Label-free sensing of cells with fluorescence lifetime imaging: the quest for metabolic heterogeneity

Molecular, morphological and physiological heterogeneity is the inherent property of cells, which governs differences in their response to external influence. The tumor cells metabolic heterogeneity is of a special interest due to its clinical relevance to the tumor progression and therapeutic outcomes. Rapid, sensitive and non-invasive assessment of metabolic heterogeneity of cells is of a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters (FDPs) of endogenous fluorophores, such as NAD(P)H. To achieve the accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this report, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity has been demonstrated. This has been achieved using a novel approach to data analysis based on the non-parametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations as compare more traditional approaches of FLIM measurements and analysis. The new approach was further validated for imaging cultured cancer cells treated with chemotherapy. Those results pave the way for an accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Using inverse Laplace transform in positronium lifetime imaging

Positronium (Ps) lifetime imaging is gaining attention to bring out additional biomedical information from positron emission tomography (PET). The lifetime of Ps in vivo can change depending on the physical and chemical environments related to some diseases. Due to the limited sensitivity, Ps lifetime imaging may require merging some voxels for statistical accuracy. This paper presents a method for separating the lifetime components in the voxel to avoid information loss due to averaging. The mathematics for this separation is the inverse Laplace transform (ILT), and the authors examined an iterative numerical ILT algorithm using Tikhonov regularization, namely CONTIN, to discriminate a small lifetime difference due to oxygen saturation. The separability makes it possible to merge voxels without missing critical information on whether they contain abnormally long or short lifetime components. The authors conclude that ILT can compensate for the weaknesses of Ps lifetime imaging and extract the maximum amount of information.

Oxygen Imaging for Non-Invasive Metastasis Detection

Sentinel lymph node (SLN) biopsy is an integral part of treatment planning for a variety of cancers as it evaluates whether a tumor has metastasized, an event that significantly reduces survival probability. However, this invasive procedure is associated with patient morbidity, and misses small metastatic deposits, resulting in the removal of additional nodes for tumors with high metastatic probability despite a negative SLN biopsy. To prevent this over-treatment and its associated morbidities for patients that were truly negative, we propose a tissue oxygen imaging method called Photoacoustic Lifetime Imaging (PALI) as an alternative or supplementary tool for SLN biopsy. As the hyper-metabolic state of cancer cells significantly depresses tissue oxygenation compared to normal tissue even for small metastatic deposits, we hypothesize that PALI can sensitively and specifically detect metastases. Before this hypothesis is tested, however, PALI’s maximum imaging depth must be evaluated to determine the cancer types for which it is best suited. To evaluate imaging depth, we developed and simulated a phantom composed of tubing in a tissue-mimicking, optically scattering liquid. Our simulation and experimental results both show that PALI’s maximum imaging depth is 16 mm. As most lymph nodes are deeper than 16 mm, ways to improve imaging depth, such as directly delivering light to the node using penetrating optical fibers, must be explored.

Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.