melanoma diagnosis presents important unmet clinical challenges . on the one hand , the ramatic mismatch between increases in detection and mortality rates ( report , 2010 ) may suggest significant overcalling ( glusac , 2011 ; swerlick and chen , 1996 ) . on the other hand , nearly one in ten patients with thin melanomas ( < 1 mm ) and no initial evidence of metastases develops recurrences ( kalady , white et al .
melanin chemistry is a natural biomarker of melanocyte activity , and is a promising marker to differentiate benign , dysplastic , and malignant pigmented lesions ( sealy et al , 1982 ; lazova and pawelek , 2009 ; marchesini et al , 2009 ; biesemeier et al , 2011 ) .
for example , a key metric for pathologists is melanocyte density and melanocyte - to - keratinocyte ratio ( herlyn and shih , 1994 ) , so it is important to differentiate cell type ( melanocyte , keratinocyte , melanophage , etc . ) .
also , melanocytes in the dermis are very concerning ( clark et al , 1969 ) , while macrophages are less concerning , even though both can be pigmented  hence the need for immunohistochemistry .
electron microscopy ( vinther et al , 2008 ; jimbow et al , 1983 ) provides spatial information , but it is not quantitative , provides no molecular contrast , and is prone to mistaking melanosome shapes for other objects if the melanosomes are not purified . in this paper , we extend our recently demonstrated pump - probe technique for quantitative imaging of eumelanin and pheomelanin ( matthews et al , 2011 ) to extract subcellular resolution and morphological information not attainable by any other method .
pump - probe imaging using the near - infrared spectrum is ideal for biological applications due to the enhanced penetration depth of near - infrared compared to ultra - violet or visible light , inherent sectioning capabilities , and low sample damage from heating ( comparable to methods using very low power continuous wave lasers ) .
we have previously shown that this method gives intrinsic contrast in a variety of biological applications , including differentiating oxy- and deoxyhemoglobin ( fu et al , 2007 ) and melanin distributions on a variety of scales , from skin cross sections ( matthews et al , 2011a ) to entire pigmented lesions in vivo ( matthews et al , 2011b ) .
eumelanin and pheomelanin have broad , featureless linear absorption spectra ; however they can be easily differentiated because their pump - probe spectra reflect many competing nonlinear processes ( including excited state absorption , stimulated emission , and ground state depletion ) .
thus the technique is effective even when highly absorbing fluorophores are present ( such as hematoxylin and eosin in previously stained slides ) ( matthews et al , 2011a ) .
specifically , we investigate a number of pigmented lesions with high spatial resolution and more spectroscopic detail , examining differences in pigment chemistry down to the level of individual melanosomes .
we refine our distinction of melanins to not just eumelanin versus pheomelanin , but also sub - classes of eumelanin .
our multi - modality microscope ( described in the materials and methods section ) shows cell structure including melanocytic dendrites and melanosomes , cell nuclei , cell membranes , and presence of collagen .
we observe melanin in macrophages that is spectroscopically different from melanin in epidermal cells , the random spatial distribution of melanin in melanocytic nests , supranuclear caps pointing perpendicular to the basal layer , and individual melanocytes and keratinocytes containing both eumelanin and pheomelanin .
melanin standards in figure 1 show that pump - probe microscopy differentiates skin phantoms containing eumelanin and pheomelanin . in fixed ,
thin tissue slices , macrophages were identified by their location in the collagen and by their coarse pigment ( massi and leboit , 2004 ) .
note that the melanin tends to be homogeneous , with mostly a uniform melanin type and that is spatially not changing rapidly .
further , we find that melanin contained in macrophages in the dermis has a slightly different spectrum than epidermal melanin . to demonstrate this we use a convenient method to capture the heterogeneity of pump - probe signatures called phasor analysis ( redford and clegg , 2005 ; robles et al , 2012 ) , which highlights differences in the spectra of individual or ensembles of spatial pixels .
this approach was previously used to differentiate components of complex fluorescence lifetime images ( redford and clegg , 2005 ) and was recently applied to spectrally separate pump - probe signals of biological and historically significant art pigments ( robles et al , 2012 ) .
phasor analysis also separates the spectra of eumelanin in the epidermis from eumelanin in the macrophages , as shown in figure 3 .
a thin skin slice is shown , along with the spectra of dermal and epidermal cells .
because they have differentiable spectra , they can be separated with phasor analysis because the pixels for each type of cell have separate locations on the phasor plot .
a high - magnification image of a single melanocyte in a melanocytic nest is shown in figure 4a , with the melanocytic nest identified by its location near the basal layer and nest - like in shape .
this is made visible by the second - harmonic generation / fluorescence channel ( shown in gray scale ) . in the nests
, the melanin tends to be spatially disorganized , containing both eumelanin and pheomelanin very close to one another . while the majority of macrophages observed in this study contained mostly eumelanin , there were exceptions to this trend .
our sample set also shows supranuclear melanin caps , which serve to protect nuclei from the damaging radiation of the sun ( kobayashi , 1998 ) .
figure 5 shows examples of supranuclear melanin caps , both cross sections of caps ( figure 5a ) and an en face cap ( figure 5b ) .
in particular , a z - stack of an en face cap shows the contour of the cap , which is rendered in three - dimensions in figure 5c .
most of the macrophages observed in this study appeared to contain eumelanin , even in cases where the epidermal melanin is composed mainly of pheomelanin .
this is consistent with previous findings that melanosomes are broken down in phagocytes but melanin is not , and that pheomelanin is likely broken down more readily due to its increased solubility ( borovansk and elleder , 2003 ) .
the exceptions to this finding were pheomelanic macrophages , found only in malignant regions of melanomas .
if a melanoma is rapidly forming melanin , then the melanin could be building up in the macrophages and allowing observation of not yet degraded pheomelanin .
additionally , melanin in macrophages tends to lack a negative signal at zero delay , possibly due to thermal effects caused by concentrated melanin absorbing the laser light and dissipating it as heat or due to effects of oxidative degradation .
this spectral difference is used to separate populations of melanin in the epidermis and in the macrophages using phasor analysis .
both eumelanin and pheomelanin can be seen in the single melanocyte ( figure 4b ) , supporting the previous finding that individual melanocytes can exhibit mixed melanogenesis ( jimbow et al , 1983 ; ito and wakamatsu , 2008 ) .
similarly , both types of melanin were observed in single keratinocytes , which is consistent with previous findings using electron microscopy ( jimbow et al , 1983 ) . in keratinocytes , the most efficient approach for
the supranuclear caps to protect nuclei would be for the caps to point toward the source of the radiation ( the skin surface ) , but cell signaling is directed from living cells , not the dead stratum corneum .
imaging cells at high magnification shows that melanin caps point perpendicular to the basal layer and not necessarily toward the skin surface ( figure 5d ) .
this supports the previous report of melanin caps covering the apical pole of a cell s nucleus and not necessarily the top of the skin ( corcuff , 2001 ) . in summary
, pump - probe microscopy has allowed the discrimination of eumelanin and pheomelanin non - destructively in individual cells .
this method can be applied to answer questions regarding the subcellular distribution of melanins in fixed samples or in vivo and identify different cell types . in this work
, it has been used to differentiate melanin in epidermal cells from melanin in macrophages , even when both melanin populations are eumelanin .
this could help differentiate macrophages from pigmented dermal melanocytes in stained or unstained biopsy slides or even in vivo .
this work also confirms previous findings that single keratinocytes and melanocytes can contain both eumelanin and pheomelanin .
the two - color pump - probe microscope used in this study is similar to the previously reported microscope in ref ( matthews et al , 2011a ) . in brief , a 720 nm pump beam is modulated at 2 mhz , and a 810 nm probe beam is un - modulated . to characterize a sample s nonlinear dynamic behavior ,
images are taken at a variety of interpulse delays including the following : time points with the probe preceding the pump ( negative delay ) for long lived signals on the order of nanoseconds , points when the pump and probe overlap for instantaneous signals , and points with the pump preceding the probe for measuring the dynamics of the excited state on the order of picoseconds .
the light that passes through the sample filters out the pump beam , such that any modulation transferred from the pump beam to the probe beam is detected using a lock - in amplifier .
the phase of the modulation is set to have positive signal for an absorptive event and negative for a transmissive event .
in addition to the pump - probe system , other microscopy modalities are incorporated : a dichroic filter separates the combined fluorescence and second harmonic generated light that returns through the objective , and is detected using a photomultiplier tube .
most images were acquired using a 60x , 0.9 na objective and a half milliwatt average power of pump and probe beam .
melanin imaging standards were prepared by embedding separately sepia melanin ( sigma - aldrich , saint louis , mi , usa ) and in - house prepared synthetic pheomelanin ( ito , 1989 ) in 1% agarose .
this preparation was molded into blocks and sliced to about 1 mm thickness , then mounted on a slide and cover - slipped .
unstained 5 m slices of formalin - fixed , paraffin - embedded skin biopsies from pigmented lesions were obtained through an institutional review board - exempted protocol .
a dermatopathologist identified the clinically concerning regions of melanoma samples to ensure the fields of view included in the study were properly categorized . for processing of the data ,
every pixel of each image was fit to spectra from melanin standards and then recolored to represent the fractional content of eumelanin ( red ) and pheomelanin ( green ) , determined by the coefficients required for the fitting .
the precision of the melanin fraction calculation depends on the exact concentration of the melanin solutions used to calculate the coefficients , which is approximate because eumelanin is insoluble in water .
 pump - probe microscopy non - destructively differentiates eumelanin and pheomelanin and can be used to quantify melanin distributions in thin biopsy slices . here 
 we have extended that work for imaging eumelanin and pheomelanin distributions on a sub - cellular scale , allowing elucidation of characteristics of different cell types . 
 the results show that melanin heterogeneity , previously found to be characteristic of melanomas , persists on the sub - cellular scale . 
 we have also found spectral changes associated with melanin located in melanophages , which could potentially differentiate invasive pigmented melanocytes from melanophages without immunohistochemical staining . 
Introduction
Results
Discussion
Materials and Methods