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Autofluorescence Imaging to Evaluate Cellular Metabolism
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Assessing cellular metabolic dynamics with NAD(P)H fluorescence polarization imaging.

Lu Ling1, Jack C Crowley2, Matthew L Tan1

  • 1Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, New York, 14853, USA.

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We developed a fast, label-free imaging method called two-photon steady-state fluorescence polarization ratiometric microscopy (FPRM) to monitor cellular metabolism. This technique tracks nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence for long-term cancer research.

Keywords:
MetabolismNADHcancertwo-photon microscopy

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Area of Science:

  • Cellular Metabolism
  • Cancer Research
  • Bioimaging Techniques

Background:

  • Altered cellular metabolism provides adaptive advantages for cancer cells.
  • There is a critical need for non-invasive, long-term methods to monitor cancer cell metabolism at various scales.
  • Existing techniques like NAD(P)H-fluorescence lifetime imaging microscopy (FLIM) can be slow and induce cytotoxic stress.

Purpose of the Study:

  • To introduce a novel, label-free imaging method for real-time, long-term monitoring of cellular metabolism.
  • To establish a faster, less cytotoxic alternative to current metabolic imaging techniques.
  • To demonstrate the utility of the new method in cancer metabolism research.

Main Methods:

  • Developed and implemented two-photon steady-state fluorescence polarization ratiometric microscopy (FPRM).
  • Utilized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence as a functional metabolic readout.
  • Established instrument-independent ratiometric parameters to correlate NAD(P)H signals with metabolic status.
  • Integrated FPRM with cell shape and migration analysis on 2D and 3D collagen matrices.

Main Results:

  • FPRM operates an order of magnitude faster than FLIM, reducing cytotoxic stress and enabling long-term monitoring.
  • The method provides high-resolution, dynamic tracking of NAD(P)H signals with subcellular detail.
  • Established ratiometric parameters correlate NAD(P)H signals with metabolic status under various perturbations.
  • Demonstrated versatility across bioengineered platforms for cancer metabolism studies.

Conclusions:

  • FPRM is a simple, rapid, and effective label-free imaging technique for monitoring cellular metabolism.
  • The method offers significant advantages for long-term studies of cancer metabolism, including reduced cytotoxicity.
  • FPRM is a versatile tool applicable to various bioengineered systems for advancing cancer research.