Fast autofluorescence imaging to evaluate dynamic changes in cell metabolism.

Abstract

Significance: Cellular metabolic dynamics can occur within milliseconds, yet there are no optimal tools to spatially and temporally capture these events. Autofluorescence imaging can provide metabolic information on the cellular level due to the intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD).

Aim: Our goal is to build and evaluate a widefield microscope optimized for rapid autofluorescence imaging of metabolic changes in cells.

Approach: A widefield, fluorescence microscope was assembled from an inverted microscope base, an light-emitting diode (LED) for excitation, and an image splitter for simultaneous but separate imaging of two bandwidths of emission (451/106 and 560/94 nm) on a single scientific complementary metal-oxide-semiconductor (sCMOS) camera. MCF-7 cells and primary murine hippocampal neurons were metabolically perturbed using cyanide and imaged to optimize illumination and camera exposure. To capture a rapid change in metabolism, MCF-7 cells were starved for 1 h and imaged while reintroduced to glucose.

Results: Significant differences in the optical redox ratio (ORR) and intensity of NAD(P)H divided by the summed intensities of NAD(P)H and FAD were quantified for cyanide-treated neurons and MCF-7 cells at illumination powers above 0.30 mW and camera exposures as low as 5 ms; however, low illumination and camera exposures hindered the ability to identify subcellular features. Minimal photobleaching was quantified for 30 s of continuous imaging for illuminations at 4.14 mW and below. Using the optimized illumination power of 4.14 mW and an exposure of 10 ms, continuous autofluorescence imaging of starved MCF-7 cells demonstrated a rapid, yet heterogeneous, increase in the ORR of cells upon exposure to glucose.

Conclusions: Ultimately, this widefield autofluorescence imaging system allowed for dynamic imaging and quantification of cellular metabolism at 99.6 Hz.