This study addresses challenges in Optical Metabolic Imaging due to dim signals, overlapping spectra, and similar lifetimes of NADH and FAD autofluorescent molecules. A Phasor-based S-FLIM-SHG microscope is introduced, enabling simultaneous acquisition of Hyperspectral Imaging (HSI), Fluorescence Lifetime Imaging Microscopy (FLIM), and Second Harmonic Generation imaging (SHG). The microscope's design efficiently detects scattered photons from complex samples and it is particularly competent at detection SHG signal. A novel 5D-snapshot (x, y, z, τ, λ). metabolic imaging method is proposed, significantly reducing acquisition times and enhancing measurement accuracy. The method's versatility is demonstrated across diverse sample types, with potential implications for advancing optical metabolic imaging capabilities.
Tumor spheroids represent valuable tools for investigating avascular solid tumors under controlled experimental conditions. In this work we investigated alterations in cellular metabolism, lipid storage, and their interactions with the extracellular matrix (ECM) using various human breast adenocarcinoma cell lines and a non-malignant breast epithelial cell line as spheroids. Three imaging methodologies, were used. The Phasor FLIM, of NADH was used to analyze metabolic signatures. Hyperspectral imaging was used to image signatures from two orthogonal solvatochromic probes: ACDAN for assessing intracellular polarity and Nile Red for detecting lipid composition. SHG images were collected through multiphoton excitation and analyzed via PLICS (Phasor analysis of Local Image Correlation Spectroscopy) to assess the cellular microenvironment and collagen remodeling within breast cancer spheroids. Notable differences emerged in NADH and lipid signatures across cell lines, spheroids, and time. Invading MDA-MB231 cells exhibited elevated hydration and lipid droplets, suggesting adaptation for invasion. These methods shed light on metastatic mechanisms and potential therapies.
The majority of cancer cells have high frequencies of DNA damage response defects which results in a deficient repair mechanism which can give rise to oncogenes that regulate cellular metabolism. In this project, the effects of tumor suppressor protein p53 and translesional synthesis protein REV3L are studied to relate DNA damage signaling and repair to cellular metabolism by using the fluorescence lifetime of the metabolic coenzyme NADH. Our results show that restoring function to p53 and silencing REV3L simultaneously suppressed the cancerous metabolic phenotype and resulted in the greatest amount of cancer cell death
The hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in cancer cells together with atypical extracellular matrix structure may be responsible for tumor cell aggressiveness and drug resistance. Here we apply the phasor approach technique in fluorescence lifetime imaging microscopy (FLIM) as a novel method to measure metabolic alteration as a function of ECM mechanics. We imaged and compared triple-negative breast cancer (TNBC) cells to non-cancerous cells on various ECM stiffness. Dysregulation of mitochondrial motion may contribute to the fueling of bioenergy demands in metastatic cancer. To measure mitochondria motion and analyze their fusion and fission events, we developed a new algorithm called “mitometer” that is unbiased, and allows for automated segmentation and tracking of mitochondria in live cell 2D and 3D time-lapse images.
Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals’ neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the spinal cords and performed fluorescent bioimaging on the sections to observe neuronal fibers going through the bridge. In parallel, to visualize myelination of regenerated axons, we exploited the characteristics of the third-harmonic generation arising from the myelin structure in these fixed sections.
Based on the Fluctuation Correlation principle we have developed a method that is capable of measuring the
stoichiometry of molecular complexes and mapping dynamic processes in living cells. The method is based on
measuring simultaneously fluctuations of the fluorescence intensity at two image channels, each detecting a different
kind of protein. This is an extension of the number and brightness analysis in which we use the use of the ratio between
the variance and the average intensity to obtain the brightness of molecules.
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