“…1A) and HWN ( Fig. 1B) regions that is typical for skin and biological tissues 23 . Figure 1C,D demonstrate typical examples of the fluorescence background subtraction in the FP (Fig.…”
Section: Resultsmentioning
confidence: 93%
“…1 A) and HWN (Fig. 1 B) regions that is typical for skin and biological tissues 23 . Figure 1 Characteristic depth-resolved Raman spectra of skin in vivo and examples of fluorescence background subtraction.…”
Section: Resultsmentioning
confidence: 93%
“…Moreover, melanin fluoresces upon two-photon excitation, allowing for its in vivo assessment and 3D imaging 21 , 22 . However, the fluorescence intensity itself does not provide specific information on the melanin type and its environment 23 , 24 . Additional contrasting of melanin can be achieved using fluorescence lifetime imaging microscopy (FLIM), as melanin exhibits a distinctive fast (< 0.2 ns) fluorescence decay 25 – 27 .…”
the fate of melanin in the epidermis is of great interest due to its involvement in numerous physiological and pathological processes in the skin. Melanin localization can be assessed ex vivo and in vivo using its distinctive optical properties. Melanin exhibits a characteristic Raman spectrum band shape and discernible near-infrared excited (NIR) fluorescence. However, a detailed analysis of the capabilities of depth-resolved confocal Raman and fluorescence microspectroscopy in the evaluation of melanin distribution in the human skin is lacking. Here we demonstrate how the fraction of melanin at different depths in the human skin in vivo can be estimated from its Raman spectra (bands at 1,380 and 1,570 cm −1) using several procedures including a simple ratiometric approach, spectral decomposition and non-negative matrix factorization. The depth profiles of matrix factorization components specific to melanin, collagen and natural moisturizing factor provide information about their localization in the skin. The depth profile of the collagen-related matrix factorization component allows for precise determination of the dermal-epidermal junction, i.e. the epidermal thickness. Spectral features of fluorescence background originating from melanin were found to correlate with relative intensities of the melanin Raman bands. We also hypothesized that NIR fluorescence in the skin is not originated solely from melanin, and the possible impact of oxidized species should be taken into account. The ratio of melanin-related Raman bands at 1,380 and 1,570 cm −1 could be related to melanin molecular organization. the proposed combined analysis of the Raman scattering signal and NIR fluorescence could be a useful tool for rapid non-invasive in vivo diagnostics of melanin-related processes in the human skin.
“…1A) and HWN ( Fig. 1B) regions that is typical for skin and biological tissues 23 . Figure 1C,D demonstrate typical examples of the fluorescence background subtraction in the FP (Fig.…”
Section: Resultsmentioning
confidence: 93%
“…1 A) and HWN (Fig. 1 B) regions that is typical for skin and biological tissues 23 . Figure 1 Characteristic depth-resolved Raman spectra of skin in vivo and examples of fluorescence background subtraction.…”
Section: Resultsmentioning
confidence: 93%
“…Moreover, melanin fluoresces upon two-photon excitation, allowing for its in vivo assessment and 3D imaging 21 , 22 . However, the fluorescence intensity itself does not provide specific information on the melanin type and its environment 23 , 24 . Additional contrasting of melanin can be achieved using fluorescence lifetime imaging microscopy (FLIM), as melanin exhibits a distinctive fast (< 0.2 ns) fluorescence decay 25 – 27 .…”
the fate of melanin in the epidermis is of great interest due to its involvement in numerous physiological and pathological processes in the skin. Melanin localization can be assessed ex vivo and in vivo using its distinctive optical properties. Melanin exhibits a characteristic Raman spectrum band shape and discernible near-infrared excited (NIR) fluorescence. However, a detailed analysis of the capabilities of depth-resolved confocal Raman and fluorescence microspectroscopy in the evaluation of melanin distribution in the human skin is lacking. Here we demonstrate how the fraction of melanin at different depths in the human skin in vivo can be estimated from its Raman spectra (bands at 1,380 and 1,570 cm −1) using several procedures including a simple ratiometric approach, spectral decomposition and non-negative matrix factorization. The depth profiles of matrix factorization components specific to melanin, collagen and natural moisturizing factor provide information about their localization in the skin. The depth profile of the collagen-related matrix factorization component allows for precise determination of the dermal-epidermal junction, i.e. the epidermal thickness. Spectral features of fluorescence background originating from melanin were found to correlate with relative intensities of the melanin Raman bands. We also hypothesized that NIR fluorescence in the skin is not originated solely from melanin, and the possible impact of oxidized species should be taken into account. The ratio of melanin-related Raman bands at 1,380 and 1,570 cm −1 could be related to melanin molecular organization. the proposed combined analysis of the Raman scattering signal and NIR fluorescence could be a useful tool for rapid non-invasive in vivo diagnostics of melanin-related processes in the human skin.
“…For different cell types, as well as for changes in cell metabolism or activation redistribution between NAD(P)H forms and its binding to different enzymes may cause pronounced changes in TPE-FLIM signatures, which enables the separation between cells at different stages of differentiation, normal and cancer cells, cells with oxidative stress, etc. 39 , 51 – 54 . In fact, it is possible that NAD(P)H fluorescence decay parameters could be characteristic for different cell types.…”
Section: Discussionmentioning
confidence: 99%
“…The intrinsic fluorescence signal of the dermis originates from all dermal components and, therefore, the component-specific fluorescence is superimposed, making the fluorescence-based separation of certain molecules challenging 38 , 39 . Recently presented multi-channel two-photon/SHG (second harmonic generation)/three-photon/THG (third harmonic generation) in vivo rat tissue imaging 40 does not separate between cell types.…”
Mast cells (MCs) are multifunctional cells of the immune system and are found in skin and all major tissues of the body. They contribute to the pathology of several diseases including urticaria, psoriasis, atopic dermatitis and mastocytosis where they are increased at lesional sites. Histomorphometric analysis of skin biopsies serves as a routine method for the assessment of MC numbers and their activation status, which comes with major limitations. As of now, non-invasive techniques to study MCs in vivo are not available. Here, we describe a label-free imaging technique to visualize MCs and their activation status in the human papillary dermis in vivo. This technique uses two-photon excited fluorescence lifetime imaging (TPE-FLIM) signatures, which are different for MCs and other dermal components. TPE-FLIM allows for the visualization and quantification of dermal MCs in healthy subjects and patients with skin diseases. Moreover, TPE-FLIM can differentiate between two MC populations in the papillary dermis in vivo—resting and activated MCs with a sensitivity of 0.81 and 0.87 and a specificity of 0.85 and 0.84, respectively. Results obtained on healthy volunteers and allergy and mastocytosis patients indicate the existence of other MC subpopulations within known resting and activated MC populations. The developed method may become an important tool for non-invasive in vivo diagnostics and therapy control in dermatology and immunology, which will help to better understand pathomechanisms involving MC accumulation, activation and degranulation and to characterize the effects of therapies that target MCs.
Fibroblasts are among the most common cell types in the stroma responsible for creating and maintaining the structural organization of the extracellular matrix (ECM) in the dermis, skin regeneration, and a range of immune responses. Until now, the processes of fibroblast adaptation and functioning in a varying environment have not been fully understood. Modern laser microscopes are capable of studying fibroblasts in vitro and ex vivo. One‐photon‐ and two‐photon‐excited fluorescence microscopy, Raman spectroscopy/micro‐spectroscopy are well‐suited non‐invasive optical methods for fibroblast imaging in vitro and ex vivo. In vivo staining‐free fibroblast imaging is not still implemented. The exception is fibroblast imaging in tattooed skin. Although in vivo non‐invasive staining‐free imaging of fibroblasts in the skin has not yet been implemented, it is expected in the future. This review summarizes the state of the art in fibroblast visualization using optical methods and discusses the advantages, limitations, and prospects for future non‐invasive imaging.This article is protected by copyright. All rights reserved.
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