Cancer progression is commonly accompanied by an altered glucose metabolism. In general, spatially resolved imaging of glucose metabolism and its subtle alterations might provide valuable diagnostic information in vivo. A classical example is positron emission tomography that exploits this feature in obtaining preferential accumulation of fluorescent analog of glucose in tumors, thereby achieving an imaging contrast. We report a novel scaling analysis of glucose metabolism in mammary epithelial (NMuMG) cells by detrended fluctuation analysis of Cerulean (cyan fluorescent protein variant) fluorescence. Fluorescence fluctuations of Cerulean are reasoned to be indicative of dynamic pH changes associated with glucose metabolism. Normal parental cells and the spontaneously transformed (cancerous) NMuMG cells displayed robust scaling exponent that reflects nonrandom fluctuations in Cerulean fluorescence. Acute dependence of cancer cells on glycolysis as compared with normal cells is exploited to yield a statistically significant difference in scaling exponent, thereby providing discrimination between normal and cancer cells in vitro. By careful design of experiments in vivo, the proposed scaling approach might even have diagnostic potential for early detection of cancer lesions in small animal models.
Early detection of primary tumors is the key for effective therapeutic intervention and successful patient survival. Small
animal models emulating human diseases are powerful tools for our comprehensive understanding of the
pathophysiology of tumor formation and metastasis to distant sites. Our long-term goal is to develop a non-invasive,
multiphoton-fluorescence lifetime imaging (MP-FLIM) modality that can precisely quantify these steps in animal tumor
models at a very early stage. The specific hypothesis is that fluorescence lifetime can be employed as reliable contrast
parameter for providing higher detection sensitivity as compared with conventional intensity-based tumor imaging
approaches and therefore it is possible to detect smaller tumor volumes (early detection) than those achieved by other
prevailing methods. We base this hypothesis on our recent observations that (1) fluorescence lifetime is "intrinsic" to
the fluorophore and its measurement is not affected by concentration and/or spectral artifacts as in intensity-based
methods, (2) multiphoton excitation can enable increased tissue penetrability and reduced phototoxicity and (3) MP-FLIM
approach can discriminate background autofluorescence from the fluorescent proteins in thick tissues thereby
achieving a ten-fold increase in signal-to-background ratio over the intensity-based approaches. We present our
preliminary data to support this hypothesis in primary tumor detection in nu/nu athymic mouse models.
Regulatory dynamics of energy metabolism in living cells entails a coordinated response of multiple enzyme networks
that operate under non-equilibrium conditions. Here we show that mitochondrial dysfunctions associated with the aging
process significantly modify nonlinear dynamical signatures in free radical generation/removal thereby altering energy
metabolism in liver cells. Combining high spatial and temporal resolution imaging and bio-energetic measurements,
our work provides experimental support to the hypothesis that mitochondria manifest nonlinear dynamical behavior for
efficiently regulating energy metabolism in intact cells and any partial or complete reduction in this behavior would
contribute to organ dysfunctions including aging process and other disease processes.
KEYWORDS: Tissues, Tumors, Luminescence, Imaging systems, Signal to noise ratio, Optical imaging, Animal model studies, Solids, In vivo imaging, Green fluorescent protein
A major need in non-invasive optical imaging of small animal models is an ability not only to visualize the solid tumors in vivo but to reproducibly quantify the tumor burden and its propensity to metastasize to other organs of the body. It is crucial to non-invasively detect the subtle molecular changes that can make a cell 'abnormal' and cancerous in its very early stage. Currently available methods for non-invasive optical imaging of solid tumors in small animals employ intensity-based detection that are severely affected by spectral artifacts and ubiquitous autofluorescence background. Thus these approaches serve merely as visualization tools and are unable to precisely quantify the size and shape of the tumors in vivo. There is a growing need to establish a reliable, reproducible and non-invasive optical imaging methodology that can provide quantitative information on solid tumors in vivo. This manuscript addresses this vital issue and proposes to employ fluorescence lifetime (rather than intensity) as a contrast parameter to discriminate tumor tissue expressing green fluorescent protein (EGFP) from surrounding autofluorescence background. In this manuscript, we present accurate lifetime measurements in intact living cells and ex vivo tissues and propose that this methodology is a potentially vital approach for whole small animal imaging.
Turbid tissues pose serious problems of strong absorption and scattering that make steady state fluorescence imaging methods less successful in imaging tissue layers deeper than a few tens of micrometers. Complications arise as one progresses from imaging cells to tissues to whole animal—which include enormous autofluorescence background in tissues and poor signal from regions of interest. Since the steady state, intensity-based methods cannot discriminate the photons arising from the fluorophores and the autofluorescence background, it is almost impractical to isolate these two signals. We describe multiphoton fluorescence lifetime imaging methods in the time domain to demonstrate fluorescence lifetime contrast in discriminating autofluorescence background from the fluorescent signals. Since the photophysical schemes of the fluorophore and autofluorescence contributions are distinct, it is feasible to isolate these two contributions in every pixel based only on their decay constants without compromising the SNR. We present preliminary lifetime measurements to characterize autofluorescence in various cell lines and ex vivo tissues obtained from mouse models. Together, these results suggest a novel direction in obtaining quantitative information from endogenous tissue fluorescence without any exogenous staining. The prospects for this approach in metabolic redox imaging and disease diagnosis are discussed.
Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.
We report the development, calibration and biomedical application of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (approximately 0.2 μm) and temporal (approximately 50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens in the context of quantitative measurement of fluorescence resonance energy transfer (FRET).
Fluorescence Resonance Energy Transfer (FRET) Microscopy has been finding substantial utility in the measurement of a number of biological processes. Most microscopic techniques that have been developed to monitor FRET measure changes in the donor and acceptor emission or fluorescent lifetime of the donor. These include measurements of sensitized emission, acceptor photobleaching and fluorescent lifetime imaging (FLI). However, which of these approaches is the best for a given experimental situation and for use with multiphoton microscopy is not clear. Using mutant GFP FRET caspase-2 substrate targeted to mitochondria, we compare FRET efficiencies measured using sensitized emission, acceptor photobleaching and FLI.
Apoptosis is a physiological process of cell death resulting from an intricate cascade of sequential protein-protein interactions. Using donor and acceptor mutant GFP fusion constructs, we have monitored the interaction between specific pro- and anti-apoptotic members of the Bcl-2 family with each other as well as proteins located in the outer mitochondrial membrane, as current hypotheses regarding apoptosis suggest that interaction of Bcl-2 family members with each other, or with other mitochondrial membrane proteins, regulates apoptosis. Our data indicate that specific interactions between pro- and anti-apoptotic Bcl-2 family members do occur in situ in the mitochondrial membrane, are altered during apoptosis and regulate cellular sensitivity to apoptosis. These findings are the first to demonstrate real time protein-protein interactions in situ at the level of individual mitochondria.
The measurement of fluorescence lifetimes offers the advantages of being independent of local intensity and concentration of the fluorophore, and can provide information regarding the molecular environment in a single living cell. Historically, measurements of fluorescence lifetimes have employed photomultipliers as detectors, providing high sensitivity but sacrificing spatial information. Fluorescence Lifetime Imaging Microscopy (FLIM) provides a 2- or 3D spatial map of the distribution of fluorescent lifetime(s) in the sample under observation. Picosecond laser pulses from a tunable dye laser are delivered to fluorophore containing living cells on the stage of a fluorescent microscope, and images of the fluorescence emission at various times during the decay of the fluorescence lifetime are collected using a high speed nanosecond-gated multichannel plate image intensifier. FLIM promises to substantially enhance the information obtainable from living cells and tissues, and will allow observations of the dynamic organization and interaction of cellular components on a spatial and temporal scale previously not possible using other microscopic techniques.
We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using fluorescence in situ hybridization image is useful for the diagnosis of many other type of diseases, the system we have developed should find numerous applications for the diagnosis of disease states.
Recent technological advances have provided an opportunity for the development of `fluorescence lifetime imaging microscopy' (FLIM). FLIM is an extremely important advance, as it allows for the first time, the sensitivity of the fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells. FLIM can be developed both on conventional and confocal fluorescence microscopes. Fluorescence lifetime detection can be performed using either time- or frequency-domain methods. In this paper, we report on the development of conventional and confocal FLIM systems currently underway in our laboratory. We also present three examples of current biological research projects in which we employ FLIM.
Alterations in plasma membrane structure and function are considered of primary importance in the pathogenesis of cell injury. Multiple microscopic techniques are employed to detail alterations in plasma membrane lipid structure during hypoxic injury in individual rat hepatocytes. Multiparameter digitized video microscopy, fluorescence quenching imaging, and fluorescence resonance energy transfer imaging are used to measure and monitor lipid domain formation and topography; laser tweezers are used to monitor the plasma membrane viscoelasticity. These microscopic techniques indicate that hypoxic injury in hepatocytes leads to alterations in plasma membrane lipid topography with the eventual formation of lipid domains. In concert with previous data generated with digitized fluorescence polarization microscopy and fluorescence recovery after photobleaching (FRAP), a model is proposed where formation of the distinct lipid domains promotes loss of the plasma membrane permeability barrier and cell death.
The analysis of fluorescent stained clusters of cells has been improved by recording multiple images of the same microscopic scene at different focal planes and then applying a three dimensional (3-D) out of focus background subtraction algorithm. The algorithm significantly reduced the out of focus signal and improved the spatial resolution. The method was tested on specimens of 10 micrometers diameter ((phi) ) beads embedded in agarose and on a 5 micrometers breast tumor section labeled with a fluorescent DNA stain. The images were analyzed using an algorithm for automatically detecting fluorescent objects. The proportion of correctly detected in focus beads and breast nuclei increased from 1/8 to 8/8 and from 56/104 to 81/104 respectively after processing by the subtraction algorithm. Furthermore, the subtraction algorithm reduced the proportion of out of focus relative to in focus total intensity detected in the bead images from 51% to 33%. Further developments of these techniques, that utilize the 3-D point spread function (PSF) of the imaging system and a 3-D segmentation algorithm, should result in the correct detection and precise quantification of virtually all cells in solid tumor specimens. Thus the approach should serve as a highly reliable automated screening method for a wide variety of clinical specimens.
Alterations in plasma membrane structure and function seem to be of primary importance in the pathogenesis of cell injury, calling for more understanding of the changes in plasma membrane lipid structure (e.g., lipid order, lateral diffusion, dependence of phase states, and viscoelasticity) during the evolution of hypoxic injury in hepatocytes using multiple fluorescent spectroscopic techniques. Following hypoxic injury, fluorescence recovery after photobleaching was used to monitor plasma membrane lipid diffusion, resonance energy transfer microscopy was used to detect the lipid topography (domain formation), and the laser trapping technique was used to measure the plasma membrane viscoelasticity. The use of these different kinds of fluorescent spectroscopic techniques coupled with the authors' previous studies using digitized fluorescence polarization microscopy which was used to measure lipid order (fluidity) allowed the delineation of alterations in membrane structure during hypoxic injury and a model of membrane architecture during hypoxic injury, which could not be obtained from the use of any of these techniques alone. A model is proposed in which gel- and fluid-phase lipid islands form during hypoxic cell injury. Formation of these lipid domains promotes cell surface bleb formation, with eventual weakening of plasma membrane integrity, bleb rupture, and cell death. 11
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