Label-free high-resolution visualization of Alzheimer’s disease (AD) neuropathological hallmark, amyloid β (Aβ) plaques, is one of the prime goals of neuroscience. Till today, traditional histological procedures, which rely on fixation and tedious staining of tissues, can only provide definitive confirmation of AD. However, recent studies have shown that label-free third harmonic generation (THG) microscopy, a virtual transition based technology, can provide structural information of biological tissues with subcellular 3D resolution. In this study, using a 1263 nm Cr: Forsterite laser source, we performed THG studies on 3xTg AD mice brain tissues in vitro, with a focus on contrast origin evaluation for plaques. Our THG study can clearly differentiate, with very high resolution, neuropathological hallmark of AD: Aβ plaques. Moreover, THG can also distinguish white and gray matter along with axons, and soma of brain. The origin of THG contrasts for various structures of brain including AD pathological hallmarks were verified through standard immunohistochemical staining procedures. Our preliminary study has successfully demonstrated the capability of THG in revealing AD histopathological features with sub-femtoliter resolution without the need of any exogenous staining of the tissues.
Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P < 0.001), while the ratio of free to protein-bound NADH ratio decreased significantly in 7- days (P < 0.001) and 14-days (P < 0.001). Thus, our results indicated a higher metabolic rate in both osteogenic and adipogenic differentiation processes when compared with undifferentiated hMSCs. This approach may be further utilized to study proliferation efficiency and differentiation potential of stem cells into other specialized cell lineages.
Seasonal as well as climate changes have immense effect on bud burst, leaf color and leaf abscission. Autumn phenology of leaves is clearly distinguishable in deciduous plant leaves where the leaf color changes from green to red (leaf senescence). In this work, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) and full-field optical coherence microscopy (FF-OCM) were applied to study mitochondrial activity and microstructural changes, respectively, in the senescence of Acer saccharum (Sugar maple) leaves. Fluorescence lifetime of reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] was recorded using 2P-FLIM to quantify the cellular metabolic changes. Compared to the green leaves, the red leaves showed a 19% increase (P < 0.05) in the average fluorescence lifetime of NAD(P)H, and a 52% decrease (p < 0.005) in the free to protein-bound NAD(P)H ratio. This infers a significant change in mitochondrial metabolic regulation in red leaves in contrast to green leaves. Additionally, en-face sectional images at 0.8 μm axial resolutions of the green and the red color Acer saccharum leaves via FF-OCM using white light emitting diode (WLED) showed a well-defined microstructure of epicuticular waxy layer in green leaves as compared to red leaves where disintegrated microstructure was observed. Our approach can potentially be used to correlate mitochondrial activity with epicuticular microstructural changes in senescing leaves and other biological tissues.
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