Time- and polarization-resolved cellular autofluorescence towards quantitative biochemistry on living cells

John Alfveby, Randi Timerman, Monica P.Soto Velasquez, Dhanushka W.P.M. Wickramasinghe, Jillian Bartusek, Ahmed A Heikal

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Native coenzymes such as the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide play pivotal roles in energy metabolism and a myriad of biochemical reactions in living cells/tissues. These coenzymes are naturally fluorescent and, therefore, have the potential to serve as intrinsic biomarkers for mitochondrial activities, programmed cell death (apoptosis), oxidative stress, aging, and neurodegenerative disease. In this contribution, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) and time-resolved anisotropy imaging of intracellular NADH for quantitative, non-invasive biochemistry on living cells in response to hydrogen-peroxide-induced oxidative stress. In contrast with steady-state one-photon, UV-excited autofluorescence, two-photon FLIM is sensitive to both molecular conformation and stimuli-induced changes in the local environment in living cells with minimum photodamage and inherently enhanced spatial resolution. On the other hand, time-resolved, two-photon anisotropy imaging of cellular autofluorescence allows for quantitative assessment of binding state and environmental restrictions on the tumbling mobility of intrinsic NADH. Our measurements reveal that free and enzyme-bound NADH exist at equilibrium, with a dominant autofluorescence contribution of the bound fraction in living cells. Parallel studies on NADH-enzyme binding in controlled environments serve as a point of reference in analyzing autofluorescence in living cells. These autofluorescence-based approaches complement the conventional analytical biochemistry methods that require the destruction of cells/tissues, while serving as an important step towards establishing intracellular NADH as a natural biomarker for monitoring changes in energy metabolism and redox state of living cells in response to environmental hazards.

Original languageEnglish (US)
Title of host publicationUltrafast Nonlinear Imaging and Spectroscopy II
EditorsIam Choon Khoo, Zhiwen Liu, Demetri Psaltis
PublisherSPIE
ISBN (Electronic)9781628412253
DOIs
StatePublished - 2014
EventUltrafast Nonlinear Imaging and Spectroscopy II - San Diego, United States
Duration: Aug 17 2014Aug 18 2014

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
Volume9198
ISSN (Print)0277-786X
ISSN (Electronic)1996-756X

Conference

ConferenceUltrafast Nonlinear Imaging and Spectroscopy II
CountryUnited States
CitySan Diego
Period8/17/148/18/14

Keywords

  • Associated anisotropy
  • Autofluorescence
  • C3H10T1/2
  • FLIM
  • Hydrogen peroxide
  • Nad(P)H
  • Oxidative stress

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