Metastasis remains the leading cause of cancer mortality but its mitochondrial dysregulation - from its morphology and motility to its metabolic influence - in modulating cancer’s progression is poorly understood. This dissertation describes the development and use of complex microphysiological culture systems to place cancer in its proper environmental context for mitochondrial phenotypic discoveries. FLIM of NADH is used to probe the metabolic differences between invasive and non- or less-invasive cancer cells and elucidate potential invasion-specific therapeutic targets. This dissertation also introduces Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer finds that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, Mitometer shows that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is then applied to investigate cellular contractility in the influence of mitochondrial phenotype. The goal of this dissertation as a whole is to accentuate the importance of creating new assays and techniques with the impact of context and translatability in mind.

Cellular functional and structural changes associated with metabolism are essential for understanding healthy tissue development and the progression of numerous diseases. Quantitatively monitoring of metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of the diseases. Unfortunately, established methods for this purpose are either destructive or require the use of exogenous agents. Recent work has highlighted the potential of endogenous two-photon excited fluorescence as a method to monitor subtle metabolic changes. In this thesis, we apply two-photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic imaging in pre-implantation embryos and other biological samples.

We exploited the intrinsically fluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. Using phasor-FLIM, we identified unique metabolic states that distinguish embryonic stem cells from differentiating progeny.

We also apply the phasor-FLIM and hyperspectral microscopy to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological and non-invasive caliber for embryo quality. We identify the unique spectroscopic trajectories at different stages of mouse pre-implantation development which can be further used to distinguish pre-implantation embryo quality using an artificial intelligence algorithm at the early compaction stage with 86% accuracy. Furthermore, we showed the heterogeneity and changes in the normal pre-implantation embryos and aneuploidy embryos treated with the spindle assembly checkpoint inhibitor during embryo division can be rapidly distinguished at blastocyst stage via spectra phasor.

Finally, we designed rapid and label-free single leukemia cell identification platform that combines high-throughput size-based separation of hemocytes, and leukemia cell identification through phasor approach and phasor-FLIM to quantify changes between free/bound NADH as an indirect measurement of metabolic alteration in living cells.

These examples illustrate the potential of fluorescence lifetime imaging microscopy for unveiling complex physiological processes. Detailed image analysis and combined microscopy modalities will continue to reveal and quantify fundamental biology that will support the advance of biomedicine.

Induced pluripotent stem cells (iPSC) have opened a new direction on study of Alzheimer’s Disease due to their pluripotency. Differentiation of iPSC to neurons has been cultured in 2D successfully with solid proof including morphological, electrophysiological and immunological, but the yield is low and unpredictable. However, 3D culture of neurons might be better approach human physiology. As a result, differentiation of iPSCs to neurons in a 3D environment has become a new research topic. We obtained 3 different iPSC cell lines from fibroblasts of, non-demented, mild cognitive impaired and demented human subjects. We differentiated these iPSCs first to neural stem cells (NSC) by neural induction, then into neurons on multi-electrode arrays to measure network electrical activity. We also cultured rat hippocampal neurons in both 2D and 3D environment of Matrigel on the MEAs to compare their average spike rate and average inter spike interval. The 3D cultures of rat neurons indicate a need for further optimization of the 3D matrix. The low yields of neuroprogenitors and neurons from human iPSC suggest a need for better control of the initial generation of the pluripotency of the iPSC.

Genome integrity is continually challenged by threats including DNA replication errors, toxic metabolic byproducts, and exposure to exogenous genotoxins. Responding to and repairing damaged DNA requires coordinating a number of critical cellular events including activating DNA repair, facilitating chromatin rearrangement, and delaying cell cycle progression. Although the factors important for DNA repair have been identified, how these activities are coordinated in the nucleus and the long-term cellular-wide consequences of DNA repair are not well characterized.

Nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in both cellular metabolism and DNA repair. Thus, analyses of NAD species could provide new insight into DNA damage signaling as well as damage-induced metabolic responses to various types of DNA lesions. Here, we use the phasor approach to fluorescence lifetime imaging microscopy in combination with genetically encoded fluorescent biosensors to measure metabolic changes in single cells with high spatiotemporal resolution. We found that the absence of the reversionless 3-like translesional synthesis DNA repair protein promotes p53-mediated upregulation of oxidative phosphorylation (oxphos) in cisplatin-treated H1299 lung carcinoma cells and increases cell sensitivity to this chemotherapeutic treatment. Furthermore, it has been well-documented that depletion of NAD+ leads to an overall metabolic collapse, but it is not clear whether or not regulating metabolism can overcome cell death pathways as a survival mechanism. We observe a PARP-dependent decrease in NAD species and an increased metabolic reliance on oxphos. In all, our analyses revealed a previously unrecognized long-term effect of DNA repair signaling on energy metabolism in DNA damaged cells.

Although the patterns of DNA repair protein redistribution following DNA damage have been systematically documented, understanding the complex response to damage requires the characterization of the molecular dynamics of these proteins with high spatiotemporal resolution. By using spatial pair cross-correlation function analysis in two-dimensions, we were able to visualize the barriers to molecular motion of DNA repair proteins in response to laser microirradiation-induced DNA damage.

In summary, my project utilized cutting-edge fluorescence dynamics techniques to reveal a connection between the DNA damage response and cellular metabolism and to develop a new method to characterize molecular diffusion in response to DNA damage.

p53 is a tumor suppressor protein that plays a very important role in determining the fate of damaged cells. Depending on the extent of damage, p53 being a transcription factor, induces target genes that are involved in cell repair mechanisms and apoptosis. In doing so, it prevents proliferation of abnormal cells that could lead to tumorigenesis. Primarily existing in its monomeric or dimeric form, when activated, it binds to DNA as a tetramer. The localization of these tetramers in cells has never been mapped. Since p53 is mutated in 50% of human cancers, its ability to tetramerize efficiently and hence bind to the DNA is disrupted leading to tumor progression. Here we use the Number and Brightness (N&B) analysis, a powerful method to measure protein oligomerization pixel by pixel from raster scanned images thereby providing spatial maps of p53 aggregates. The research described here shows, for the first time, the oligomerization maps of the p53 protein and its mutant counterparts to establish the crucial role of p53 tetramers in tumor suppression. In addition, p53 also regulates the metabolism of the cell by modulating important metabolic pathways upon cellular stress. To determine whether this switch is indicative of the balance between apoptosis and DNA repair, the phasor approach to lifetime imaging microscopy (FLIM) was employed to detect the free and bound lifetime of reduced nicotinamide adenine dinucleotide (NADH). The shifts in lifetime are informative of the level of stress and give an indication whether the cell is undergoing cell cycle arrest or apoptosis. The ratios of free and bound NADH obtained from this data may be used as a marker of transcriptional activity. The N&B and FLIM results together provide an insight to p53 activation and this information can be further exploited to improve the field of cancer research.

Neurodegenerative diseases occur when brain cells (neurons) start to deteriorate. Changes in these cells will lead to dysfunction and eventual cell death. This will lead to mild symptoms like problems with coordination, psychiatric disorders, or memory loss; and as more neurons die the symptoms also progressively worsen. World Health Organization (WHO) indicates up to 1 billion people worldwide are affected by various types of neurodegenerative diseases. In this study, I focused on Huntington disease (HD), a model to study neurodegeneration that is caused by a glitch in a single gene called huntingtin gene (HTT). Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. We all carry HTT; however, the normal length of DNA trinucleotide, CAG, that codes for glutamine are between 10-35. The expanded repeats of above 40 or more will lead to HD. Using advanced functional imaging technique called Two-Photon Fluorescence Lifetime Imaging Microscopy (2P-FLIM), and spectral and temporal phasor approach, spectro-temporal phasor map in living mammalian cells and animal tissue was obtained. Using this sophisticated imaging technique, I have developed new methods and identified novel biomarkers that can help detect Huntington disease early on. This can also help for evaluating the efficacy of treatment. The novel method established in this work is noninvasive and can be performed at the single cell level. Phasor transformation used here simplifies the FLIM and spectral measurements by providing a graphical global view of the process at each pixel and avoids some of the complexity of the multi-exponential analysis. In this way, using a fit free approach that can be applied to both time and frequency domain measurements, Fluorescence Lifetime and spectral emission can be analyzed. It is hoped that this work shed a light on understanding the mechanism of Huntington disease and for new drug discovery and early diagnosis of the disease. The approach introduced in this work can also be applied as a method for understating similar neurodegenerative diseases. This work is supported in part by NIH grant P41 GM103540, NSF BEST IGERT and UC PDY grant.