Label-Free Imaging

Cancer researchers consider metabolic changes to be one of the key hallmarks of cancer. Consequently, imaging and assessing cellular metabolic changes has become critical to advancing cancer research. The assessment of such cellular metabolic responses can be achieved by two-photon imaging two endogenous, auto-fluorescent redox cofactors, reduced NAD(P)H, and oxidized FAD involved in multiple metabolic processes. In doing so, fluorescence lifetime imaging microscopy (FLIM) estimates the time the fluorophore is in the excited state before returning to the ground state and differentiates between free and bound NAD(P)H and FAD. Imaging the fluorescence intensity of NAD(P)H and FAD and measuring their fluorescence lifetime provides quantitative information about the cellular metabolism (oxidative phosphorylation and glucose catabolism) of cancer tissue. Combining FLIM with MEM allows for single-cell resolution. This is essential to collecting accurate and reliable results because various populations of cells may respond differently to for example a drug treatment.

Researchers' interest in monitoring cellular metabolic changes extends to cells participating in the immunological response to cancer. Using two-photon FLIM, researchers have found that immune cells — such as microphages and T-cells — and microglia cells in the brain respond to their microenvironment by taking adaptive or injurious phenotypes. For example, after activation, T-cells have increased metabolic demands. This metabolic state of increased aerobic glycolysis is required for T-cells to maintain their function. FLIM allows researchers to distinguish between quiescent and activated populations of T-cells, providing information they can then use to characterize immune cells in-vivo and in clinical applications focused on bio-manufactured cell lines for immune therapies.

Changes in collagen organization are also associated with cancer. Oddly, the extracellular collagen matrix has been found to promote the progression of many types of cancer. The visualization of the spatial organization of collagen is based on a two-photon non-linear optical effect known as second-harmonic generation (SHG). SHG is a scattering process where two photons of the same wavelength interact simultaneously with certain biological structures, leading to the emission of a single photon with twice the energy and half the wavelength. Using this approach, researchers have found that it is possible to predict the prognosis of breast, ovarian, and pancreatic cancers depending on the alignment of collagen fibers with respect to each other or with respect to associated tumor boundaries.