Melanin diagnostics with nonlinear optics: a mini-review

Optical methods are widely used to perform fundamental studies of living systems and solve problems of biomedical diagnostics. Along with the classical spectroscopy, methods of nonlinear optics (e.g., multiphoton microscopy) are also applied in biophotonics. The potential of nonlinear optical methods for visualisation and analysis of the properties of endogenous chromophore molecules are considered in this minireview. Melanin - a pigment with specific spectral features of photophysical properties in the visible and near-IR ranges - is taken as an example. It is discussed what information about its localisation in tissues and structural organisation can be obtained by nonlinear optical methods: multiphoton fluorescence microscopy (including fluorescence lifetime imaging), third harmonic generation, pump - probe spectroscopy, and coherent anti- Stokes Raman spectroscopy.

Elastic Laminar Reorganization Occurs with Outward Diameter Oxidase for Stabilization

When a large artery becomes occluded, hemodynamic changes stimulate remodeling of arterial networks to form collateral arteries in a process termed arteriogenesis. However, the structural changes necessary for collateral remodeling have not been defined. We hypothesize that deconstruction of the extracellular matrix is essential to remodel smaller arteries into effective collaterals. Using multiphoton microscopy, we analyzed collagen and elastin structure in maturing collateral arteries isolated from ischemic rat hindlimbs. Collateral arteries harvested at different timepoints showed progressive diameter expansion associated with striking rearrangement of internal elastic lamina (IEL) into a loose fibrous mesh, a pattern persisting at 8 weeks. Despite a 2.5-fold increase in luminal diameter, total elastin content remained unchanged in collaterals compared with control arteries. Among the collateral midzones, baseline elastic fiber content was low. Outward remodeling of these vessels with a 10–20 fold diameter increase was associated with fractures of the elastic fibers and evidence of increased wall tension, as demonstrated by the straightening of the adventitial collagen. Inhibition of lysyl oxidase (LOX) function with β-aminopropionitrile resulted in severe fragmentation or complete loss of continuity of the IEL in developing collaterals. Collateral artery development is associated with permanent redistribution of existing elastic fibers to accommodate diameter growth. We found no evidence of new elastic fiber formation. Stabilization of the arterial wall during outward remodeling is necessary and dependent on LOX activity.

Digital Image Analysis Tools Developed by the Indiana O’Brien Center

The scale and complexity of images collected in biological microscopy have grown enormously over the past 30 years. The development and commercialization of multiphoton microscopy has promoted a renaissance of intravital microscopy, providing a window into cell biology in vivo. New methods of optical sectioning and tissue clearing now enable biologists to characterize entire organs at subcellular resolution. New methods of multiplexed imaging support simultaneous localization of forty or more probes at a time. Exploiting these exciting new techniques has increasingly required biomedical researchers to master procedures of image analysis that were once the specialized province of imaging experts. A primary goal of the Indiana O’Brien Center has been to develop robust and accessible image analysis tools for biomedical researchers. Here we describe biomedical image analysis software developed by the Indiana O’Brien Center over the past 25 years.

Improving Multiphoton Microscopy by Combining Spherical Aberration Patterns and Variable Axicons

Multiphoton (MP) microscopy is a well-established method for the non-invasive imaging of biological tissues. However, its optical sectioning capabilities are reduced due to specimen-induced aberrations. Both the manipulation of spherical aberration (SA) and the use of axicons have been reported to be useful techniques to bypass this limitation. We propose the combination of SA patterns and variable axicons to further improve the quality of MP microscopy images. This approach provides enhanced images at different depth locations whose quality is better than those corresponding to the use of SA or axicons separately. Thus, the procedure proposed herein facilitates the visualization of details and increases the depth observable at high resolution.

Whole-brain optical access in small adult vertebrates with two- and three-photon microscopy

Although optical microscopy has allowed us to study the entire brain in early developmental stages, access to the brains of live, adult vertebrates has been limited. Danionella, a genus of miniature, transparent fish closely related to zebrafish has been introduced as a neuroscience model to study the entire adult vertebrate brain. However, the extent of optically accessible depth in these animals has not been quantitatively characterized. Here, we show that two- and three-photon microscopy can be used to access the entire depth of the adult wild type Danionella dracula brain without any modifications to the animal other than mechanical stabilization. Three-photon microscopy provides high signal to background ratio and optical sectioning through the deepest part of the brain. While vasculature can be observed with two-photon microscopy, the deeper regions have low contrast. We show that multiphoton microscopy is ideal for readily penetrating the entire adult brain within the geometry of these animals' head structures and without the need for pigment removal. With multiphoton microscopy enabling optical access to the entire adult brain and a repertoire of methods that allow observation of the larval brain, Danionella provides a model system for readily studying the entire brain over the lifetime of a vertebrate.

Multimodal Analyses of Stable Vitiligo Skin Identify Tissue Interactions That Control Disease Persistence

Vitiligo is an autoimmune skin disease that is characterized by the progressive destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells but why white patches in stable disease persist is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. Here, we combine non-invasive multiphoton microscopy (MPM) imaging and single-cell RNA sequencing (scRNA-seq) to identify distinct subpopulations of keratinocytes in lesional skin of stable vitiligo patients. We show that these keratinocytes are enriched in lesional vitiligo skin and differ in metabolism, an observation corroborated by both MPM and scRNA-seq. Systematic investigation of cell-cell communication show that CXCL is the prominent signaling change in this small population of keratinocytes, which secrete CXCL9 and CXCL10 to create local inflammatory cytokine loops with T cells to drive stable vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative keratinocyte differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cellcell communication networks and keratinocyte cell states that perpetuate inflammation and prevent repigmentation.One Sentence SummaryCommunication between keratinocytes, immune cells, and melanocytes maintain depigmented patches in stable vitiligo.