Endogenous Two-photon Excited Fluorescence Research Articles

Mitochondrial Function and Metabolism of Cultured Skeletal Cells.

Measuring cellular metabolism accurately is necessary to understand bioenergetic pathways in cells. The major ATP generating pathways in cells are oxidative phosphorylation and glycolysis. We have recently analyzed and published bioenergetic pathways active in osteoblasts undergoing differentiation in response to various substrates. Based on those studies, here we provide step-by-step procedures to isolate, culture, plate and run a seahorse assay for measuring cellular metabolism. Furthermore, we provide an example of oxygen consumption and extracellular acidification rate traces obtained from MC3T3E1-C4 cells using the XFe96 seahorse analyzer. One of the limitations of studying bioenergetics in bone cells is the current lack of techniques to analyze bioenergetics in vivo in live animals. There are currently techniques that have been developed using third harmonic generation to study osteocytes using three-photon microscopy along with metabolic changes using endogenous two-photon excited fluorescence. However, these sophisticated techniques are not widely available. The relative ease with which one can obtain data pertaining to metabolic parameters using the XF technology makes it a very attractive technique to utilize on a monolayer of adherent cells.

Label-free Imaging of Tissue Architecture during Axolotl Peripheral Nerve Regeneration in Comparison to Functional Recovery

Human peripheral nerves hold the potential to regenerate after injuries; however, whether a successful axonal regrowth was achieved can be elucidated only months after injury by assessing function. The axolotl salamander is a regenerative model where nerves always regenerate quickly and fully after all types of injury. Here, de- and regeneration of the axolotl sciatic nerve were investigated in a single and double injury model by label-free multiphoton imaging in comparison to functional recovery. We used coherent anti-Stokes Raman scattering to visualize myelin fragmentation and axonal regeneration. The presence of axons at the lesion site corresponded to onset of functional recovery in both lesion models. In addition, we detected axonal regrowth later in the double injury model in agreement with a higher severity of injury. Moreover, endogenous two-photon excited fluorescence visualized macrophages and revealed a similar timecourse of inflammation in both injury models, which did not correlate with functional recovery. Finally, using the same techniques, axonal structure and status of myelin were visualized in vivo after sciatic nerve injury. Label-free imaging is a new experimental approach that provides mechanistic insights in animal models, with the potential to be used in the future for investigation of regeneration after nerve injuries in humans.

Nerve regeneration in the cephalopod molluscOctopus vulgaris:label-free multiphoton microscopy as a tool for investigation

Octopus and cephalopods are able to regenerate injured tissues. Recent advancements in the study of regeneration in cephalopods appear promising encompassing different approaches helping to decipher cellular and molecular machinery involved in the process. However, lack of specific markers to investigate degenerative/regenerative phenomena and inflammatory events occurring after damage is limiting these studies. Label-free multiphoton microscopy is applied for the first time to the transected pallial nerve of Octopus vulgaris Various optical contrast methods including coherent anti-Stokes Raman scattering (CARS), endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) have been used. We detected cells and structures often not revealed with classical staining methods. CARS highlighted the involvement of haemocytes in building up scar tissue; CARS and TPEF facilitated the identification of degenerating fibres; SHG allowed visualization of fibrillary collagen, revealing the formation of a connective tissue bridge between the nerve stumps, likely involved in axon guidance. Using label-free multiphoton microscopy, we studied the regenerative events in octopus without using any other labelling techniques. These imaging methods provided extremely helpful morpho-chemical information to describe regeneration events. The techniques applied here are species-specific independent and should facilitate the comparison among various animal species.

Mapping metabolic changes by noninvasive, multiparametric, high-resolution imaging using endogenous contrast.

Monitoring subcellular functional and structural changes associated with metabolism is essential for understanding healthy tissue development and the progression of numerous diseases, including cancer, diabetes, and cardiovascular and neurodegenerative disorders. Unfortunately, established methods for this purpose either are destructive or require the use of exogenous agents. Recent work has highlighted the potential of endogenous two-photon excited fluorescence (TPEF) as a method to monitor subtle metabolic changes; however, mechanistic understanding of the connections between the detected optical signal and the underlying metabolic pathways has been lacking. We present a quantitative approach to detecting both functional and structural metabolic biomarkers noninvasively, relying on endogenous TPEF from two coenzymes, NADH (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). We perform multiparametric analysis of three optical biomarkers within intact, living cells and three-dimensional tissues: cellular redox state, NADH fluorescence lifetime, and mitochondrial clustering. We monitor the biomarkers in cells and tissues subjected to metabolic perturbations that trigger changes in distinct metabolic processes, including glycolysis and glutaminolysis, extrinsic and intrinsic mitochondrial uncoupling, and fatty acid oxidation and synthesis. We demonstrate that these optical biomarkers provide complementary insights into the underlying biological mechanisms. Thus, when used in combination, these biomarkers can serve as a valuable tool for sensitive, label-free identification of changes in specific metabolic pathways and characterization of the heterogeneity of the elicited responses with single-cell resolution.

Label-free Imaging of Myocardial Remodeling in Atrial Fibrillation Using Nonlinear Optical Microscopy: A Feasibility Study.

Atrial fibrillation, characterized by rapid disorganized electrical activation of myocardium, is caused by and accompanied by remodeling of myocardial tissue. We applied nonlinear optical microscopy (NLOM) to visualize typical myocardial features and atrial fibrillation effects in order to test anon-destructive imaging technology that in principle can be applied in vivo.Coherent anti-Stokes Raman scattering, endogenous two-photon excited fluorescence, and second harmonic generation were used to inspect unstained human atrial myocardium from three patients who underwent surgical Cox-MAZE procedure with amputation of left atrial appendage. Using NLOM techniques, we collected detailrich pictures of unstained tissue that enable comprehensive characterization of myocardial characteristics like myocyte structure, collagen and lipofuscin deposition, intercalating disc width, and fatty degradation. Development of in vivo application of the NLOM technique may represent a revolutionary approach in characterizing atrial fibrillation associated myocardial remodeling with important implications for therapy individualization and monitoring.

Multimodal nonlinear optical microscopy reveals critical role of kinesin-1 in cartilage development.

We developed a multimodal nonlinear optical (NLO) microscope system by integrating stimulated Raman scattering (SRS), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging. The system was used to study the morphological and biochemical characteristics of tibial cartilage in a kinesin-1 (Kif5b) knockout mouse model. The detailed structure of fibrillar collagen in the extracellular matrix of cartilage was visualized by the forward and backward SHG signals, while high resolution imaging of chondrocytes was achieved by capturing endogenous TPEF and SRS signals of the cells. The results demonstrate that collagen fibrils in the superficial surface of the articular cartilage decreased significantly in the absence of Kif5b. The distorted morphology along with accumulated intracellular collagen was observed in the Kif5b-deficient chondrocytes, indicating the critical roles of kinesin-1 in the chondrocyte morphogenesis and collagen secretion. The study shows that multimodal NLO imaging method is an effective approach to investigate early development of cartilage.

Label-free multiphoton microscopy reveals altered tissue architecture in hippocampal sclerosis.

The properties and structure of tissue can be visualized without labeling or preparation by multiphoton microscopy combining coherent anti-Stokes Raman scattering (CARS), addressing lipid content, second harmonic generation (SHG) showing collagen, and two-photon excited fluorescence (TPEF) of endogenous fluorophores. We compared samples of sclerotic and nonsclerotic human hippocampus to detect pathologic changes in the brain of patients with pharmacoresistant temporomesial epilepsy (n = 15). Multiphoton microscopy of cryosections and bulk tissue revealed hippocampal layering and micromorphologic details in accordance with reference histology: CARS displayed white and gray matter layering and allowed the assessment of axonal myelin. SHG visualized blood vessels based on adventitial collagen. In addition, corpora amylacea (CoA) were found to be SHG-active. Pyramidal cell bodies were characterized by intense cytoplasmic endogenous TPEF. Furthermore, diffuse TPEF around blood vessels was observed that co-localized with positive albumin immunohistochemistry and might indicate degeneration-associated vascular leakage. We present a label-free and fast optical approach that analyzes pathologic aspects of HS. Hippocampal layering, loss of pyramidal cells, and presence of CoA indicative of sclerosis are visualized. Label-free multiphoton microscopy has the potential to extend the histopathologic armamentarium for ex vivo assessment of changes of the hippocampal formation on fresh tissue and prospectively in vivo.

Abstract 5118: In-vivo non-invasive mitochondrial evaluation of 3D human skin

Abstract Alterations in metabolism are considered one of the hallmarks of cancer and have been associated with changes in mitochondrial organization. The latter is typically studied in two-dimensional cell cultures, using exogenous stains, and qualitative or at best semi-quantitative approaches. We have recently developed a method that relies on the automated analysis of high resolution intrinsic NADH two-photon excited fluorescence (TPEF) images of three-dimensional specimens to assess quantitatively mitochondrial clustering levels. The purpose of this study was to assess whether this approach could be extended to the analysis of endogenous TPEF images acquired in vivo from human subjects. In-vivo depth-resolved TPEF and SHG images were acquired in vivo using a MPTflex clinical tomograph (Jenlab) at 790nm excitation. The TPEF signal was detected over 410nm-650nm, whereas SHG signal was detected in the 385nm-405nm range. In this proof of principle study a patient with a basal cell carcinoma lesion and a healthy volunteer were imaged. All in vivo measurements were conducted according to an approved institutional protocol. Image processing was performed in ImJ and Matlab. A series of analytical steps were implemented to exclude from the analysis the nuclei and interstitial space and also the collagen and melanin-associated fluorescence. After the cytoplasmic isolation, we proceeded with an automated, thresholding and digital object cloning method we have previously described. The power spectral density of the corresponding two-dimensional fourier transform of each processed image was fit to a simple inverse power law decay expression to extract a parameter indicative of the levels of mitochondrial clustering, β. Increased β in our study represents more clustered mitochondrial formations. The detailed depth-dependent analysis demonstrates distinct patterns of the clustering for the healthy and diseased tissue examined. An increase in mitochondrial clustering from the superficial to the basal layer is detected for the healthy sample, an outcome in accordance to previously results observed in fresh biopsies from healthy cervical epithelia. Since both the cervical and the skin epithelium are of similar histological type our finding is not startling but rather encouraging. The mitochondrial clustering values of the BCC lesion on the other hand, are depth-invariant, which implies an undifferentiated cellular phenotype, finding consistent with both the cancerous nature of the lesion and previously published in vitro and ex-vivo studies. In conclusion, we demonstrate that characterization of mitochondrial organization is feasible within three dimensional tissues of human subjects. This approach offers the potential to observe dynamically subtle changes in mitochondrial dynamics that occur during cancer development or in response to treatment, which would enrich our understanding of tissue metabolism and could further enhance current diagnostic approaches. Citation Format: Dimitra Pouli, Mihaela Balu, Bruce Tromberg, Irene Georgakoudi. In-vivo non-invasive mitochondrial evaluation of 3D human skin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2015-5118

Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord.

Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.

NI-78 * LABEL-FREE MULTIPHOTON MICROSCOPY: A NOVEL TOOL FOR THE IMAGING OF BRAIN TUMORS

Changes in tissue composition caused by brain tumor growth involve a series of complex biochemical alterations which can be imaged on unstained native tissue using multiphoton microscopy: We used coherent anti-Stokes Raman scattering (CARS) imaging that resonantly excites the symmetric stretching vibration of CH2 groups at 2850 cm−1 and visualizes lipid content in combination with imaging of endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to discern different types of tumors from normal tissue in unstained, native brain samples. Experimental brain tumors were induced in nude mice NMRI nu/nu (n = 25) by stereotactic implantation of glioblastoma (U87), melanoma (A375) and breast cancer (MCF-7) cell lines. Label-free multiphoton microscopy of brain cryosections provided exhaustive information of the tumor morphochemistry. The tumor border was defined with cellular resolution by a strong reduction of CARS signal intensity to 61% (glioblastoma), 71% (melanoma) and 68% (breast cancer). This reduction of lipid content within the tumor was confirmed by Raman spectroscopy. Micrometastases infiltrating normal tissue (size 50 - 200 µm) were identified in glioblastoma and melanoma. Additionally, multiphoton microscopy proved a reduction of CARS signal intensity in all human glioblastoma samples analyzed (to 72%, n = 6). Additionally, relevant SHG and TPEF signals were detected in human primary and secondary brain tumor samples and enabled to image variations in tumor associated vasculature, fibrosis, necrosis and nuclear size and density. All primary or secondary brain tumors investigated were characterized by a lower intensity of the CARS signal, therefore offering a simple tool for objective tumor detection and delineation. The combination of techniques allows retrieving a quantity of information on native unstained tissue which is comparable to H&E staining. Therefore, label-free multiphoton microscopy has the potential to become a technology routinely used intraoperatively for inspection and by neuropathology for frozen section equivalent assessment of brain tumors.

Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues.

Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.

Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development.

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.

Endogenous Two-Photon Fluorescence Imaging Elucidates Metabolic Changes Related to Enhanced Glycolysis and Glutamine Consumption in Precancerous Epithelial Tissues

Alterations in the balance between different metabolic pathways used to meet cellular bioenergetic and biosynthetic demands are considered hallmarks of cancer. Optical imaging relying on endogenous fluorescence has been used as a noninvasive approach to assess tissue metabolic changes during cancer development. However, quantitative correlations of optical assessments with variations in the concentration of relevant metabolites or in the specific metabolic pathways that are involved have been lacking. In this study, we use high-resolution, depth-resolved imaging, relying entirely on endogenous two-photon excited fluorescence in combination with invasive biochemical assays and mass spectrometry to demonstrate the sensitivity and quantitative nature of optical redox ratio tissue assessments. We identify significant differences in the optical redox ratio of live, engineered normal and precancerous squamous epithelial tissues. We establish that while decreases in the optical redox ratio are associated with enhanced levels of glycolysis relative to oxidative phosphorylation, increases in glutamine consumption to support energy production are associated with increased optical redox ratio values. Such mechanistic insights in the origins of optical metabolic assessments are critical for exploiting fully the potential of such noninvasive approaches to monitor and understand important metabolic changes that occur in live tissues at the onset of cancer or in response to treatment.

Heart valve stenosis in laser spotlights: insights into a complex disease.

Degenerative heart valve disease is a life-threatening disease affecting about 3% of the population over 65 years. Up to date, cardiac surgery with heart valve replacement is the only available therapy. The disease is characterized by degenerative disorganization of the heart valve structure and alterations in the residing cell populations. Causes and mechanisms of disease genesis are still not fully understood and until now pharmacological therapies are not available. Thus there is enormous interest in new technologies that enable a better characterization of structure and composition of diseased valves. Currently most research techniques demand for extensive processing of extracted valve material. We present a novel approach combining coherent anti-Stokes Raman scattering, endogenous two-photon excited fluorescence and second harmonic generation. Cusp constituents can be examined simultaneously, three-dimensionally and without extensive manipulation of the sample enabling impressive insights into a complex disease.

Improved Fourier-based characterization of intracellular fractal features

A novel Fourier-based image analysis method for measuring fractal features is presented which can significantly reduce artifacts due to non-fractal edge effects. The technique is broadly applicable to the quantitative characterization of internal morphology (texture) of image features with well-defined borders. In this study, we explore the capacity of this method for quantitative assessment of intracellular fractal morphology of mitochondrial networks in images of normal and diseased (precancerous) epithelial tissues. Using a combination of simulated fractal images and endogenous two-photon excited fluorescence (TPEF) microscopy, our method is shown to more accurately characterize the exponent of the high-frequency power spectral density (PSD) of these images in the presence of artifacts that arise due to cellular and nuclear borders.

Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields.

BackgroundElectric fields are integral to many biological events, from maintaining cellular homeostasis to embryonic development to healing. The application of electric fields offers substantial therapeutic potential, while optimal dosing regimens and the underlying mechanisms responsible for the positive clinical impact are poorly understood.MethodsThe purpose of this study was to track the differentiation profile and stress response of human bone marrow derived mesenchymal stem cells (hMSCs) undergoing osteogenic differentiation during exposure to a 20 mV/cm, 60 kHz electric field. Morphological and biochemical changes were imaged using endogenous two-photon excited fluorescence (TPEF) and quantitatively assessed through eccentricity calculations and extraction of the redox ratio from NADH, FAD and lipofuscin contributions. Real time reverse transcriptase-polymerase chain reactions (RT-PCR) were used to track osteogenic differentiation markers, namely alkaline phosphatase (ALP) and collagen type 1 (col1), and stress response markers, such as heat shock protein 27 (hsp27) and heat shock protein 70 (hsp70). Comparisons of collagen deposition between the stimulated hMSCs and controls were examined through second harmonic generation (SHG) imaging.ResultsQuantitative differences in cell morphology, as described through an eccentricity ratio, were found on days 2 and days 5 (p < 0.05) in samples exposed to the electric field. A delayed but two fold increase in ALP and col1 transcript was detected by week 2 (p < 0.05) in differentiating hMSCs exposed to an electric field in comparison to the nonstimulated controls. Upregulation in stress marker, hsp27, and type 1 collagen deposition were correlated with this response. Increases in NADH, FAD, and lipofuscin were traced in the stimulation group during the first week of field exposure with differences statistically significant on day 10 (p < 0.05). Changes in hsp27 expression correlate well with changes in lipofuscin detected in the stimulation group, suggesting a connection with oxidative stress. Both differentiation factors and electrical stimulation improved hMSC differentiation potential to bone based on calcium deposition on day 28.ConclusionsElectrical stimulation is a useful tool to improve hMSC osteogenic differentiation, while heat shock proteins may reveal underlying mechanisms, and optical non-invasive imaging may be used to monitor the induced morphological and biochemical changes.

Selective corneal imaging using combined second-harmonic generation and two-photon excited fluorescence

A multiphoton microscope employing second-harmonic generation (SHG) and two-photon excited fluorescence (TPF) is used for high-resolution ex vivo imaging of rabbit cornea in a backscattering geometry. Endogenous TPF and SHG signals from corneal cells and extracellular matrix, respectively, are clearly visible without exogenous dyes. Spectral characterization of these upconverted signals provides confirmation of the structural origin of both TPF and SHG, and spectral imaging facilitates the separation of keratocyte and epithelial cells from the collagen-rich corneal stroma. The polarization dependence of collagen SHG is used to highlight fiber orientation, and three-dimensional SHG tomography reveals that approximately 88% of the stromal volume is occupied by collagen lamellae.