Revealing the cellular metabolism and microstructural changes in vivo in senescing Acer saccharum leaves using two-photon FLIM and full-field OCM

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 mu m 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.