Using Becker & Hickl TCSPC FLIM systems with fast TCSPC modules and fast detectors, we found ultra-fast fluorescence decay components in biological material, such as mushroom spores, pollen grains, plant tissue, and malignant melanoma. The component decay times are on the order of 10 to 50 ps, the amplitudes range from about 60% to more than 99%. In general, the shortest lifetimes and the largest amplitudes of the fast component were found in strongly coloured material, such as black mushroom spores and melanoma tissue. However, the lifetime not always correlates with the colour. In particular, a fast decay component was not found in tissue from basal cell papilloma, although it is dark brown. This may open a way to identify melanoma tissue and melanoma cells, and thus provide a new tool to investigate melanoma progression.
Lichen sclerosus (LS) is the most common vulvar dermatosis, which is characterized by damage to the connective tissue of the dermis. The affected area in lichen sclerosus is characterized by a sequential change in the main components of the connective tissue - collagen and elastin fibers. The affected area is polymorphic and remains poorly defined from a histological point of view. Among histopathologists, there are no unequivocal opinions on changes in the connective tissue of the dermis in LS. However, an assessment of the degree of dermis damage is important for the timely diagnosis of the condition and adequate treatment. Nonlinear microscopy includes second-harmonic generation (SHG) and twophoton autofluorescence (TPEF). SHG allows to selectively examine the signal from heterotypic collagen fibers of the dermis that contain type 1 collagen. TPEF allows to identify elastic fibers of the connective tissue matrix. It has been demonstrated that nonlinear microscopy allows visualizing the changes in the microstructure of collagen and elastin fibers. Three histological patterns were revealed as a result of the analysis of the nonlinear optical microscopy of the classical VLS. These histological patterns cannot be distinguished using histological stains and indicate a polymorphism of connective tissue changes. Nonlinear microscopy makes it possible to assess the changes in tissue structure, which is important for the histological interpretation of changes in the dermis and to clarify histological classification system in the future.
To develop new methods for the diagnosis and treatment of such a widespread disease as steatosis, there is still a lack of fundamental biological knowledge about various aspects of the functioning of the liver tissue at the cellular level. In our work, we assessed the metabolic state of hepatocytes, as well as the collagen content in the liver tissue with induced steatosis using the modern label-free minimally invasive methods of multiphoton laser scanning microscopy with TPEF mode (Two-Photon Excited Fluorescence) and SHG mode (Second Harmonic Generation), equipped with FLIM (Fluorescence-Lifetime Imaging Microscopy). Using multiphoton microscopy, it was shown that during the development of steatosis, it is possible to identify areas with a reduced NAD(P)H autofluorescence signal in damaged hepatocytes. Using SHG we showed a gradual accumulation of collagen in the liver tissue with induced steatosis, however, extensive areas of fibrosis were not detected even at the advanced stages of the pathology. Using FLIM, we studied the specific features of the energy metabolism of hepatocytes based on data on the lifetimes of various forms of NAD(P)H and their relative contributions. It has been revealed that there is a gradual decrease in the intensity of oxidative phosphorylation, accompanied by the rise in the intensity of lipogenesis in the liver tissue with induced steatosis. Such results are consistent with the data of histological analysis. The results obtained in this work can be useful for developing new criteria for express intraoperative assessment of liver pathology at the cellular level in the clinic.
One of the promising approaches to the treatment of injured tissue is the application of bioengineering techniques based on the introduction of cells into the damage area. An application of cells alone does not provide a complete replacement of the tissue defect. Therefore, scaffolds are used allowing organization of the cells into a structure, which is capable of the total reproduction of the damaged tissue integrity. Great number of factors, which influence the cell behavior and tissue formation at the injury site when using scaffolds are known. Here, we analyzed the effect of structural heterogeneity of scaffolds on cellular behavior and metabolism. All scaffolds were obtained by two-photon polymerization technic. It was found that colonization of heterogeneous scaffolds was insignificant less than homogeneous ones. However, there were not dead cells on heterogeneous matrix. We found that the level of free and bound NAD(P)H for the cells on the heterogeneous and homogeneous scaffolds was differ. This can indicate a different contribution of glycolysis and oxidative phosphorylation intensity in stem cells seeded on two types of scaffolds.
KEYWORDS: Fluorescence lifetime imaging, Oxygen, 3D modeling, Luminescence, Biological research, 3D displays, Tissues, Stem cells, Microscopy, Medical research
3D models based on cells differentiated from patient-specific induced pluripotent stem cells (iPSCs) are widely used to identify disease phenotypes, to accurately analyze dysfunctions at the level of human tissues and organs, to screen new drugs, which makes them more promising tool for biomedical research tasks than monolayer cultures, which is associated with their proximity to in vivo. The metabolic activity with oxygenation level of cells, assessed by optical imaging methods, can be used as markers of cell viability, proliferative activity and the degree of differentiation in 3D culture conditions. In this paper we used fluorescence and phosphorescence lifetime imaging microscopies (FLIM and PLIM) to study the metabolic status and the oxygenation level of derived from iPSCs neural stem cells (NSC) cultured in 3D condition. An analysis of the fluorescence intensities and FLIM data showed that NSCs in monolayer and at the periphery of large spheroids have more glycolytic phenotypes, NSCs in the center of large spheroids and NSCs grouped into small spheroids have more oxidative state. For determination of the relative oxygen level in spheroids PLIM of BPTDM stained neurospheres was carried out. As it was supposed, oxygen transport in the spheroid depended on it size. In neurospheres with an average size 600 μm O2 distribution is radial, with the lowest concentration in the center. Thus, the metabolic status and oxygenation level of the NSC in the spheroid composition was assessed in a life-time and noninvasive manner.
Breast cancer is a genetically heterogeneous disease characterized by various biomolecular and morphological features that affect the diagnosis, prognosis, and treatment response. In this study we combined cross-polarization optical coherence tomography (CP OCT) and multiphoton tomography (MPT), based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) to visualize tumor stroma and tumor cells in specimens of a human breast tissue. The data obtained by both techniques were compared with histopathology. The CP OCT and MPT images were quantitatively assessed to distinguish a breast normal tissue from a cancer as well as between a low and a high grade of cancer. Quantitative assessment of the CP OCT image included the calculation of signal attenuation coefficients separately for co- and cross- polarization channels and the formation the color-coded en-face distribution maps of these coefficients. The attenuation coefficient in cross- polarization channel showed better difference between breast cancer of low and high grades and distinguish them from normal tissue. The SHG images were processed using texture analysis techniques to quantify the density of collagen fibers in normal tissue and tumor. Thus, both imaging techniques have great potential to distinguish nontumorous and tumorous human breast tissue of varying degrees of malignancy and could provide identifying breast cancer margins for in surgery.
The changes in cell metabolism can affect the epigenome-modifying enzymes activity during iPSCs differentiation and thus control the functional potential of the final cell. Therefore, for therapeutic applications, the restoration of a fully functional mitochondrial network specific for the cell types derived from iPSCs will be required to support the energy and other mitochondrial factors. Recently, FLIM method allows to study the metabolic changes that accompanying cell differentiation noninvasively and without additional labels. In this study, we investigated the metabolic changes in iPSCs during neural differentiation using two-photon fluorescence microscopy and FLIM. Cellular metabolism was examined by monitoring the optical redox ratio (FAD/NAD(P)H), the fluorescence lifetime contributions of the free and bound forms of NADH and NADPH. Given that neural differentiation is also accompanied by synthetic processes and oxidative stress, this process was included in the scope of this work. We demonstrated an increased contribution of protein-bound NADH and NADPH in neuron associated with metabolic switch to oxidative phosphorylation and the biosynthetic processes or oxidative stress, respectively. We also found that the optical redox ratio FAD/NAD(P)H decreased during neural differentiation, and this was likely to be explained by the intensive lipid membrane synthesis or ROS generating and the enhanced NADPH production associated with them. The biochemical analysis was carried out to verify the metabolic status of iPSCs and their neural derivatives. Based on the data on glucose consumption, lactate and ATP amount we registered the trend to the metabolic pathways redistribution towards the oxidative phosphorylation in neuron.
Early diagnosis of melanoma is an ongoing challenge in dermatology and oncology. There is a special subgroup of melanocytic lesions that can be clinically and dermoscopically indistinguishable from early melanoma. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of dermoscopic equivocal melanocytic lesions based on combination of multiphoton tomography (MPT) and optical coherence angiography (OCA). A multiphoton optical score (MPOS) for quantitative assessment of the melanoma features revealed by MPM was developed. OCA images were processed to calculate the vessel densities and the total lengths thin and thick vessels. Histopathological analysis separated the equivocal lesions into benign, melanoma in situ, and invasive melanoma. The MPOS value of benign lesions was significant lower than for the malignant ones. Quantitative analysis of OCA images revealed that the invasive melanoma type has the highest vessel density. The combined use of multiphoton tomography with MPOS calculation and quantification of optical coherence angiography data demonstrated a potential to discriminate all dermoscopic equivocal melanocytic lesions in vivo.
The differentiation of endothelial cells from human iPSC has incontestable advantages in diseases research and therapeutic applications. However, the safe use of iPSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Shifts in cellular metabolism associated with intracellular pH changes affect the enzymes that control epigenetic configuration, which impact chromatin reorganization and gene expression changes during reprogramming and differentiation. FLIM-based metabolic imaging of NADH and FAD is a powerful tool for measuring mitochondrial metabolic state and widely used diagnostic method for identification of neoplastic diseases, skin diseases, ocular pathologies and stem cells differentiation. Therefore, in this study, we used the potential of FLIM-based metabolic imaging and fluorescence microscopy of NADH and FAD to study the metabolic changes during iPSC differentiation in endothelial cells. The evaluation of the intracellular pH was carried out with the fluorescent pH-sensor SypHer-2 and fluorescence microscopy to obtain complete information about metabolic status of iPSC and their endothelial derivatives. Based on the FAD/NAD(P)H optical redox ratios increase and the contributions rise of the NAD(P)H fluorescence lifetime in iPSC during endothelial differentiation, we demonstrated an contribution increase of OXPHOS to cellular metabolism. Based on the shift toward more acidic intracellular pH in endothelial cell derived from iPSCs we verified their oxidative state.
Raster-scan optoacoustic angiography at 532 nm wavelength with 50 μm lateral resolution at 2 mm diagnostic depth was used for quantitative characterization of neoangiogenesis in colon cancer models. Vessels of subcutaneously growing murine colon carcinoma (СT26) was imaged from 5th to 13th day of growth. The values of vascular density were calculated from the optoacoustic data. Inhomogeneous distribution of areas with high and low vascularization was demonstrated in the tumors. During tumor development vessel growth from the periphery to the center of the tumor was shown. Increase of vascular density precedes the increase of tumor volume. The obtained results may be important for the investigation of tumor development and for improvement of cancer treatment strategies.
In this study we combined cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), based on second harmonic generation, and two-photon-excited fluorescence to visualize collagen fibers and tumor cells in the various morphological subtypes of breast cancer. The ability of CP OCT to visualize tissue birefringence and cross-scattering adds new information about the microstructure of such breast cancers, while the MPT provides verification of this microstructure. Mammary glands, both normal and tumorous, were assessed by MPT and CP OCT to establish the relationships between spatial organization features of the cellular component and the intercellular matrix. It was shown, that such multimodal optical imaging has great potential for distinguishing various breast cancer morphological subtypes and could provide useful tools for identifying positive breast cancer margins for surgery.
The clinical diagnosis of melanocytic lesions is an ongoing medical challenge. Non-invasive tools and technologies can help to distinguish equivocal lesions. The aim of the study was to improve the in vivo diagnostic possibilities for the differentiation of benign and malignant melanocytic lesions based on combination of three imaging label-free modalities (multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography). Thirty-two melanocytic lesions were studied, using multiphoton tomography, fluorescence lifetime imaging and optical coherence angiography. Multiphoton tomography features of benign melanocytic nevi were characterized by the normal morphology of both the keratinocytes and the nevus cell nests surrounded by collagen. Dysplastic nevi were characterized by their increased intercellular distances and enlarged cell nuclei. Melanomas showed the presence of melanocytes and dendritic structures in all layers of the epidermis. Analysis of the metabolic state revealed that melanomas and dysplastic nevi were characterized by enhanced glycolysis. Optical coherence angiography shows that benign nevi had regular vascular networks and equal numbers of thin and thick vessels. Vascular networks of dysplastic nevi were characterized thin curved vessels. Thick irregular spiral vessels formed a dense microvascular network of melanomas.
The main purpose of this work is to evaluate the possibility to distinguish in vivo benign papilloma, severe dysplasia and squamous cell carcinoma by establishing quantitative image characteristics of multiphoton tomography (MPT) and multimodal optical coherence tomography images (MM OCT). Specific features of papillomatous outgrowths at different stages were revealed using 7,12-dimethylbenz[a]anthracen (DMBA)-induced hamster oral carcinoma. Analysis of MPT images included assessment of nuclear-cytoplasmic (NC) ratio, nuclear density and heterogeneity parameter F. Crosspolarization OCT images were quantified via the integral depolarization factor (IDF). Analysis of OCT microvascular maps enabled differential analysis based on the number of smallest-diameter blood vessels present in a particular pathology. Both MPT and MM OCT metrics showed some difference between benign papilloma, dysplastic papilloma, and squamous cell carcinoma tissue states. The results suggested that combined use of MPT and MM OCT have great potential for in vivo differentiation between benign and malignant papillomas.
Nonlinear optical microscopy combined with fluorescence lifetime imaging is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a noninvasive investigation of the biological tissue with subcellular resolution. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. In this study features of tumor metabolism in different systems of organization (from cell culture to patient lesion) was showed. The observed differences in the relative contributions of free NAD(P)H and FAD testify to an increased a glycolytic metabolism in cancer cells compare to fibroblasts. In 3D spheroids, the cells of the proliferating zone had greater a1 and lower tm values than the cells of the quiescent zone, which likely is a consequence of their higher glycolytic rate. During the growth of colorectal cancer in the experimental mouse model, the contribution of the free component of NAD(P)H was increased. Dysplastic nevus and melanoma is characterized by raised contribution of free NADH compare to healthy skin. Therefore, melanoma cells had very short value of τ1.
In the case of infiltrative brain tumors the surgeon faces difficulties in determining their boundaries to achieve total resection. The aim of the investigation was to evaluate the performance of multimodal OCT (MM OCT) for differential diagnostics of normal brain tissue and glioma using an experimental model of glioblastoma. The spectral domain OCT device that was used for the study provides simultaneously two modes: cross-polarization and microangiographic OCT. The comparative analysis of the both OCT modalities images from tumorous and normal brain tissue areas concurrently with histologic correlation shows certain difference between when accordingly to morphological and microvascular tissue features.
In the current paper we present preliminary data demonstrating therapeutic efficiency of local laser hyperthermia of
mouse tumors with gold nanoparticles. Measuring the tumor temperatures both superficial and inner by means of
standard NIR-thermograph and original acoustic thermometer correspondingly we show that the gold nanoparticles
increase thermal sensitivity of tumor tissue. Transmission electron microscopy and histopathology of the tumor tissue
indicated that the mechanism of apoptotic death of tumor cells is triggered following the laser treatment. 5 days after the
treatment tumor growth inhibition was 104 %.
The study was performed on 16 CBA-line female mice with transplanted cervical cancer. 0.2 ml of gold
nanoparticle solution with a concentration of 109 particles/ml were injected into the animals intravenously. The particles
were 200-250 nm in size; the plasmon-resonance related extinction maximum was at the wavelength of 850-950 nm.
Accumulation of the nanoparticles into tumor node was visualized by the method of optical coherence tomography
(OCT). When the accumulation of nanoparticles in the tumor was maximal, hyperthermia was accomplished using the
LSP-AZOR laser setup generating cw radiation at 810 nm. The duration of exposition was 20 min. The therapeutical
effect was assessed by the rate of tumor growth inhibition (TGI, %).
Determining the instant when nanoparticle concentration in tumor tissue reaches its maximum enables more
efficient laser impact. The use of nanoparticles decreases laser irradiation power and ensures local action.
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