shows the results , 
 along with the approximate locations and bandwidths of the raman peaks 
 and their overlap with the bandwidth of the pump and probe beams . 
 clearly , the magnitude of the instantaneous negative pump 
probe 
 signal is figure 4b does not track with the 
 raman spectrum of eumelanin , further confirming that stimulated raman 
 is not a major contribution to the pump  probe response , and 
 that differences in raman spectra can not account for the differences 
 in pump  probe response caused by adding iron to eumelanin .
( a ) corresponding 
 wavenumber bandwidths for 805 nm probe wavelength with various pump 
 wavelengths . overlaid
( b ) pump  probe response of iron - loaded 
 eumelanin at varying wavenumber differences .
probe 
 response of melanins scaled linearly with the product of pump and 
 probe intensities , at low optical intensities , by the standard method 
 of fitting a line to a plotting of the log of signal magnitude with 
 respect to the log of optical intensity .
here we performed 
 a more detailed experiment to separate signal components that are 
 linear with respect to the product of pump and probe intensities from 
 those that are quadratic with respect to either the pump or probe . 
 for each sample , we acquired a set of data at nine different pump 
 and probe power levels ( restricting intensity to below that which 
 causes a noticeable change in the pump  probe signal upon repeated 
 scanning ) .
figure 5a shows that the form of 
 the edta - washed eumelanin response does not change appreciably under 
 optical intensities comparable to the results in refs ( 1 ) and ( 2 ) ( our objective na greater 
 by a factor of 3 , making the intensity greater by a factor of 9 , for 
 the same power ) and remains stable until approximately 0.3 mw total 
 power ( data not shown ) , at which visible damage ensues .
the power 
 dependence of the other two pigments is very different at higher intensities . 

figure 5b shows that for the iron - loaded eumelanin , 
 as the total power level increases , the ground state bleaching signal 
 initially increases and then it decreases as it competes with an increasing 
 excited state absorption signal .
synthetic pheomelanin also has an 
 excited state absorption that grows in with increasingly high power 
 levels .
this excited state absorption competes with the ground state 
 bleaching signal and shortens its apparent lifetime .
probe responses 
 of edta - washed sepia eumelanin , iron - loaded sepia eumelanin , and synthetic pheomelanin at varying 
 power levels .
pump wavelength is 720 nm ; probe 
 wavelength is 815 nm . at high intensities , the pump  probe response of iron - loaded 
 eumelanin changes irreversibly .
the responses of pheomelanin and edta - washed 
 eumelanin do not undergo irreversible changes in the power limits 
 investigated thus far , so we focus our attention to eumelanins containing 
 iron . an irreversible change to the pump  probe response ( observed 
 as both a change in pump  probe response form and a decrease 
 in the magnitude of the pump  probe signal , herein referred 
 to as pump  probe photobleaching ) is likely caused by chemical 
 changes to the melanins . because high power can also enhance melanin 
 fluorescence
, we investigate how the 
 pump  probe response changes correlate with melanin fluorescence 
 enhancement .
figure 6 shows images of iron - loaded 
 eumelanin at four power levels . due to the spatial heterogeneity of 
 these results , the pump  probe data in figure 6 are rendered as false - color images , using phasor analysis .
each pixel is colored to indicate the degree 
 of similarity to the pump  probe response of edta - washed eumelanin 
 ( red ) or iron - loaded eumelanin ( green ) .
fluorescence intensity ( collected 
 with a pmt , 680 nm dichroic , a 600 nm short - pass filter , and a bg-39 
 filter to reject near - ir scattered light ) is overlaid in white .
melanin 
 granules with notable behavior over the course of the power study 
 are highlighted .
the white box ( with zoom inset ) shows a melanin granule 
 that first shifts from green to yellow , then pump  probe photobleaches , 
 and finally begins to fluoresce .
the magenta arrow shows a granule 
 of melanin where the pump  probe response form gradually shifts 
 from similar to iron - loaded eumelanin to resembling that of edta - washed 
 eumelanin .
when an image is taken at low power after exposure to high 
 power ( not shown ) , the melanin granules maintain their new pump 
additionally , changes 
 to the melanin morphology , such as the piece of melanin shrinking 
 as power increases from 0.3 to 0.7 mw ( white box in figure 6 ) are irreversible . note that the pump 
kerimo et al . also found that melanin fluorescence activation 
 takes variable amounts of optical intensity to induce and that melanin 
 fluorescence was prone to photobleach under these conditions .
fluorescence 
 enhancement was also found in a fraction of the melanin sampled and 
 most often seen in melanin granules undergoing morphological changes .
images of iron - loaded sepia eumelanin at various total power levels taken in succession with 
 720 nm pump , 815 nm probe .
the white box ( with zoom inset ) shows a melanin granule that first 
 pump  probe photobleaches and then begins to fluoresce .
probe 
 response form gradually shifts from similar to iron - loaded eumelanin 
 to resembling that of edta - washed eumelanin .
physiological iron content is much lower than in our iron - loaded 
 sepia eumelanin . to test whether our 
 finding from figure 6 applies to eumelanin 
 with physiologically relevant metal ion content , we imaged a black 
 hair cross section , embedded in paraffin wax .
the samples were imaged 
 using a 20 , 0.8na zeiss objective , starting at 0.1 mw total 
 powers until reaching 2 mw , measured at the sample ( figure 7b , part 1 ) .
then the samples were reimaged in decreasing 
 power intervals ( figure 7b , part 2 ) . the average 
 spectrum for each image is shown in figure 7a , where the solid lines correspond to part 1 and the dashed lines 
 to part 2 .
as power levels increase , the pump  probe response 
 curves have increasing levels of excited state absorption and decreasing 
 levels of ground state bleaching , taking on more edta - washed eumelanin 
 form ( figure 2 ) .
when the samples were reimaged 
 at decreasing power levels , the response curves did not return to 
 their original forms , indicating that high power levels cause irreversible 
 damage .
( a ) pump  probe response of black hair as a function of power . 

solid lines are taken with increasing power levels , and the dashed 
 lines are taken with decreasing power levels after reaching the maximum 
 power of 2 mw .
figure 7b displays the data according 
 to the way the ratio of the positive peak to the negative peak changes 
 with power level .
note that from 0.1 to 1.1 mw , the spectra 
 trace one path , and for powers > 1.1mw , they trace another .
interestingly , 
 the spectra continue on this new path after decreasing the power again 
 in part 2 , clearly indicating irreversible changes occur above 1 
 mw average power , which is the same as that reported by kerimo et 
 al .
found that activation occurs even with continuous wave laser exposure , 
 indicating that peak power is less significant than average power .
the similar average power required for these 
 two phenomena suggests they may have a common cause .
we simultaneously 
 acquired fluorescence while imaging the hair but did not find bulk 
 fluorescence activation under these conditions .
not purging the sample with nitrogen most likely caused the melanins 
 undergoing fluorescence activation to photobleach .
, blue circles are taken with increasing power levels , 
 and the red circles are taken with decreasing power levels after reaching 
 the maximum power of 2 mw . though we did not observe fluorescence activation in the 
 hair cross sections , it is clear from figure 6 that iron - loaded eumelanin fluoresces when photodamaged , and from 
 figures 6 and 7 that 
 photodamage manifests itself by shifting the pump  probe response 
 toward that of edta - washed eumelanin . to test how iron affects melanin 
 fluorescence ,
we collected fluorescence images for iron - loaded and 
 edta - washed eumelanin at different power levels . shown in figure 9 , at low power levels , edta - washed eumelanin is 
 fluorescent , whereas iron - loaded eumelanin is not , but iron - loaded 
 eumelanin fluoresces when exposed to higher optical intensity .
multiphoton 
 fluorescence of dry melanins at 0.4 and 0.8 mw total power with 720 
 nm pump and 815 nm probe .
figure 10 shows 
 the pump  probe response of a black hair cross section being 
 gradually oxidized chemically via hydrogen peroxide .
the data are 
 normalized to the negative instantaneous signal because the focal 
 position was optimized at the beginning of every scan due to fluctuations 
 in the flow cell focal position and because signal decreased throughout 
 the experiment due to gradual photobleaching and the amount of melanin 
 decreasing due to oxidative dissolution .
power levels were kept low 
 ( 0.1 mw for each beam ) in the interest of isolating the effects of 
 chemical oxidation from photodamage , so no fluorescence was obtained , 
 but it has been previously reported that chemical oxidation increases 
 uv - induced fluorescence . using higher 
 power could have caused detrimental melanin pump  probe photobleaching , 
 intensity - related changes to the pump  probe signal independent 
 of the chemical oxidation , or even accelerated the chemical changes 
 to the melanin .
similarly to figure 7 , the excited state absorption increases and ground 
 state bleaching decreases as the melanin becomes increasingly damaged . 

as the experiment proceeds , we see a response more closely resembling 
 that of edta - washed eumelanin , suggesting that one of the effects 
 of chemical oxidation is to break the iron  eumelanin bond . pump 
probe 
 response of a black hair cross section as it is chemically oxidized . 

data are normalized to the negative peak because the focal position 
 changed throughout the experiment and to account for pump 
suspecting that the iron - eumelanin 
 bonds were breaking through oxidation ( figure 10 ) or high power ( figure 7 ) , we considered 
 consequences that this bond breakage may have that would modify the 
 pump  probe response : changing the unit spectra of individual 
 chromophores , and/or changing the melanin aggregate size .
previously suggested that the pump  probe response differences 
 between eumelanin and pheomelanin could be attributed to melanin aggregate 
 size differences .
edta - washing reduces particles to 138 nm ; subsequent iron - loading 
 increases particle size to 143 nm .
if 
 iron affects the response of eumelanin through aggregation , then we 
 would expect that increasing aggregate size and increasing iron content 
 would have the same effect .
we test this with several different filtered 
 sizes of eumelanin particles at pu = 720 nm , pr = 815 nm , where loading eumelanin with iron changes the 
 response from excited state absorption ( positive ) to ground state 
 bleaching ( negative ) .
figure 11 shows that 
 increasing eumelanin aggregation has the opposite effect on the response 
 when compared with iron loading : small aggregates have a ground state 
 bleaching response , and large aggregates have an excited state absorption . 

therefore , iron modifies the pump  probe response through some 
 mechanism other than aggregation .
because sample preparation can 
 dramatically change the size of melanin aggregates , poorly controlled sample preparation methods could cause 
 the aggregate size to be a confounding factor in experiments where 
 the metal content is varied .
although sample preparation methods were 
 identical for edta - washed eumelanin and iron - loaded eumelanin , additional 
 edta - washing experiments were performed on natural melanin ( previously purified directly from sepia officinalis as opposed to sigma - aldrich ) , from 
 another bottle of sigma sepia melanin , 
 and on melanin in a black hair cross - section ( data not shown ) to verify 
 the trend observed in figure 2a , b . in both 
 cases ,
probe 
 response under the pu = 720/pr = 810 nm experimental conditions : the instantaneous negative signal 
 decreased and the excited state absorption signal increased , although 
 the negative signal did not always completely disappear . from this 

we conclude that sample preparation procedures are not the source 
 of pump  probe signal differences in figure 2 .
care should be taken when the chemical composition of melanins 
 is varied to ensure the aggregate size is well - controlled .
previously we found that the 
 two major classes of melanin , eumelanin , and pheomelanin , have sufficient 
 differences in their wavelength - dependent pump  probe responses 
 to be leveraged for imaging contrast .
we have also 
 found that the iron content of eumelanin dramatically changes the 
 pump  probe response . even though 
 iron only causes slight changes to the linear optical absorption spectrum . here
, we have characterized the response of 
 these melanins for a range of pu from 700 to 725 
 nm ( figure 2 ) and ruled out differences in 
 raman resonances and changes in aggregation as mechanisms of iron s 
 influence on the pump  probe response of eumelanin ( figures 3 , 4 , and 11 ) . turning our attention to the time - delayed ( t > 250
fs ) response , we consider how these melanins 
 differ , and what underlying causes might account for the differences . 
 a positive time - delayed response
( signaling a decrease in detected 
 probe intensity when the pump is on ) may be unambiguously interpreted 
 as an excited state absorption , whereas a negative response may be 
 attributed to both stimulated emission and excited state absorption 
 ( both of which can increase the detected probe intensity when the 
 pump is on ) .
stimulated emission is ruled out when pr < pu , but even when this is not the case , it 
 is unlikely to be a significant contributor to the pump 
probe 
 response of melanins because the requirements for stimulated emission 
 are very restrictive compared with ground state bleaching .
stimulated 
 emission will be observed only for relaxation pathways that specifically 
 involve energy levels that have dipole - allowed transitions to the 
 lower - energy states that are both resonant with pr and are aligned with the probe polarization vector .
ground state 
 bleaching , on the other hand , will be observed regardless of the relaxation 
 pathways . in melanins ,
optical excitations relax via a large number 
 of energy transfer pathways , as evidenced by rapid nonradiative relaxation 
 and rapid fluorescence anisotropy decay .
it is unlikely that a significant fraction of these relaxation pathways 
 meet the relatively strict conditions for stimulated emission .
therefore , 
 we attribute the negative signals observed here solely to ground state 
 bleaching . with the exception of edta - washed eumelanin
, we have 
 observed that the transient response of melanins at pr 810815 nm crosses over from a response dominated 
 by ground state absorption to one dominated by excited state absorption 
 as we tune pu from 750 to 700 nm ( see figure 2 of this manuscript and figure 7 of ref ( 2 ) ) . for the sake of discussion
, 
 we define the crossover wavelength pu , x as the pump 
 wavelength at which the probe ground state bleach and excited state 
 absorption signals are balanced ( i.e. , t>250fss(t )  dt = 0 ) .
a summary of the approximate crossover pump wavelengths 
 for various melanins , with an 810815 nm probe , is shown in 
 table 1 .
natural sepia eumelanin , which has some native iron content , crosses over at pu , x = 750 nm .
adding iron content by saturating with fecl3 shifts pu , x to approximately 715725 
 nm , suggesting that the net effect of bound iron is to shift pu , x to shorter wavelengths .
if that is the case , we expect 
 that removing the iron with edta would shift pu , x to > 750 nm .
this is consistent with our observations that the 
 response of edta - washed eumelanin is dominated by excited state absorption 
 in all our measurements with pr = 817 nm , pu  750 nm ( data not shown ; our opo is not tunable 
 beyond 750 nm , preventing us from locating pu , x ) .
the observation that bound iron content shifts pu , x to shorter wavelengths may be explained hypothetically within the 
 framework that considers the broad optical absorption of melanins 
 to be the sum of a large number of heterogeneous chromophores . though 
 the sum optical absorption spectrum is broad and featureless , each 
 underlying chromophore exhibits peaks whose locations and widths depend 
 on the number and arrangement of its dhi(ca ) units ( or benzothiazine 
 units in the case of pheomelanin ) , local chemical environment , stacking 
 structure , and metal ions .
only a subset 
 of these chromophores ( those having strong optical absorption at pu ) are excited initially in a pump  probe experiment . 

the time - delayed probe response reflects competition between ground 
 state bleaching and excited state absorption , depending on a number 
 of factors .
if excited state absorption were absent , the ground state 
 bleach signal would depend on pr in the same manner 
 as a transient spectral hole burning measurement : the ground state bleach intensifies as pr approaches pu , and scanning pr would reveal the sum absorption spectrum of the subset of chromophores 
 excited by pu .
but excited - state absorption is not 
 absent for pu and pr in the near - infrared , 
 and this response replaces ground state bleaching as pu is tuned away from pr . although ground state bleaching 
 is restricted to probe interactions with the pump - selected subpopulation
, 
 excited state absorption might involve excited state transitions either 
 within the pump - excited chromophore or from a pump - excited chromophore 
 to a nearby chromophore that has an available energy level in the 
 vicinity of e  hc/pu + hc/pr .
in that case , 
 given the high degree of heterogeneity within melanins , it is likely 
 that any near - ir pu , pr combination 
 will access an excited state absorption .
thus we expect excited state 
 absorption to dominate the response for pr outside 
 the spectral hole burned by the pump , and to be overwhelmed by an 
 increasingly dominant ground state bleach as we tune pr toward pu , consistent with our primary findings 
 here in figure 2 and in figure 7 of ref ( 2 ) .
this hypothesis for ground state bleach / excited state absorption 
 competition in melanins is illustrated by the four scenarios sketched 
 in figure 12 , showing combinations of chromophores 
 having narrow and broad absorption spectra with pump / probe wavelengths 
 having small and large detuning pp = |pu  pr| . in figure 12a
, the probe is within the narrow spectral hole burned by 
 the pump , and the response is dominated by ground state bleaching . 

figure 12b shows that as the probe is tuned 
 outside the spectral hole , ground state bleaching is weak , and excited 
 state absorption to a neighboring chromophore occurs .
figure 12c , d show the same pump and probe wavelengths , but 
 with chromophores that have broader absorption bands . in this case
, 
 the larger pp still results in ground state 
 bleaching , with no crossover to excited state absorption .
this implies 
 that the spectral widths of the pump - excited chromophores determine 
 the crossover wavelength pu , x we reported here .
schematic 
 diagram showing the proposed explanation of the connection between 
 pu , x and the individual oligomer absorption spectrum . 

the black curve represents the broad melanin absorption spectrum composed 
 of the sum of individual oligomer absorption spectra .
the dotted orange lines represent the absorption 
 spectra of a neighboring chromophore . under this framework , the pump  probe data can be interpreted 
 in a manner that is consistent with the idea that melanin s 
 broad , featureless absorption spectrum is the result of the sum of 
 many individual chromophores .
smaller chromophores , with fewer dhi(ca ) 
 subunits , account for the strong uv  vis absorption , whereas 
 larger chromophores , with extensive electron delocalization and red - shifted , 
 broadened absorption bands , account for the near - ir absorption tail .
this implies that longer - wavelength pu will access broader - absorbing chromophores , and the probe 
 response will be increasingly dominated by ground state bleaching . 

indeed , pump  probe measurements with pu  
 700750 nm and pp < 100 nm exhibit 
 a mixture of excited state absorption and ground state bleaching ( figure 2 here and figure 7 of ref ( 2 ) ) , whereas measurements 
 with longer - wavelength pu  800815 
 nm and comparable pp are dominated by ground 
 state bleaching ( figure 3 here , and figures 
 2 , 4 , and 8 of ref ( 2 ) ) .
this framework also allows us to infer the cause of iron 
 effect on the pump  probe response of eumelanin .
the observed 
 blue shift in pu , x that occurs with increasing iron 
 content ( figure 2 and table 1 ) corresponds with a broadening of available pp over which ground state bleaching is dominant , indicating 
 that iron serves to broaden the absorption spectra of the underlying 
 eumelanin chromophores  just as predicted by density functional 
 theory calculations on proposed eumelanin protomolecules .
chemical oxidation 
 could damage iron - containing melanin through the fenton cycle : iron 
 present in melanin catalyzes the production of free radicals in the 
 presence of hydrogen peroxide . because 
 these free radicals would form first near the iron - containing melanin 
 chromophores and subsequently degrade these particles , the pump 
probe 
 response would become more dominated by the iron - free chromophores 
 as oxidation progresses , as we observed in figure 10 .
figure 6 also shows the general pattern 
 we have observed for the pattern of damage for iron - loaded eumelanin : 
 first , the pump  probe response changes , then the granule pump  probe 
 photobleaches and undergoes morphological changes , and finally ( in 
 some cases ) the granule begins to fluoresce .
this is in agreement 
 with previously reported melanin fluorescence activation , which also 
 follows morphological changes to the melanin .
iron - catalyzed oxidative degradation may cause these morphological 
 changes , which happen first to melanins containing the highest concentration 
 of iron .
high optical intensity , on the other hand , may damage the 
 iron  melanin bond itself .
high optical intensity 
 may cause the melanin to release iron , which would cause a pump 
probe 
 response dominated by iron - free melanin , as we observed in figures 6 and 7 .
the power threshold 
 of 1 mw average power reported here and by kerimo et al .
suggest that in applications 
 where higher average power is used , the melanin may be altered or 
 damaged .
multiphoton and fluorescence lifetime microscopy of the epidermis 
 applies between 20 and 45 mw of average power to the skin , and although the exact power delivered depends 
 on the imaging depth , this power level could easily damage epidermal 
 melanins .
probe 
 response , iron quenches melanin fluorescence by forcing fluorescence 
 to compete with additional nonradiative relaxation pathways .
these 
 additional pathways manifest themselves by broadening the absorption 
 spectrum , as previously discussed .
melanin ligand - to - metal 
 charge transfer ( lmct ) band , which is between 700 and 800 nm , which 
 relaxes via a nonradiative mechanism .
degradation of iron - containing melanin particles and/or the 
 iron  melanin bond is a possible mechanism to explain the previously 
 reported observations that oxidative damage and high optical intensity cause melanin 
 fluorescence enhancement .
chemical oxidation , before it completely 
 breaks down all the melanin units capable of absorbing nir , will first 
 break down iron - containing melanin , leaving intact only iron - free 
 melanin , which fluoresces more readily because they have fewer nonradiative 
 relaxation pathways . the explanation that high optical intensity causes 
 the release of redox - active iron ions is particularly compelling because 
 lai et al . attempted to prevent oxidative photobleaching by performing 
 experiments under nitrogen gas . despite this attempt to prevent oxidation , 

the black hair melanin continued to photobleach , most likely because black hairs contain iron , which would catalyze melanin oxidation .
chemical oxidation may also enhance melanin fluorescence via breaking 
 large melanin aggregates into smaller aggregates .
the strong fluorescence of small aggregates suggests narrow unit spectra ; however , ground 
 state bleach dominates the pump  probe response , which instead 
 suggests broad unit spectra .
however , in this case , our assumption 
 that melanins always have a possible excited state absorption and 
 a possible ground state bleach is no longer valid ; this assumption 
 was based on the idea that eumelanins are heterogeneous mixtures of 
 oligomers and that absorbed energy could always be passed to a nearby 
 oligomer that will allow an excited state absorption .
selecting only 
 the smallest melanin aggregates from the bulk mixture reduces number 
 of possible melanin aggregate configurations , making the unit absorption 
 spectra much more similar to each other . making the melanins
more 
 homogeneous decreases the chances of an excited state absorption at 
 any possible wavelength combination .
obvious lifetime differences between 
 the iron - loaded and edta - washed eumelanins at shorter wavelengths 
 could discriminate them ; however , relying on lifetime differences 
 requires much higher snr pump  probe response curves than simply 
 using signals with opposite signs .
competing 
 processes contribute to the pump  probe response different amounts 
 based on the exact power delivered . when these processes have different 
 signs , such as those contributing to the melanin pump 
probe 
 response curves , the power effectively modifies the lifetime that 
 is observed . referring back to figure 5 , the 
 increasing dominance of the excited state absorption in the cases 
 of pheomelanin and iron - loaded eumelanin shortens the apparent lifetime 
 of the ground state bleach .
if one were to calculate a lifetime for 
 the ground state bleach based on observations at a single power level , 
 the result would be a function of the relative contributions of the 
 two processes and their respective lifetimes .
a better option 
 is combining information from two different pump wavelengths , such 
 as 705 and 720 nm . under these conditions , a pixel that has a negative 
 instantaneous signal at both wavelengths is pheomelanin , a pixel that 
 has an excited state absorption at both wavelengths is edta - washed 
 eumelanin , and a pixel that has a negative signal at 720 nm but an 
 excited state absorption at 705 nm is iron - loaded eumelanin . making 
 the two pump wavelengths closer , such as at 710 and 715 nm , makes 
 the experiment easier to achieve in practice , but it is a poor choice 
 because at these wavelengths , the signal becomes very weak in the 
 case of iron - loaded eumelanin as the ground state bleach and excited 
 state absorption nearly cancel one another . for differentiating 
 melanins with the two wavelength combinations ,
power levels must be 
 kept sufficiently low to prevent an excited state absorption from 
 growing into the iron - loaded eumelanin and pheomelanin pump 
probe 
 response , as shown in figure 5 . because melanins 
 have such broad absorption spectra , the near - infrared pump 
probe 
 experiments might access energy levels comparable to ground - state 
 transitions in the blue / ultraviolet spectral region if two photons 
 are absorbed simultaneously from the pump or the probe .
such contributions 
 to the response are nonlinear in either the pump or the probe , and 
 might be the origin of the excited state absorption observed in iron - loaded 
 eumelanin and pheomelanin that grows in with increasing optical intensity . 
 in imaging experiments , high power levels incident on iron - loaded 
 eumelanin or pheomelanin
could incorrectly suggest the presence of 
 eumelanin lacking metals . additionally , increasing the power past 
 saturation will cause unnecessary damage to the samples without improving 
 the snr .
however , the differences in the power dependencies may be 
 leveraged for discriminating melanins when the laser setup requires 
 that the wavelength is fixed .
in 
 this report , we have shown that at sufficiently low power , iron - loaded 
 eumelanin , edta - washed eumelanin , and synthetic pheomelanin can be 
 differentiated by imaging at two pump  probe combinations : 705 
 and 720 nm pump with 815 nm probe .
this is possible because the dominant 
 response of the iron - loaded eumelanin switches sign around 715 nm , 
 whereas for native iron concentrations in sepia eumelanin , the sign switch occurs at 750 nm and pheomelanin has 
 a switch at 700 nm .
analysis of 
 the pump and probe intensity dependence shows that at higher powers , 
 excited state absorption components grow in to the pump 
this could make discriminating melanins more difficult , 
 especially in thick specimens and in vivo , where the exact optical 
 power delivered to the focal spot can not be precisely determined .
the differences in the pump  probe responses between these 
 melanins can not be attributed to stimulated raman scattering .
rather , 
 the wavelength at which the melanins switch from a ground state bleaching 
 signal to an excited state absorption signal depends on the broadness 
 of the absorption bands of the individual chromophores being excited . 

pump  probe response curves suggest that chemical oxidation 
 and photodamage both narrow the absorption bands of iron - loaded eumelanin , 
 possibly by damaging the iron  melanin bond .
fluorescence data 
 support this argument , as fluorescence is enhanced by chemical and photo - oxidation , as well as by removing metals . therefore , it is likely that the 
 previously reported  activation  of eumelanin fluorescence 
 comes from the dissociation of redox - active metal ions and rapid degradation 
 of metal - containing melanin , both of which can reduce fluorescence 
 quenching , in addition to the release of free ptca to produce smaller 
 eumelanin particles . characterizing 
 the pu , x for melanin in tissue
has potential value 
 for understanding how melanin chemistry is altered in different types 
 of skin malignancies .
drawing conclusions about melanin chemistry 
 will be challenging because many factors contribute to the pu , x .
for example , we previously reported that the pump  probe 
 response of eumelanin is different in dermal cells than in epidermal 
 cells .
the response differences suggest 
 that dermal cells contain less iron , but considering the results contained 
 herein , one can not eliminate the possibility that the melanin is under 
 oxidative stress or simply forming large melanin aggregates . distinguishing 
 between the many factors that contribute to changes to the pump 
probe 
 signal will require further research , such as establishing pu , x for edta - washed eumelanin and measuring the effects of 
 other metals , particularly redox - active metals such as copper .
additional 
 time - resolved fluorescence measurements may also lead to a better 
 understanding of the effect of metals and degradation on radiative 
 relaxation pathways .
we will also extend this work to include pheomelanin 
 with varying metal iron content .
 ultrafast pump  probe measurements 
 can discriminate the two forms of melanin found in biological tissue 
 ( eumelanin and pheomelanin ) , which may be useful for diagnosing and 
 grading melanoma . 
 however , recent work has shown that bound iron content 
 changes eumelanin s pump  probe response , making it more 
 similar to that of pheomelanin . here 
 we record the pump  
 probe 
 response of these melanins at a wider range of wavelengths than previous 
 work and show that with shorter pump wavelengths the response crosses 
 over from being dominated by ground - state bleaching to being dominated 
 by excited - state absorption . 
 the crossover wavelength is different 
 for each type of melanin . in our analysis 
 , we found that the mechanism 
 by which iron modifies eumelanin s pump  probe response 
 can not be attributed to raman resonances or differences in melanin 
 aggregation and is more likely caused by iron acting to broaden the 
 unit spectra of individual chromophores in the heterogeneous melanin 
 aggregate . 
 we analyze the dependence on optical intensity , finding 
 that iron - loaded eumelanin undergoes irreversible changes to the pump  
 probe 
 response after intense laser exposure . simultaneously acquired 
 fluorescence 
 data suggest that the previously reported  activation  
 of eumelanin fluorescence may be caused in part by the dissociation 
 of metal ions or the selective degradation of iron - containing melanin . 
Introduction
Experimental Methods
Results
Discussion
Conclusion