Time-resolved optical imaging provides a molecular snapshot of altered metabolic function in living human cancer cell models

Abstract

A fluorescence lifetime imaging microscopy (FLIM) method was developed and applied to investigate metabolic function in living human normal esophageal (HET-1) and Barrett's adenocarcinoma (SEG-1) cells. In FLIM, image contrast is based on fluorophore excited state lifetimes, which reflect local biochemistry and molecular activity. Unique FLIM system attributes, including variable ultrafast time gating (>/= 200 ps), wide spectral tunability (337.1 - 960 nm), large temporal dynamic range (>/= 600 ps), and short data acquisition and processing times (15 s), enabled the study of two key molecules consumed at the termini of the oxidative phosphorylation pathway, NADH and oxygen, in living cells under controlled and calibrated environmental conditions. NADH is an endogenous cellular fluorophore detectable in living human tissues that has been shown to be a quantitative biomarker of dysplasia in the esophagus. Lifetime calibration of an oxygen-sensitive, ruthenium-based cellular stain enabled in vivo oxygen level measurements with a resolution of 8 muM over the entire physiological range (1 - 300 muM). Starkly higher intracellular oxygen and NADH levels in living SEG-1 vs. HET-1 cells were detected by FLIM and attributed to altered metabolic pathways in malignant cells.

Imaging a photodynamic therapy photosensitizerin vivowith a time-gated fluorescence tomography system

“…The mean lifetime increased from approximately ½4.3 AE 0.2 ns to ½5.0 AE 0.2 ns, resulting in a ∼16% of increase at the middle slice (Z ¼ 9 mm). As much as ∼700 ps change in lifetime is expected to be due to PDT-induced physiological changes such as changes in oxygenation (see Sud et al) 57 and changes in the microenvironment of HPPH, such as changes in pH (see Scully et al 55 and Sud et al). 57 Lifetime increase is related to oxygenation decrease, which is highly possible since oxygen is being consumed during PDT.…”

“…As much as ∼700 ps change in lifetime is expected to be due to PDT-induced physiological changes such as changes in oxygenation (see Sud et al) 57 and changes in the microenvironment of HPPH, such as changes in pH (see Scully et al 55 and Sud et al). 57 Lifetime increase is related to oxygenation decrease, which is highly possible since oxygen is being consumed during PDT. PDT can induce early blood flow increase (see Becker et al), 72 and interstitial fluid pressure decrease, resulting in a decrease in tumor acidity and increase in pH, which is related to lifetime increase.…”

“…34,54,55 As such, changes in fluorophore lifetime can be potential noninvasive biomarkers for investigating and visualizing the local microvascular changes, photophysical processes, and photochemical reactions at molecular and cellular levels. [55][56][57][58][59] For example, microscopic TD imaging has been applied for studying photobleaching and lifetime dynamics of PDT photosensitizers in cells. 55,56,60,61 In this paper, we report the application of TD-FDOT for 3-D reconstructions of the fluorescence yield and lifetime of the photosensitizer HPPH in vivo before and after PDT.…”

Enhancing precision in time-domain fluorescence lifetime imaging

“…24 In addition, this system had a large temporal dynamic range ͑750 ps to ϱ͒, 50 ps lifetime discrimination, and spatial resolution of 1.4 m, which made it very suitable for studying a variety of endogenous and exogenous fluorophores in biological samples. 2,4,[25][26][27][28] Fluorescence lifetime maps were determined by first acquiring fluorescence intensity images at four delays and then calculating the lifetime values from the intensity images on a pixel-by-pixel basis ͑described in Sec. 3.2͒.…”

3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

“…The lifetime of typical phosphorescence sensors for pO 2 range between hundreds of ns to several hundred μs. In simple biological samples that do not require 3D imaging, single-photon wide field phosphorescence lifetime imaging has been implemented efficiently [47][48][49][50]. However, 3D PLIM using point scanning confocal or multiphoton excitation is always slow since the required pixel residence time must be substantially longer than the phosphorescence lifetime (as long as a fraction of a millisecond).…”