Basic and Applied R&D


Monitoring gene expression in individual living cells

In this project, we transfect cells with expression vectors of the enhanced green fluorescent protein (EGFP) and with expression vectors of chimeras between EGFP and other proteins. After transfection, EGFP expressing cells are loaded onto the Cell Retainer (CR) in order to measure the fluorescent signals from each single cell and to monitor the differences between single cells in the culture. Fluorescence measurements include the kinetics of translocation and degradation of the EGFP-tagged proteins induced by a biologically relevant trigger.

A shorter procedure for the development of EGFP-expressing stable cell lines is being established by using the CR technology, by loading transfected cells onto the CR, after one week of antibiotic selection. The laser tweezers are used to unload positive EGFP-expressing cells from the CR and seed them in a fresh tissue culture dish, each cell in a single well.

 

Live cell sensors

This study develops a cell based methodology for simultaneous analysis of cellular functionality, secreted metabolites and molecular content at the individual cell resolution.

In this project, two sets of cells are used. One set acts as "sensing cells" for the extra-cellular medium (to sense molecules secreted or diffused from other cells), while the other set includes the tested cells that produce specific effects or molecules. The positioning of cells in the Cell Retainer can be either controlled or random. Controlled positioning of "sensing cells" is done consecutively by optical tweezers, while random positioning is a simultaneous (thus faster) procedure in which a suspension of mixed target and sensing cells is loaded onto the CR and their spatial distribution in the array is statistically determined by controlling their initial concentration ratio in the mixed cell suspension.

The resulting methodology can be applied in drug discovery, pharmacological screening, cell therapy and personal diagnosis. For example, this technique can be used in monitoring the intercellular diffusion of nitric oxide (NO) and reactive oxygen species (ROS).

 

Monitoring changes in membrane potential by calculating energy transfer efficiency via fluorescence polarization measurements

One of the most important functions of the cell membrane is to control material transport into and out of the cell. In most cases, control over membrane permeability is executed by changes in the membrane surface electric potential. Hence, the ability to detect these changes may provide a convenient and precise tool for monitoring membrane activity under the influence of various agents, such as drugs, mitogens, or toxins. Such monitoring tools, the correct interpretation of the measured signal, and the mapping ability in various cellular areas at an individual cell level, are of obvious importance.

In this project, a unique method is being developed to monitor the membrane potential in living cells by fluorescence resonance energy transfer (FRET) between Coumarin and Oxanol, via fluorescence polarization measurements. We measure changes in the degree of fluorescence polarization of the donor resulting from changes in membrane potential, due to which the proximity between the donor and acceptor changes. This permits its FRET mapping in various cellular areas at an individual cell level, yielding spatial information regarding membrane potential alterations.

 

Differential temporal and spatial biophysical aspects of the fluorescence of cellular fluorophores

In a heterogeneous environment, such as cellular media, different zones may possess various physiochemical features, which dissimilarly influence the spectroscopic characteristics of fluorescent molecules hosted in the different zones. These differently-influenced fluorophores (even from the same type) may be simultaneously monitored and eventually correlated via Time Resolved Measurements (TRM), if their fluorescence life time (FLT) - iF and/or rotational correlation time - iR differ by at least 0.5 and about 1 nsec correspondingly.
This study is the first to develop high resolution TRM-based microscopy for single cells, which will validly, quantitatively and directly probe the cellular fluorophore's local environment, e.g. refractive index, viscosity, pH, ionic strength, etc. before, during and after cell treatment.

More importantly, using both FLT and fluorescence anisotropy decay (FAD), the weight of each emitting group may be best assessed, and thus simultaneously traced, whereby the chemical-physical mechanisms involved may be interpreted and local cellular alterations can be monitored.

In the current study, TRM based microscopy is combined with the CR methodology to enable the investigation of drug - protein, and protein – protein interactions, as well as dimerisation of receptors, protein phosphorylation and conformational changes upon stimulation. For example, the mechanism of binding of the anti-inflammatory drug methotrexate with cellular proteins is investigated in lymphocytes derived from healthy donors and rheumatoid arthritis patients.

 

The development and utilization of Fluorescence Resonance Energy Transfer (FRET) imaging based on Fluorescence Polarization (FP) measurements

FRET has become a widely used spectroscopic tool for detecting molecular interactions and molecular proximity in solutions, as well as in membranes. Yet, FRET is generally assessed via absolute fluorescence intensity measurements, which are sensitive to the emission band used. On the other hand, FP measurement is ratiometric, simple, predictive and insensitive to inner-filter effects. Moreover, the determining of fluorescence energy transfer efficiency (E) through FP is simple to perform, conveniently adaptable to the commonly used fluorescent microscopy, and readily interpretable.

The Jerome Schottenstein Center was the first to perform FP-based FRET measurements, and we are advancing and adapting this scientific success for cell based assays, in order for that to become a routine biophysical imaging tool in High Content Analysis. As a model, FRET efficiency (E) is assessed between fluorescein and rhodamine conjugated ConA attached to receptors in the lymphocyte membrane.

This technique can be further used to monitor time and spatial dependency of essential activities in living cells, such as protein synthesis by ribosomes or post translational protein modifications, as well as protein interactions within the plasma membrane. For the study of protein synthesis and translocation, wide-field deconvolution microscopy is used; whereas total internal reflection microscopy is used to monitor processes occurring in the membrane adjacent to the Cell Retainer pico-well bottom.

 

Quantitative analysis of tumor metastatic potential at the individual cell resolution via diffusion characteristics

One of the most important stages in the development of primary breast cancer tissue into a secondary metastatic tissue is the dismantling of the extracellular matrix (ECM), which connects the tissue cells. In the process of breast cancer invasion and metastasis, ECM and components of the degraded basal membrane play a critical role. The matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes, which are involved in the degradation of many different components of the ECM in breast cancer. MMPs occur in high levels in cancer cells and are secreted by them. The aim of this study is to quantify the "potential invasiveness” levels of cancer cells, in spreading cancer. This is done by monitoring the amount, the kinetics, the secretion, and the spread of the MMP enzyme, at a single cell resolution. First, the activity of purified MMP enzyme is measured using gelatin substrate, which is a denatured type of collagen, a major component of the ECM. We use a highly quenched gelatin substrate which, upon digestion by MMP, releases bright fluorescent peptides. The increase in fluorescence upon degradation is proportional to the proteolytic activity.

By monitoring the fluorescence time dependency, both the action rate and diffusion parameters (through the gel) can be assessed. In order to do this in a controlled environment, special geometrical arrangements of gel are molded with pre-designed symmetries, in order to simplify the analytical treatment of the differential diffusion equation under the initial and boundary conditions of these specially designed arrangements. From the comparison between the analytical solutions, derived under the specific substrate arrangement, the equation parameters are assessed and their relevancy and specificity for normal cells, cancer cells, as well as treated cells, are evaluated. Finally, similar experiments are performed directly on individual cancer cells which are positioned in the Cell Retainer in order to characterize their metastatic potential utilizing these novel individual cell-based MMP diffusion parameters. Initial results indicate the heterogeneity of cells’ MMP diffusion parameters.

Analysis of laser scattering patterns as a measure of morphological shape changes:
This study aims to conduct relatively long-term, real-time monitoring of subtle, early changes in cellular volume and conformation (due to alterations in water content, which usually precedes many other cellular events that are commonly detected by fluorophores), induced by mitogens, antigens, allergens, and hormones.

This is done by developing a single cell 'wide angle laser light scattering analyzer'. There are several advantages to such an approach. First, the use of fluorescent probes might be fully avoided (in many cases fluorophores interfere with cell physiology, bleach, quench, passively leak, etc.). Secondly, the laser light used does not interact with the cell medium it is just scattered by it, consequently enabling long term measurements. Thirdly, as we have previously shown, calculating subtle spatial dimension changes via inverse Fourier transform of the object wide angle detected power spectrum (in Fourier's plane), may be more accurate with reference to image analysis or forward scattered light of flow-cytometric analysis.

 

Computer-controlled Optical tweezers

In this research, a comprehensive system of computer-controlled optical tweezers is being designed and incorporated within a fluorescence inverted microscope. This hardware has the capability of supporting all experimental Cell Retainer configurations, and it is capable of trapping and relocating individual cells within the pico-wells as well as from a given pico-well to auxiliary structures, such as inlet/outlet ports or reservoirs, in combined CRs. Dedicated software controls laser intensity, beam location and coordinates over the CR, stage velocity, etc. The system is used for manipulating cells, separating individual cells from a population and inter-cellular force measurements.

 

Monitoring drug effects and pathological pathways in living cells by non-interfering Optical Tweezers

The trapping force of optical tweezers (OTW) depends on the Clausius-Mossotti coefficient, as well as on the elevated particle volume. The fact that low intensity light OTWs do not interfere with cell physiology, makes them a convenient long-term measurement tool for tracing alterations in cells dielectric features and volume, under bio-modulation. The principal aim of this study is to trace dielectric changes taking place upon pharmaceutical treatments or cell interactions. Beside the direct scientific value of this research, one major applicative outcome may be the addition of a non-interfering monitoring agent to the arsenal of fluorescence techniques which, unfortunately, interfere with cell physiology, especially in long term measurements.

 

Identifying intracellular EGFP-Bid Translocation by quantitative fluorescence measurements

Protein translocation inside the cell is a known phenomenon which characterizes many biological systems and processes (e.g. cancer). This phenomenon is currently detected by attaching fluorescent protein probes to the examined protein, and imaging via fluorescence microscopy.

This study identifies protein translocation by measuring fluorescence derivative parameters, rather than by image analysis. In this study, Jurkat T cells are transfected with enhanced green fluorescent protein EGFP-Bid. Bid is a cytoplasmic protein which translocates into the mitochondria, under stimulated apoptosis. After translocation, the EGFP-Bid concentration in the mitochondria is elevated; hence a change in mitochondrial viscosity is expected. Changes in the mitochondrial viscosity and in the EGFP-Bid concentration can cause alterations in photo-physical properties of the EGFP fluorophore, such as fluorescence polarization, fluorescence polarization decay and fluorescence lifetime. Measuring these parameters provides a simple quantitative way to identify translocation, without using cumbersome, time consuming image analysis.

 

The biophysical mechanism of FP alteration in EGFP expressed in living cells, following stimulation

In the current study, fluorescence polarization (FP) measurements are used to monitor the expression of intracellular proteins in living cells, under physiological and pathological conditions. In recent experiments, we found that the FP of enhanced green fluorescent protein (EGFP), expressed in Jurkat-EGFP transfected cells, was increased after treatment with the mitogen phytohemagglutinin (PHA).
The aim of this study is to understand the biophysical mechanism underlying the FP increase. Especially, the involvement of Homo-Fluorescence-Resonance-Energy-Transfer (Homo-FRET) between two EGFP molecules is examined under different conditions, such as disassembly of EGFP dimers, changes in cell volume, and changes in ion concentrations (Ca2+, Na+).
Furthermore, a protocol for quantitative measurements of FP, FP-decay and fluorescence lifetime, are established to bypass cumbersome image analysis.

 

Lysosomal involvement in apoptosis induced by oxidative stress

Apoptosis induced by oxidative stress is associated with mitochondrial permeability transition and lysosomal rupture.

In this research, we use kinetic measurements of individual living promonocytic cells for a continuous monitoring of the alterations in lysosomal stability and in mitochondrial membrane potential. Lysosomal damage and mitochondrial membrane potential are detected in single cells, by observing changes in Fluorescence intensity (FI) and polarization (FP) of the vital fluorophores Acridine Orange (AO) and TMRM, respectively

 

Cell-mediated cytotoxicity

Cellular cytotoxicity plays a prominent role in various physiological and pathological states. The assessment of cytolytic activity is important for monitoring immuno-competence in cancer, infectious diseases, and autoimmune diseases. Cell-mediated cytotoxicity tests are often used in mixed lymphocyte cultures, when identifying possible transplant matches.

In the current study, cytotoxicity is tested as a fundamental biochemical pathway leading to cell death/apoptosis. Utilizing the Cell Retaining methodology, real time kinetics of cytotoxicity are measured at an individual cell level. In the current model, target cells (Jurkat T cells) are co-incubated with cytotoxic effector cells (NK-92) in 100 micron Cell Retainers, and cell death is observed. In addition, immuno-phenotypic or other post-fixation analyses are conducted, to test the composition and fate of target and effector cells.