Assessing cellular metabolic dynamics with NAD(P)H fluorescence polarization imaging

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.