Microscopy Technique Monitors Biomarkers of Subcellular Alterations

Unfortunately, established methods for this purpose either are destructive or require the use of exogenous agents.

To avoid these shortcomings, investigators at Tufts University (Medford/Sommerville, MA, USA) developed a quantitative approach to detecting both functional and structural metabolic biomarkers noninvasively based on two-photon excited fluorescence (TPEF).

TPEF is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in depth. It differs from traditional fluorescence microscopy, in which the excitation wavelength is shorter than the emission wavelength, as the wavelengths of the two exciting photons are longer than the wavelength of the resulting emitted light. Two-photon excitation microscopy typically uses near-infrared excitation light, which can also excite fluorescent dyes. However, for each excitation, two photons of infrared light are absorbed. Using infrared light minimizes scattering in the tissue. Due to the multiphoton absorption, the background signal is strongly suppressed. Both effects lead to an increased penetration depth for these microscopes.

The newly developed technique relied on endogenous TPEF from two coenzymes, NADH (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). The investigators performed multi-parametric analysis of three optical biomarkers within intact, living cells and three-dimensional tissues: cellular redox state, NADH fluorescence lifetime, and mitochondrial clustering. They monitored the biomarkers in cells and tissues subjected to metabolic perturbations that triggered changes in distinct metabolic processes, including glycolysis and glutaminolysis, extrinsic and intrinsic mitochondrial uncoupling, and fatty acid oxidation and synthesis.

Results published in the March 7, 2018, online edition of the journal Science Advances revealed that these optical biomarkers provided complementary insights into the underlying biological mechanisms. Thus, when used in combination, these biomarkers could serve as a valuable tool for sensitive, label-free identification of changes in specific metabolic pathways and characterization of the heterogeneity of the elicited responses with single-cell resolution.

“Taken together, these three parameters begin to provide more specific, and unique metabolic signatures of cellular health or dysfunction,” said senior author Dr. Irene Georgakoudi, professor of biomedical engineering at Tufts University. “The power of this method is the ability to get the information on live cells, without the use of contrast agents or attached labels that could interfere with results.”

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