Whole-object fluorescence lifetime setup for efficient non-imaging quantitative intracellular fluorophore measurements.

In the present study we introduce a Whole-Object Fluorescence Life Time (wo-FLT) measurement approach for ease and a relatively inexpensive method of tracing alterations in intracellular fluorophore distribution and in the physical-chemical features of the microenvironments hosting the fluorophore. Two common fluorophores, Rhodamine 123 and Acridine Orange, were used to stain U937 cells which were incubated, with and without either Carbonyl cyanide 3-chlorphenylhydrazon or the apoptosis inducer H(2)O(2). The wo-FLT, which is a non-imaging quantitative measurement, was able to detect several fluorescence decay components and corresponding weights in a single cell resolution. Following cell treatment, both decay time and weight were altered. Results suggest that the prominent factor responsible for these alterations and in some cases to a shift in emission spectrum as well, is the intracellular fluorophore local concentration. In this study it was demonstrated that the proposed wo-FLT method is superior to color fluorescence based imaging in cases where the emission spectrum of a fluorophore remains unchanged during the investigated process. The proposed wo-FLT approach may be of particular importance when direct imaging is impossible.

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. II: functional activity of cryopreserved cells.

BACKGROUND: The cryopreservation and thawing processes are known to induce many deleterious effects in cells and might be detrimental to several cell types. There is an inherent variability in cellular responses among cell types and within individual cells of a given population with regard to their ability to endure the freezing and thawing process. The aim of this study was to evaluate the fate of cryopreserved cells within an optical cryo apparatus, the individual-cell-based cryo-chip (i3C), by monitoring several basic cellular functional activities at the resolution of individual cells. RESULTS: In the present study, U937 cells underwent the freezing and thawing cycle in the i3C device. Then a panel of vital tests was performed, including the number of dead cells (PI staining), apoptotic rate (Annexin V staining), mitochondrial membrane potential (TMRM staining), cytoplasm membrane integrity and intracellular metabolism (FDA staining), as well as post-thawing cell proliferation assays. Cells that underwent the freezing - thawing cycle in i3C devices exhibited the same functional activity as control cells. Moreover, the combination of the multi-parametric analysis at a single cell resolution and the optical and biological features of the device enable an accurate determination of the functional status of individual cells and subsequent retrieval and utilization of the most valuable cells. CONCLUSIONS: The means and methodologies described here enable the freezing and thawing of spatially identifiable cells, as well as the efficient detection of viable, specific, highly biologically active cells for future applications.

The individual-cell-based cryo-chip for the cryopreservation, manipulation and observation of spatially identifiable cells. I: methodology.

BACKGROUND: Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient. RESULTS: The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing--thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing--thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior. CONCLUSIONS: The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing--thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.

Fluorescence resonance energy transfer imaging via fluorescence polarization measurement.

Fluorescence resonance energy transfer (FRET) has become a widely used spectroscopic tool for detecting molecular interactions and molecular proximity in solution, as well as in membranes. On the other hand, fluorescence polarization (FP) is a convenient measure: ratiometric and simple to execute. This work presents a novel methodology for determining energy transfer efficiency (E) via FP measurement. The methodology is based on the fact that a donor's fluorescence lifetime is shortened due to FRET and, consequently, its FP increases. As a model, the present work evaluates the E between fluorescein and rhodamine conjugated ConA attached to the receptors in the lymphocyte membrane. It shows not only that FRET imaging via FP is possible, but also that it is inexpensive, simple to perform, conveniently adaptable to the commonly used fluorescent microscopy, and readily interpretable.

Influence of fluorescence anisotropy on fluorescence intensity and lifetime measurement: theory, simulations and experiments.

The significance of fluorescence anisotropy in fluorescence intensity and lifetime measurements, and erroneous measurements and interpretations resulting from its disregard, are thoroughly discussed, formulated and quantified. In all fluorescence-related measurements--including excitation and emission spectra, relative fluorescence intensity (FI), fluorescenc life time (FLT), fluorescence resonance energy transfer (FRET), etc., with the exception of fluorescence polarization and anisotropy--it is generally true that the higher the fluorescence anisotropy, the greater the distortion of fluorescence measurements. Quantifiable distortions occur when fluorescence measurements are conducted without considering the influence of fluorescence anisotropy. Here, this influence is described by numerous newly developed mathematical expressions which are simulated and experimentally confirmed utilizing single and binary fluorescent solutions of fluorophores with different spectroscopic characteristics. A marked agreement is shown between the theory and experimental data, clearly indicating the legitimacy of the physical suppositions and the mathematical expressions presented in this paper. Practical and instructive implications are discussed. The following findings are of special applicative importance: 1) the existence of an infinite number of couples of Magic Angles; 2) the deviation between two equally fluorescing particles having different fluorescence anisotropies; 3) the relation between the detected fluorescence intensity and anisotropy when measured under various setups of emission and excitation polarizers; 4) the dependence of the artificial normalized steady-state weight of a single-exponentially decaying fluorophore on its fluorescence anisotropy.

Polymer live-cell array for real-time kinetic imaging of immune cells.

Direct quantitative experimental investigations of the function of lymphocytes and other immune cells are challenging due to the cell mobility and the complexity of intercellular communications. In order to facilitate such investigations, an in vitro system is required that is noninvasive and provides kinetic data on cellular responses to challenges such as drug treatments. The present work reports the development of a disposable, inexpensive polymer-made device, the Polymer Live Cell Array (PLCA), for real-time, kinetic analysis of immune cells. The PLCA proved to be optically and biologically compatible, thus individual immune cells can be observed and treated independently without being tethered. The cells share a common space which facilitates cellular communications via secreted molecules or via direct intercellular interactions. These properties facilitate real-time, non-intrusive, repeated measurements of immune cells under multiple experimental treatments.