Fluorescence microscopy is a core biomedical imaging tool that provides high-resolution images of molecular contrast in living samples. Stokes coined the term “fluorescence” in 1852 for the “remarkable phenomena of light” observed in the materials that emitted light at a different color than the absorbed light.¹ At that time, refraction (or refrangibility), internal dispersion, circular dichroism, and other phenomena of light were well studied. Stokes examined flower petals, leaves, turmeric, calcium fluoride, and many other compounds. In the 20th century, fluorescence was redefined as a short-lived emission of photons caused by the incidence of higher energy photons and became a popular tool for studying molecular dynamics and characterizing compounds.

Fluorescence lifetime imaging microscopy (FLIM) is a functional imaging technique that probes the excited state lifetime of a fluorophore. The excited state lifetime can be altered by the fluorophore microenvironment, including factors such as local pH, temperature, viscosity, and oxygen concentration.¹ FLIM, which relies on the temporally resolved fluorescence signal, is advantageous over fluorescence intensity or spectral measurements for small-volume tissue imaging because it is independent of fluorophore concentration, and minimally affected by tissue absorption and scattering, and fluctuations in excitation intensity.² Multiphoton-excited FLIM can generate 3-D images of fluorophore lifetimes with cellular and subcellular resolution.¹ The near-infrared (NIR) excitation used in multiphoton microscopy³ enables greater penetration depths⁴ and improved tissue viability⁵ compared to single-photon excitation. An intuitive application of multiphoton FLIM is to exploit the endogenous fluorescence contrast from electron carriers, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) already present in tissue for the early detection of cancer.

Purpose: Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed. Experimental Design: Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively. Results: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer. Conclusions: These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Microfluidic platform reveals the effects of tumor environmental stress on natural killer cell biology.

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ₂) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ₂ offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.