Two-Photon-Excited FLIM of NAD(P)H and FAD-Metabolic Activity of Fibroblasts for the Diagnostics of Osteoimplant Survival.

Bioinert materials such as the zirconium dioxide and aluminum oxide are widely used in surgery and dentistry due to the absence of cytotoxicity of the materials in relation to the surrounding cells of the body. However, little attention has been paid to the study of metabolic processes occurring at the implant-cell interface. The metabolic activity of mouse 3T3 fibroblasts incubated on yttrium-stabilized zirconium ceramics cured with aluminum oxide (ATZ) and stabilized zirconium ceramics (Y-TZP) was analyzed based on the ratio of the free/bound forms of cofactors NAD(P)H and FAD obtained using two-photon microscopy. The results show that fibroblasts incubated on ceramics demonstrate a shift towards the free form of NAD(P)H, which is observed during the glycolysis process, which, according to our assumptions, is related to the porosity of the surface of ceramic structures. Consequently, despite the high viability and good proliferation of fibroblasts assessed using an MTT test and a scanning electron microscope, the cells are in a state of hypoxia during incubation on ceramic structures. The FLIM results obtained in this work can be used as additional information for scientists who are interested in manufacturing osteoimplants.

Review of optical methods for noninvasive imaging of skin fibroblasts-From in vitro to ex vivo and in vivo visualization.

Fibroblasts are among the most common cell types in the stroma responsible for creating and maintaining the structural organization of the extracellular matrix 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/microspectroscopy are well-suited noninvasive 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 noninvasive 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 noninvasive imaging.

Measurement and Modeling of the Optical Properties of Adipose Tissue in the Terahertz Range: Aspects of Disease Diagnosis.

In this paper, the measurement and modeling of optical properties in the terahertz (THz) range of adipose tissue and its components with temperature changes were performed. Spectral measurements were made in the frequency range 0.25-1 THz. The structural models of main triglycerides of fatty acids are constructed using the B3LYP/6-31G(d) method and the Gaussian03, Revision B.03 program. The optical density (OD) of adipose tissue samples decreases as temperature increases, which can be associated mostly with the dehydration of the sample. Some inclusion of THz wave scattering suppression into the OD decrease can also be expected due to refractive index matching provided by free fatty acids released from adipocytes at thermally induced cell lipolysis. It was shown that the difference between the THz absorption spectra of water and fat makes it possible to estimate the water content in adipose tissue. The proposed model was verified on the basis of molecular modeling and a comparison with experimental data for terahertz spectra of adipose tissue during its heating. Knowing the exact percentage of free and bound water in adipose tissue can help diagnose and monitor diseases, such as diabetes, obesity, and cancer.

In Vivo Quantification of the Effectiveness of Topical Low-Dose Photodynamic Therapy in Wound Healing Using Two-Photon Microscopy.

The effect of low-dose photodynamic therapy on in vivo wound healing with topical application of 5-aminolevulinic acid and methylene blue was investigated using an animal model for two laser radiation doses (1 and 4 J/cm2). A second-harmonic-generation-to-auto-fluorescence aging index of the dermis (SAAID) was analyzed by two-photon microscopy. SAAID measured at 60-80 µm depths was shown to be a suitable quantitative parameter to monitor wound healing. A comparison of SAAID in healthy and wound tissues during phototherapy showed that both light doses were effective for wound healing; however, healing was better at a dose of 4 J/cm2.

The In Vivo Quantitative Assessment of the Effectiveness of Low-Dose Photodynamic Therapy on Wound Healing Using Optical Coherence Tomography.

The effect of low-dose photodynamic therapy on in vivo wound healing was investigated using optical coherence tomography. This work aims to develop an approach to quantitative assessment of the wound's state during wound healing including the effect of low-dose photodynamic therapy using topical application of two different photosensitizers, 5-aminolevulinic acid and methylene blue, and two laser doses of 1 J/cm2 and 4 J/cm2. It was concluded that the laser dose of 4 J/cm2 was better compared to 1 J/cm2 and allowed the wound healing process to accelerate.

In vivo non-invasive staining-free visualization of dermal mast cells in healthy, allergy and mastocytosis humans using two-photon fluorescence lifetime imaging.

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.

Application of multiphoton imaging and machine learning to lymphedema tissue analysis.

The results of in-vivo two-photon imaging of lymphedema tissue are presented. The study involved 36 image samples from II stage lymphedema patients and 42 image samples from healthy volunteers. The papillary layer of the skin with a penetration depth of about 100 µm was examined. Both the collagen network disorganization and increase of the collagen/elastin ratio in lymphedema tissue, characterizing the severity of fibrosis, was observed. Various methods of image characterization, including edge detectors, a histogram of oriented gradients method, and a predictive model for diagnosis using machine learning, were used. The classification by "ensemble learning" provided 96% accuracy in validating the data from the testing set.