Photon Excited Fluorescence Research Articles

Liver fibrosis analysis using digital pathology.

Digital pathology has enabled the noninvasive quantification of pathological parameters. In addition, the combination of digital pathology and artificial intelligence has enabled the analysis of a vast amount of information, leading to the sharing of much information and the elimination of knowledge gaps. Fibrosis, which reflects chronic inflammation, is the most important pathological parameter in chronic liver diseases, such as viral hepatitis and metabolic dysfunction-associated steatotic liver disease. It has been reported that the quantitative evaluation of various fibrotic parameters by digital pathology can predict the prognosis of liver disease and hepatocarcinogenesis. Liver fibrosis evaluation methods include 1 fiber quantification, 2 elastin and collagen quantification, 3s harmonic generation/two photon excitation fluorescence (SHG/TPE) microscopy, and 4 Fibronest™.. In this review, we provide an overview of role of digital pathology on the evaluation of fibrosis in liver disease and the characteristics of recent methods to assess liver fibrosis.

Revealing degradation mechanisms of archaeological flax textiles through the evolution of fibres’ parietal polymers by synchrotron deep-UV fluorescence

Autofluorescence generated by plant cells and tissues provides a label-free method for investigating them without altering their structure and biochemistry. While in the literature, research mainly focuses on investigating fresh plant tissues, this study examines flax yarns from five archaeological artefacts to understand how cell wall fibres degrade and how their biochemical composition is modified after natural ageing over millennia. Using Synchrotron-based deep UV fluorescence (S-DUV), we aim to correlate fibre autofluorescence with the environmental conditions of discovery (clothes of votive statuettes buried in a sanctuary or shrouds and bandages for mummification rituals found in tombs) or their intended purposes (fishing net, which underwent wet-dry cycles during its use). Out of the five archaeological objects studied, the two yarns sampled from the buried fabrics were found encrusted with minerals, but with significant differences in the fibres’ degradation. The yarn sampled from the fishing net displayed a great variability of the dataset, with some fibres biochemically similar to the buried samples while others were closer to the funerary linen shrouds. Lignin's predominant presence at 480 nm in the fishing net suggests extensive cellulose-enriched cell wall layer degradation while the lignin-enriched layer is still preserved. Additionally, second harmonic generation imaging microscopy (SHG) and 2-photon excitation fluorescence (2PEF) were used alongside S-DUV analyses to evaluate the flax fibres’ ultrastructure. SHG confirmed S-DUV findings and indicated poor crystalline cellulose preservation in one linen shroud, explaining the yarn's brittleness.

Persistent steatosis correlates with decreased fibrosis regression during anti-HBV treatment in patients with chronic HBV infection.

Despite the increasing prevalence of steatosis in patients with chronic hepatitis B (CHB), whether the changes in steatosis impact fibrosis regression during antiviral therapy remain unclear. We aimed to identify the association between histological changes of steatosis and fibrosis in patients undergone antiviral treatment. Patients with paired liver biopsies before and after 78 weeks of antiviral therapy were enrolled in this study. Liver fibrosis was assessed by the Ishak score combined with Beijing Classification predominantly progressive, indeterminate, and predominately regressive score. Steatosis was evaluated by the nonalcoholic fatty liver disease activity score. Collagen in each site was quantitated by second harmonic generation/two photon excitation fluorescence technology. Serum proteomic changes after treatment were characterized by mass-based spectrometry. A total of 239 CHB patients were included and divided into four groups according to the changes in steatosis: 162 (67.8%) had no steatosis throughout, 24 (10.0%) developed new-onset steatosis, 21 (8.8%) had initial steatosis which disappeared, and 32 (13.4%) had persistent steatosis. The persistent steatosis group showed the lowest rate of fibrosis regression (14/32, 43.8%). Persistent steatosis correlated with decreased fibrosis regression significantly after adjusting for age, sex, fibrosis stage, and metabolic factors at baseline, as well as the viral response (adjusted odds ratio = 0.380, 95% confidence interval 0.145-0.996, p = 0.049). This decreased fibrosis regression was associated with accumulated collagen in the perisinusoidal area. Patients with persistent steatosis showed unique changes in glycolipid metabolism according to the serum proteomic atlas. Persistent steatosis correlated with decreased fibrosis regression during antiviral therapy in patients with CHB.

Quantitative Assessment of Hepatic Steatosis Using Label-Free Multiphoton Imaging and Customized Image Processing Program

Nonalcoholic fatty liver disease is rapidly becoming one of the most common causes of chronic liver disease worldwide and is the leading cause of liver-related morbidity and mortality. A quantitative assessment of the degree of steatosis would be more advantageous for diagnostic evaluation and exploring the patterns of disease progression. Here, multiphoton microscopy, based on the second harmonic generation and 2-photon excited fluorescence, was used to label-free image the samples of nonalcoholic fatty liver. Imaging results confirm that multiphoton microscopy is capable of directly visualizing important pathologic features such as normal hepatocytes, hepatic steatosis, Mallory bodies, necrosis, inflammation, collagen deposition, microvessel, and so on and is a reliable auxiliary tool for the diagnosis of nonalcoholic fatty liver disease. Furthermore, we developed an image segmentation algorithm to simultaneously assess hepatic steatosis and fibrotic changes, and quantitative results reveal that there is a correlation between the degree of steatosis and collagen content. We also developed a feature extraction program to precisely display the spatial distribution of hepatocyte steatosis in tissues. These studies may be beneficial for a better clinical understanding of the process of steatosis as well as for exploring possible therapeutic targets.

From static to dynamic: live observation of the support system after ischemic stroke by two photon-excited fluorescence laser-scanning microscopy.

Ischemic stroke is one of the most common causes of mortality and disability worldwide. However, treatment efficacy and the progress of research remain unsatisfactory. As the critical support system and essential components in neurovascular units, glial cells and blood vessels (including the blood-brain barrier) together maintain an optimal microenvironment for neuronal function. They provide nutrients, regulate neuronal excitability, and prevent harmful substances from entering brain tissue. The highly dynamic networks of this support system play an essential role in ischemic stroke through processes including brain homeostasis, supporting neuronal function, and reacting to injuries. However, most studies have focused on postmortem animals, which inevitably lack critical information about the dynamic changes that occur after ischemic stroke. Therefore, a high-precision technique for research in living animals is urgently needed. Two-photon fluorescence laser-scanning microscopy is a powerful imaging technique that can facilitate live imaging at high spatiotemporal resolutions. Two-photon fluorescence laser-scanning microscopy can provide images of the whole-cortex vascular 3D structure, information on multicellular component interactions, and provide images of structure and function in the cranial window. This technique shifts the existing research paradigm from static to dynamic, from flat to stereoscopic, and from single-cell function to multicellular intercommunication, thus providing direct and reliable evidence to identify the pathophysiological mechanisms following ischemic stroke in an intact brain. In this review, we discuss exciting findings from research on the support system after ischemic stroke using two-photon fluorescence laser-scanning microscopy, highlighting the importance of dynamic observations of cellular behavior and interactions in the networks of the brain's support systems. We show the excellent application prospects and advantages of two-photon fluorescence laser-scanning microscopy and predict future research developments and directions in the study of ischemic stroke.

Prolonged Lifetimes of Histologic Autofluorescence in Ectopic Retinal Pigment Epithelium in Age-Related Macular Degeneration.

The purpose of this study was to investigate histologic autofluorescence lifetimes and spectra of retinal pigment epithelium (RPE) on the transition from normal aging to RPE activation and migration in age-related macular degeneration (AMD). Autofluorescence lifetimes and spectra of 9 donor eyes were analyzed in cryosections by means of 2-photon excited fluorescence at 960 nm. Spectra were detected at 483 to 665 nm. Lifetimes were measured using time-correlated single photon counting in 2 spectral channels: 500 to 550 nm (short-wavelength spectral channel [SSC]) and 550 to 700 nm (long-wavelength spectral channel [LSC]). Fluorescence decays over time were approximated by a series of three exponential functions. The amplitude-weighted mean fluorescence lifetime was determined. Markers for retinoid activity (RPE65) and immune function (CD68) were immunolocalized in selected neighboring sections. We identified 9 RPE morphology phenotypes resulting in 399 regions of interest (ROIs) for spectral and 497 ROIs for lifetime measurements. RPE dysmorphia results in a shorter wavelength peak of spectral emission: normal aging versus RPE migrated into the retina (intraELM) = 601.7 (9.5) nm versus 581.6 (7.3) nm, P < 0.001, whereas autofluorescence lifetimes increase: normal aging versus intraELM: SSC 180 (44) picosecond (ps) versus 320 (86) ps, P < 0.001; and LSC 250 (55) ps versus 441 (76) ps, P < 0.001. Ectopic RPE within the neurosensory retina is strongly CD68 positive and RPE65 negative. In the process of RPE degeneration, comprising different steps of dysmorphia and migration, lengthening of autofluorescence lifetimes and a hypsochromic shift of emission spectra can be observed. These autofluorescence changes might provide early biomarkers for AMD progression and contribute to our understanding of RPE-driven pathology.

Label-free second harmonic generation imaging of cerebral vascular wall in local ischemia mouse model in vivo

Second harmonic generation (SHG) imaging is label-free and non-invasive, and it has been extensively applied in multiple biological and medical studies, but not in the brain in vivo. In this study, we modified classical two photon excited fluorescence (TPEF) system to perform in vivo simultaneous TPEF and SHG imaging in the local ischemia mouse model. In cerebral vascular walls, we found strong SHG signal, which co-localized with collagen. In the continuous 2 days’ in vivo imaging, this SHG signal remained stable in the local ischemic blood vessel in the initial 4 h, then its signal abruptly increased and got spatially thickened 5 h after thrombosis, and this tendency continued in the following 48 h. This study provides direct and precise timeline of rapid collagen change in cerebral vascular walls in vivo, and reveals the subtle but significant temporal-spatial dynamics of this structural signal during local ischemia. Thus, this cerebral in vivo SHG imaging provides a powerful tool to identify the early and subtle pathological change of collagen around clinical key therapeutic time window.

Artificial Intelligence Improves Pathologist Agreement for Fibrosis Scores in Nonalcoholic Steatohepatitis Patients

Artificial Intelligence Improves Pathologist Agreement for Fibrosis Scores in Nonalcoholic Steatohepatitis Patients

Applications of triarylborane materials in cell imaging and sensing of bio-relevant molecules such as DNA, RNA, and proteins.

Triarylboranes have been known for more than 100 years and have found potential applications in various fields such as anion sensors and optoelectronics, for example in organic light emitting diodes (OLEDs), field effect transistors (OFETs), and organic photovoltaic devices. However, biological applications, such as bioimaging agents and biomolecule sensors have evolved much more recently. This review summarises progress in this relatively young field and highlights the potential of triarylboranes in biological applications.

Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy.

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells’ nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.

Crystal structure and optical property of a Cadmium(II) complex based on triphenylamine derivative—Theoretical and experimental investigation

A triphenylamine derivative (E)-N,N-diphenyl-4-(2-(pyridin-2-yl)vinyl)benzenamine (L) was synthesized, and the corresponding cadmium complex CdI2L2 crystal was obtained and solved by single crystal X-ray diffraction analysis. The experimental resutls of one photon absorption (OPA) and one photon excited fluorescence (OPEF) indicate that coordination of CdI2 only has slight effect on the spectrum peak position relative to the free ligand. Besides the Stokes shift, the full width half maximum of OPEF and the variation of the “peak span between solvents” from OPA to OPEF can also indicate the electron transition direction of the excitation process. The laser experiments show that both the ligand and complex have considerable two photon excited fluorescence (TPEF) emission. The experimental Stokes shift is correlated with the computational solvent polarity rMPI, and the results illustrate that the curve of “experimental Stokes shift vs. solvent rMPI” is exactly the Boltzmann S-curve. The computational results also indicate that the static interaction between the CdI2L2 molecules in crystal has strong effect on OPA properties. Considering the weak coordination bond strength of the complex CdI2L2, the S1→S0′ fluorescence emission may occur on both ligands, although the S0→S1′ excitation only occurs on the right ligand.

Femtosecond Lasers in Cornea & Refractive Surgery

Since the introduction of femtosecond laser (FS) systems for corneal flap creation in laser-assisted in-situ keratomileusis there have been numerous applications for FS laser in corneal surgery. This manuscript details the utility of FS lasers in corneal surgical procedures including refractive laser surgeries, intracorneal ring segment tunnels, presbyopic treatments, and FS-assisted keratoplasty. We also review the role of FS lasers in diagnostic procedures such as two photon excitation fluorescence and second harmonic generation.

In-vivo assessment of a tissue engineered vascular graft computationally optimized for target vessel compliance

Tissue engineered vascular grafts (TEVGs) have the ability to be tuned to match a target vessel’s compliance, diameter, wall thickness, and thereby prevent compliance mismatch. In this work, TEVG compliance was manipulated by computationally tuning its layered composition or by manipulating a crosslinking agent (genipin). In particular, these three acelluluar TEVGs were compared: a compliance matched graft (CMgel - high gelatin content); a hypocompliant PCL graft (HYPOpcl - high polycaprolactone content); and a hypocompliant genipin graft (HYPOgen - equivalent composition as CMgel but hypocompliant via increased genipin crosslinking). All constructs were implanted interpositionally into the abdominal aorta of 21 Sprague Dawley rats (n=7, males=11, females=10) for 28 days, imaged in-vivo using ultrasound, explanted, and assessed for remodeling using immunofluorescence and two photon excitation fluorescence imaging. Compliance matched grafts remained compliance-matched in-vivo compared to the hypocompliant grafts through 4 weeks (p<0.05). Construct degradation and cellular infiltration was increased in the CMgel and HYPOgen TEVGs. Contractile smooth muscle cell markers in the proximal anastomosis of the graft were increased in the CMgel group compared to the HYPOpcl (p=0.007) and HYPOgen grafts (p=0.04). Both hypocompliant grafts also had an increased pro-inflammatory response (increased ratio of CD163 to CD86 in the mid-axial location) compared to the CMgel group. Our results suggest that compliance matching using a computational optimization approach leads to the improved acute (28 day) remodeling of TEVGs. To the authors’ knowledge, this is the first in-vivo rat study investigating TEVGs that have been computationally optimized for target vessel compliance.

Depth-resolved Mueller matrix polarimetry microscopy of the rat cornea.

Mueller matrix polarimetry (MMP) is a promising linear imaging modality that can enable visualization and measurement of the polarization properties of the cornea. Although the distribution of corneal birefringence has been reported, depth resolved MMP imaging of the cornea has not been archived and remains challenging. In this work, we perform depth-resolved imaging of the cornea using an improved system that combines Mueller matrix reflectance and transmission microscopy together with nonlinear microscopy utilizing second harmonic generation (SHG) and two photon excitation fluorescence (TPEF). We show that TPEF can reveal corneal epithelial cellular network while SHG can highlight the presence of corneal stromal lamellae. We then demonstrate that, in confocal reflectance measurement, as depth increases from 0 to 80 μm both corneal depolarization and retardation increase. Furthermore, it is shown that the spatial distribution of corneal depolarization and retardation displays similar complexity in both reflectance (confocal and non-confocal) and transmission measurement, likely due to the strong degree of heterogeneity in the stromal lamellae.

Two photon excited fluorescence and hyper Rayleigh scattering of Protoporphyrin IX

The two-photon excited fluorescence (TPEF) cross-section of Protoporphyrin IX (PpIX) in solution is determined at different excitation wavelengths. Maxima observed at 620 and 634 nm are associated with two different forms of PpIX already observed in one-photon excited fluorescence studies. In order to further provide insights into these two forms, first hyperpolarizabilities are also reported for these two forms with corresponding depolarization ratio further demonstrating the existence of specific symmetry differences. The static and the frequency dependent first hyperpolarizability were also calculated using Gaussian 09 and compared to the experimental data. This work shows that PpIX can exhibit two forms that might be targeted in applications like oncology clinical agents.

Multiphoton Microscopic Study of the Renal Cell Carcinoma Pseudocapsule: Implications for Tumour Enucleation

OBJECTIVETo utilize Multiphoton Microscopy (MPM) as a novel imaging technique to characterize and quantify collagen at the Renal Cell Carcinoma Pseudocapsule, to assess for both intra-tumoral and inter-tumoral variation of collagen characteristics. MPM combines Second Harmonic Generation and Two Photon Excitation Fluorescence to image extracellular matrix architecture. METHODSTwenty partial nephrectomy specimen tissues were retrieved, cut into 5-micron sections, mounted on slides and deparaffinized. The pseudocapsules (PCs) were imaged with 2X and 20X objective at selected Regions of Interest. Corresponding clinical information was retrieved. PC thickness was determined. Collagen parameters measured included quantification by the Collagen Area Ratio, and qualitative measurements by the Collagen Fiber Density and Collagen Reticulation Index. RESULTSThe boundaries between tumor, PC and normal renal parenchyma were distinguished by MPM without need for staining. In the thickest areas of the PC, collagen content and density were quantitatively higher compared to the thinnest areas. Median Collagen Area Ratio was higher in the thickest compared to the thinnest areas of the PC (P = .01). Clear Cell RCC specimens had a consistently higher Collagen Fiber Density in both the thickest and thinnest areas compared to non-Clear Cell RCC specimens (P = .02). CONCLUSIONSWe demonstrated the ability of MPM to quantify collagen characteristics of PCs without fluorescent labeling. Tumor enucleation for Renal Cell Carcinoma along its PC remains debatable with regards to oncological safety. Even with a complete and intact PC, the PC is not a homogenous structure, and varies in its thickness and its collagen characteristics within, and between tumors.

In-Vivo Assessment of a Tissue Engineered Vascular Graft Computationally Optimized for Target Vessel Compliance

Compliance mismatch between a bypass graft and the host vasculature can lead to in-vivo graft failure. Specifically, hypocompliant (stiff) grafts are believed to provide a hemodynamic environment that promotes graft failure via thrombus formation and the development of intimal hyperplasia. Tissue engineered vascular grafts(TEVGs) offer an opportunity to control the compliance of a graft to match that of the native target vasculature and minimize these undesirable outcomes. Our group has recently developed a technique to computationally and experimentally optimize the compliance of a TEVG to a wide range of target values. In this work, acellular TEVGs were created using our validated computational optimization approach that were either compliance matched (CMgel- high gelatin content) or hypocompliant (HYPOpcl- high polycaprolactone content) relative to rat abdominal aorta.A third acellular hypocompliant graft (HYPOgen- increased genipin crosslinking) was also fabricated using the CMgel material composition but crosslinked with an increased genipin concentration such that it's compliance matched that of HYPOpcl grafts. All TEVGs were implanted interpositionally into the abdominal aorta of 21 Sprague Dawley rats(n=7 for each group, males=11, females=10) for 28 days, imaged in-vivo using ultrasound, explanted, and assessed for cellular/extracellular markers using immunofluorescence and two photon excitation fluorescence imaging. In-vivo ultrasound demonstrated that CMgel grafts remained compliance-matched while hypocompliant grafts remained stiff throughout the 4 week implantation period (p<0.05). Overall, the constructs with higher gelatin content (CMgel & HYPOgen) showed increased cellular infiltration and degradation compared to the HYPOpcl grafts. The compliance matched grafts showed an increase in the amount of contractile smooth muscle cell markers in the proximal portion of the graft compared to hypocompliant grafts (0.058±0.015 CMgel vs 0.033±0.009 HYPOpcl(p=0.007) & 0.022±0.018 HYPOgen, p=0.04). The middle section (along the axial length) of compliance matched grafts displayed an increase in the ratio of CD163 to CD68 macrophages (CMgel=1.83±1.82) compared to the HYPOpcl grafts (0.13±0.06, p=0.03)suggesting a reduced pro-inflammatory response in these optimized grafts. Our results suggest that computational optimization may improve the acute (28 day) in-vivo remodeling of a biopolymer TEVG. To the authors' knowledge,this is the first in-vivo rat study comparing electrospun acellular grafts that have been computationally optimized for target levels of compliance.

Two Photon Excited Fluorescence Microscopy Metrics are Sensitive to Early Phenotypic Changes in Calcific Aortic Valve Disease In Vitro and Ex Vivo

Two Photon Excited Fluorescence Microscopy Metrics are Sensitive to Early Phenotypic Changes in Calcific Aortic Valve Disease In Vitro and Ex Vivo

New advances in biomedical applications of multiphoton imaging technology

In contrast to single photon excitation fluorescence imaging, laser scanning confocal imaging, and wide-field imaging, the multi-photon imaging has advantages of minimal invasion and deeper penetration by using near-infrared (NIR) laser source. Moreover, it can carry out three-dimensional high-spatial-resolution imaging of biological tissues due to its natural optical tomography capability. Since its advent, multi-photon imaging has become a powerful tool in biomedicine and achieved a series of significant discoveries in cancer pathology, neurological diseases and brain functional imaging. In the past decade, as a major form of multi-photon imaging techonoogy, two-photon excited fluorescence microscopy imaging has a great potential in biomedical applications. In order to satisfy the practical biomedical applications, multi-photon imaging technologies have made significant breakthroughs in improving the deficiencies of traditional 2PEF in multi-color imaging, functional imaging, live imaging and imaging depth, such as multicolor two-photon excitation fluorescence microscopy, two-photon fluorescence lifetime imaging microscopy, two-photon fiber endoscopic imaging, and three-photon microscopy imaging technology. For example, multicolor two-photon excitation fluorescence microscopy is demonstrated to achieve simultaneous imaging of multiple fluorophores with multiple wavelenth excitation lasers or continuous spectrum. In addition, the two-photon fluorescence lifetime microscopic imaging provides a method to achieve high-resolution three-dimensional imaging of biological tissue with multi-dimensional information including fluorescence intensity and lifetime. In addition, two-photon optical fiber endoscopic imaging with small system size and mimal invasion is developed and used to image the tissue inside the deep organ. Finally, two-photon excitation fluorescence microscopy technique still has relatively strong scattering for brain functional imaging in vivo. Therefore, the imaging depth is limited by the signal-to-background ratio. Three-photon microscopic imaging technique can achieve higher imaging depth and a desired signal-to-noise ratio by extending the wavelength from 1600 nm to 1820 nm because the attenuation of the excitation light in this wavelenth range is much smaller. In this article, we briefly introduce the principles and applications of these multi-photon imaging technologies, and finally provide our view for their future development.

Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques

Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive ‘fit-free’ approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and longer spectral emission (580–625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to phasors with longer lifetimes and shorter spectral emission (550–585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells.