Aim 1:
   There is growing evidence, however, that a dynamic interplay between cancer cells and stromal components, both cellular and non-cellular, influences tumor growth, angiogenesis, metastasis, and therapeutic resistance.1-3 The PDAC stroma is a histopathological hallmark in that it is extremely fibrotic and can remarkably account for up to 90% of the tumor mass.4 A mainstay tissue component of most tumor stromas, including PDAC, is the ECM protein collagen.5 Recently, a number of groups have shown that collagen fiber organization is uniquely altered in various cancer types and may carry biological and clinical significance.6 To date, visualization of collagen-based changes has been greatly accelerated by Second Harmonic Generation (SHG) imaging, a laser scanning microscopy technique that can provide high-resolution, quantifiable images of discrete collagen fibers without the need for exogenous staining. For example, researchers at UW’s LOCI have used SHG to identify unique collagen organizational patterns in breast cancer coined “tumor-associated collagen signatures” (TACS). One of these signatures, TACS-3, describes bundles of straightened, aligned collagen fibers oriented perpendicular to the tumor boundary. Mechanistically, it is hypothesized that these fibers act as pathways that facilitate cancer cell migration away from the tumor and towards vasculature during the metastatic process. It has also been shown that the detection of TACS-3 in routine breast cancer histopathology slides can predict disease recurrence and patient survival. Due to the emerging clinical significance of collagen reorganization, translational technologies are being developed to better detect and quantify changes  
Second Harmonic Generation -  Structure - Ti:Sapphire, Bulk tissues
Matrix stiffness has been shown to promote a malignant phenotype in tumor cells, enhance micrgration and invasion. 
Tumor Associate Collagen Signatures - predictable way during tumor progrssion. 
TACS - aligned collagen perpendicular to the tumor boundary- correlates in predictable wats during tumor progrssion.
creates highway to migrate in vivo. 
we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle,   
Aim 2:
Optical metabolic imaging probes the fluorescence intensity and lifetime of NAD(P)H and FAD. NAD(P)H and FAD are coenzymes used in multiple cellular metabolism processes including glycolysis and oxidative phosphorylation. The end points of OMI include the redox ratio, NAD(P)H fluorescence lifetime, FAD fluorescence lifetime, and a combination variable, the OMI index. The redox ratio is the intensity of NAD(P)H fluorescence relative to the intensity of FAD fluorescence and provides information on the relative amounts of electron donors and acceptors in the cell.6,10 The redox ratio is sensitive to shifts in metabolic pathways.4,10 The fluorescence lifetimes report changes in the microenvironment of NAD(P)H and FAD and are especially sensitive to the binding state of the fluorophore, as well as local temperature, pH, and proximity to quenchers such as molecular oxygen.11 Both NAD(P)H and FAD fluorescence lifetimes can be either short or long, depending on the binding state of NAD(P)H and FAD (free or bound to an enzyme complex).12,13 Previous studies have shown that OMI end points are sensitive to metabolism differences between cancer subtypes.3,4,14 In addition, the OMI end points provide dynamic readouts of cellular metabolism and detect premalignant transformations within tissues,5,15 classify subtypes of breast cancer cells,4,14 and detect response to anticancer drugs.3
Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration
Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore.  
Fluorescence lifetime imaging microscopy (FLIM) is a technique utilized to measure the fluorescence lifetime of molecules. The fluorescence lifetime is the average time a molecule spends in an excited state before returning to the ground state. This measurement is a highly quantitative value that can be used to determine molecular dynamics with nanoscale resolution. FLIM is a standard technique for determining the lifetimes of fluorescent molecules in cells, tissues, and whole animal models.
Two-photon fluorescence lifetime imaging microscopy (TPFLIM) represents a non-invasive imaging technique to visualize alterations in metabolic state, by tracking intrinsic fluorophores present in the cell, such as nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH). NAD(P)H is a key coenzyme in glycolysis and oxidative energy metabolism that acts as a principal electron carrier in energy transduction and biosynthetic processes [25]. NADH plays a key role in catalyzing catabolic reactions in the mitochondria and cytosol while NADPH is a cofactor in anabolic reactions and plays a key role as a cellular antioxidant [25]. An imbalance of these co-factors can provide evidence for differential energy flow. Although, the coenzyme exists in an oxidized [NAD(P)+] and a reduced [NAD(P)H] form, only the reduced form is intrinsically fluorescent [26]. With two photon excitation at 730–750 nm wavelengths, the cellular intrinsic fluorescence is dominated by NAD(P)H species [27]. Careful selection of wavelengths for excitation and emission can enable non-destructive detection with spatial and temporal resolution [28]. The fluorescent lifetime represents the time a molecule spends in its excited state before returning to its ground state. Observations of the changes in NAD(P)H fluorescence lifetime give additional insight into the cofactor’s microenvironment, including its conformation, interactions with other molecules in the system, and changes in pH [28], [29]. Subpopulations of NAD(P)H exhibit distinct lifetimes corresponding to the free and protein bound forms of NAD(P)H [28]. Assuming a model fitting bi-exponential fluorescence decay enables monitoring of the ratio of free to protein bound NAD(P)H. The relative quantities of the free and bound forms of this coenzyme can give insight into the metabolic state of the cell as the functional roles of the cofactors are dependent on the proteins with which they interact. The mean lifetime of protein bound NAD(P)H (t2) is ,2.3–3.0 ns, while the free form has a short lifetime (t1) of ,0.3–0.4 ns [28]. Measurements of the fluorescence intensity and lifetime of NAD(P)H provides a unique visualization of cellular metabolic signatures.
First, the ability to move, second the capacity to degrade tissue matrix (ECM), third the aptitude to survive in blood and finally the physical quality of being able to establish itself in a new tissue environment.