Assessing cellular metabolic dynamics with two-photon NAD(P)H fluorescence polarization microscopy.

Altered metabolism enables adaptive advantages for cancer cells, driving the need for improved methods for noninvasive long-term monitoring of cellular metabolism. Here, we present a fast live-cell NADH imaging method that provides a real-time measurement of the fractional level of unbound NADH and show that it is a robust indicator of a cell's metabolic status. The method, two-photon fluorescence polarization ratiometric microscopy (FPRM), is easy and inexpensive to implement and more than an order of magnitude faster than fluorescence lifetime imaging microscopy (FLIM), a common means of assessing bound and unbound NADH levels. We show that FPRM returns instrument-independent ratiometric parameters that correlate with the expected metabolic changes arising from pharmaceutical and environmental perturbations. By correlating FPRM-returned parameters with cell morphology and migration in two- and three-dimensional collagen matrices, we demonstrate the technique's versatility in typical bioengineered platforms used in cancer metabolism research.