Calibration of fluorescent optical sensors for accurate, quantitative intracellular measurements in vivo suffers from lack of a representative medium that appropriately simulates the molecular complexity of the cytosol. We present a novel protocol for accurate intracellular oxygen sensing via fluorescence lifetime imaging microscopy (FLIM) using cell lysate-FLIM measurements to correct the in vitro calibration of a fluorescent oxygen sensor, and we describe electron paramagnetic resonance (EPR) validation studies. Lysate-FLIM studies provided biochemical information, while EPR provided a "gold standard" for intracellular oxygen estimation. Oxygen levels were evaluated in living human normal squamous and adenocarcinoma esophageal epithelial cells, and good agreement was observed between oxygen levels derived from the optical protocol and EPR. The proposed protocol introduces the concept of a living cell line as a reference for estimating unknown oxygen levels in other cell lines and accounts for high degrees of variability between different cell lines.
For the first time, a fluorescence lifetime calibration method for an oxygen-sensitive dye ruthenium tris(2,2-dipyridyl) dichloride hexahydrate (RTDP) is applied to image oxygen levels in poly(dimethyl siloxane) (PDMS) bioreactors containing living C2C12 mouse myoblasts. PDMS microsystems are broadly used in bioengineering applications due to their biocompatibility and ease of handling. For these systems, oxygen concentrations are of significance and are likely to play an important role in cell behavior and gene expression. Fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited state lifetimes, which reflect local biochemistry. Unique attributes of the widefield, time-domain FLIM system include tunable excitation (337.1 to 960 nm), large temporal dynamic range (600 ps), high spatial resolution (1.4 µm), calibrated detection (0 to 300±8 µM of oxygen), and rapid data acquisition and processing times (10 s). Oxygen levels decrease with increasing cell densities and are consistent with model outcomes obtained by simulating bioreactor oxygen diffusion and cell proliferation. In single bioreactor loops, FLIM detects spatial heterogeneity in oxygen levels with variations as high as 20%. The fluorescence lifetime-based imaging approach we describe avoids intensity-based artifacts (including photobleaching and concentration variations) and provides a technique with high spatial discrimination for oxygen monitoring in continuous cell culture systems.
Intracellular oxygen levels were measured <i>in vivo </i>under physiological-temperature controlled conditions by monitoring the fluorescence lifetime of the oxygen sensitive dye ruthenium tris(2,2'-dipyridyl) dichloride hexahydrate (RTDP). We employed fluorescence lifetime imaging microscopy (FLIM) and an independent oxygen sensor to calibrate changes in RTDP lifetime with corresponding changes in oxygen level. The FLIM method reproducibly quantified oxygen levels in living human cells cultured from a (Barrett's) adenocarcinoma columnar cell line (SEG-1). Approaches such as those developed here should prove useful for studying oxygen gradients in 3-D biological specimens and for monitoring cellular metabolic status.
In contrast to intensity-based fluorescence microscopy, fluorescence lifetime imaging microscopy (FLIM) bases image contrast on fluorophore excited-state lifetime. This technique is sensitive to the fluorophore's local environment (temperature, ion concentration, dissolved gas concentration, and molecular associations), while being independent of factors impacting fluorescence intensity (fluorophore concentration, photobleaching, scattering, and absorption). We present design features of a novel UV-visible-NIR wide-field time-domain FLIM system with optical sectioning (10 μm), high temporal discrimination (50 ps), and large temporal dynamic range (750 ps - 1 μs), and apply the system to probe cellular metabolic function and detect molecular activity <i>in vivo</i>.