Fluorescent imaging tracks metabolism of cells in real time

Cornell researchers have designed a new, faster form of two-photon fluorescence imaging for observing cell metabolism in real time, a method that could enable quicker screening of new therapeutic treatments. The findings were published Sept. 4 in Science Advances. The co-lead authors are former postdoctoral researcher Lu Ling, Ph.D. '20, and doctoral student Jack Crowley, M.S. '22.

"We want to look at cancer metabolism, but it's a very dynamic process. Everything can happen within minutes or hours, so there's not really a good way to track each cell's dynamics with the tools we had at that time," said Ling, who initiated the project while working in the lab of Claudia Fischbach, the James M. and Marsha McCormick Family director of the Meinig School of Biomedical Engineering and the Stanley Bryer 1946 professor of biomedical engineering in the Cornell Duffield College of Engineering.

"The idea was, can we do something better than the standard methods?" Ling said.

Light exposure limits prolonged imaging

Fischbach and Ling reached out to Warren Zipfel, associate professor of biomedical engineering in Duffield Engineering, who specializes in using optical microscopy for biomedical research. Fischbach was particularly interested in fluorescence lifetime imaging microscopy (FLIM), which has been a common way of monitoring metabolism based on the fluorescence of NADH in cells. (NADH is a vital small molecule that transports the raw energy harvested from food directly into the cellular machinery that produces power.)

FLIM works by exciting molecules and measuring how long they stay excited before releasing their energy as fluorescent light. The time delay of detected fluorescence photons reveals information about the molecule's local environment and behavior.

But Zipfel argued that FLIM was not ideal for correlating cell movement with changes in metabolism because the cells need to be imaged at multiple time points over extended periods, and too much light exposure is a serious stress for the cells.

Polarization reveals molecular rotation

In the method they developed instead, two-photon fluorescence polarization ratiometric microscopy (FPRM), a laser-scanning microscope scans polarized, focused light across cells and then uses a polarizing beam splitter to split the fluorescence simultaneously into two detectors. This provides a measure of how fast the emitting molecules are rotating.

Slower rotation—with the emitted fluorescence being highly polarized—indicates more of the NADH molecules may be bound to proteins and therefore might indicate energy generation in that part of the cell. Meanwhile, unbound NADH molecules rotate more freely, with a depolarized emission.

"This level of bias toward a polarized state is our readout in place of this photon timing experiment that takes much more complicated instrumentation," said Crowley, who took over the project when Ling left to continue her postdoctoral research at the University of California, Berkeley. "It's a way to tell us similar information to what we could acquire using FLIM, but a lot faster with a lot less light."

While the FPRM instrumentation is not as complicated as FLIM instrumentation, the experiments produced data that were challenging to interpret and required new analysis and calibration methods as well as numerous control experiments.

Expanding experiments in three-dimensional tissues

In addition to cancer cells, two-photon microscopy is well suited for imaging complex three-dimensional tissue systems, including organoids and model organisms.

"If you study cells in three-dimensional environments, they behave more like they do in the human body, or in the disease state that we try to model with our research," Crowley said. "So, any opportunity to provide new measurement strategies, especially ones that are simpler and lower cost, means that more people can envision diverse experiments that watch dynamic responses in intricate three-dimensional tissue-engineered structures. And I think that is going to be amazing for research more broadly."