2.1.

Materials

Phytohemagglutinin (PHA) (Wellcome Ltd., London, UK) was reconstituted in 5 mL of double-distilled water and further diluted 1:10. For stimulation, 0.1 mL of this solution was added to 1 mL cell suspension (final concentration 90 μg/mL). Fluorescein solutions (1, 5, 10, and 100 μM) were prepared by dissolving fluorescein (Sigma, St. Louis, MO, USA) in phosphate buffered saline (PBS) solutions having different pH (5, 6. 5, 7. 4, 8) and viscosity [different glycerol (Gly) PBS concentrations: 0, 30%, 60%, and 80% Gly]. Polyscience fluorescent beads of fluoresbrite (fluorescein), 5.7±0.1 μm in diameter, with low fluorescent intensity [2000–50 000 molecules of equivalent soluble fluorochromes (MESF)] were used, from Bangs Laboratories, Inc. (Fishers, IN, USA).

2.2.

Cell Preparation

For separation of peripheral blood mononuclear cells (PBMC), 20 mL of heparinized blood, obtained from healthy volunteers, was diluted 1:1 in PBS, pH 7.2. Blood was then layered in a 10 mL density gradient solution (1.077 g/cm³, Ficol Paque, Pharmacia, Upsala, Sweden), and centrifuged at 550 g for 30 min. Cells accumulated at the interphase, between the plasma and ficol layers, were collected and washed three times with complete PBS and resuspended in PBS at a final concentration of 6×10⁶ cells/mL. More than 70% of the cells were defined as T lymphocytes, and viability, determined by trypan blue exclusion, was always higher than 90%. Human Jurkat T-lymphoblast cell line was grown in a humidified atmosphere containing 5% CO ₂, in a RPMI 1640 medium (Biological Industries, Beit Haemek, Israel), supplemented with 10% (v/v) heat inactivated fetal calf serum (Biological Industries, Beit Haemek, Israel), 2 mM L-glutamine, 10 mM HEPES buffer solution, 1 mM sodium pyruvate, 50 U/mL penicillin, and 100 μg/mL streptomycin.

2.3.

Mitogenic Stimulation and Apoptosis Induction

Freshly prepared PBMC (10⁷ cells/mL) were incubated at 37 °C with 90 μg/mL PHA for 45 min for mitogenic activation.

Apoptosis was induced by incubating Jurkat T-lymphoblast cells in complete RPMI 1640 medium in the presence of 50 μM H ₂ O ₂ at 37 °C for various time periods, followed by a quick rinsing of the H ₂ O ₂, to determine the initial time of the apoptotic process.

2.4.

Fluorescein Diacetate Staining

An aliquot of 100 μL of unstained cell suspension (5×10⁶ cells/mL) was loaded on the picoliter well-per-cell array. Initial scanning was then performed in order to detect background scattering and auto-fluorescence. This undesired signal was recorded per measurement location and subtracted from the total emission signal (after staining) in order to obtain the correct fluorescence signal.

Cells were washed twice with incomplete RPMI 1640 medium without phenol red, containing 10 mM HEPES buffer solution. An aliquot of 100 μL of cell suspension (5×10⁶ cells/mL) was added to 50 μL of fluorescein diacetate staining solution (Sigma, St. Louis, MO, USA, F7378) in PBS, and incubated at room temperature for 5 min. At the end of incubation, cells were loaded onto the picoliter well-per-cell array (see the following explanation), washed three times with fresh buffer to remove excess staining solution, and measured.

Figure 1 shows (a) light and (b) fluorescent micrographs of the same individual living PBMC stained with fluorescein diacetate, positioned in the picoliter well-per-cell array. As it can be seen in Fig. 1, the diameter of cells, as observed by light and fluorescent microscopy, is the same. This signifies that the dye uniformly stains the entire cell.

Figure 1

(a) Light and (b) fluorescent micrographs of individual living PBMC, stained with fluorescein diacetate, positioned in the picoliter well-per-cell array.

Plasma membrane integrity of fluorescein diacetate stained cells was checked by re-staining the same cells with propidium iodide (PI). At the end of fluorescein diacetate measurement, the cells were washed twice with fresh buffer and a solution containing PI (2.5 μg/mL) was added on top of the pretested localized cells for 5 min. Cells were then washed twice and remeasured. Positive PI cells were excluded from the analysis.

To confirm apoptosis, Annexin V staining was performed. One million cells were washed with PBS and resuspended in binding buffer (Genzime, Cambridge, MA, USA). FITC Annexin V was added at a final concentration of 1 μg/mL. After 10 min of incubation in the dark, at room temperature, cells were analyzed by FACS (Beckton-Dickenson, FACS-Calibur, Mountainview, CA, USA).

2.5.

Measurement System

Measurements of FAD and τ_F were performed on live cells by microscopy, based on the frequency domain methodology.

Schematic depiction of the frequency domain time resolved microscope (FDTRM) is shown in Fig. 2. The FDTRM comprises two main stand-alone components: the optical system (an upright, epi-fluorescent Zeiss microscope Z-1) and a modified frequency domain FLT and FAD ISS K-2 spectrofluorimeter.

Figure 2

Schematic representation of the FDTRM apparatus. The laser light passes through the Pockel cell and excites the sample. The Pockel cell and the detectors (PMT) are modulated by two frequency synthesizers and two RF amplifiers. All the data are collected by the personal computer controlling the entire measurement process (P—Polarizer).

Briefly, an excitation of a 488 nm polarized beam from a continuous wave (cw) argon ionic laser (model 161B-070, Spectra-Physics, Mountain View, CA, USA) is directed to the light source entrance of the spectrofluorimeter, passing through an electro-optic Pockel’s cell (Laser Metrics KD^*P 1044, Winterpark, FL, USA) which modulates the beam at the frequency range from 1 kHz to 500 MHz. In most experiments, ten frequencies were used, ranging from 2 to 200 MHz in logarithmic scale. A small portion of the modulated excitation beam is sampled by a reference detector (Hamamatsu R928 PMT, Hamamatsu City, Japan), while the rest of it exits the spectrofluorimeter. Then it is doubly refracted by a mirror stirrer, impinges on the microscope illuminator (the epifluorescent arrangement) which directs the beam through the objective lens [×40, numerical aperture (NA)=0.60, LWD CDPlan 40 PL, Olympus, Japan] that focuses the beam to, at most, a 15 μm diameter spot on the interrogation plane, where it illuminates the microscopic fluorescent sample under investigation. The Olympus objective was fitted to the Zeiss optical arrangement.

The diverging cone of the emitted fluorescence is then collected and collimated by the same objective, filtered by an emission filter (530±5 nm) and thereafter impinges on a Glan-Tomphson polarization analyzer, which splits the emission into two polarization (FA) components—parallel (I_∥) and orthogonal (I₊) emission field vectors—relative to the excitation field. Two modulated emission detectors (Hamamatsu R928 PMT) then simultaneously detect the 2 FA components, from which FAD can be calculated, as well as τ_R. For τ_F measurements, the same setup is used, but whereas one of the emission detectors is used for the measurement of the fluorescence signal, the other serves to measure the reference scattered excitation signal.

The induced photocurrent is then fed back to the ISS spectrofluorimeter electronic and computer units for calculation of the requested parameters. During measurements, the modulation frequency is changed automatically according to a predesigned menu. The integrated system is fully computer controlled. For FLT measurements, the polarizers of the system were configured to measure at the magic angle.

2.6.

Individual Cell Measurement

50 μL of cell suspension are loaded on top of the specially designed picoliter well-per-cell array (Patent No. WO 03/035824). Single cells are then settled into the single cell wells (SCW) array by sedimentation. After sedimentation, the excess unsettled cells are washed out of the picoliter well-per-cell array. The bottom of a SCW possesses high optical quality. As a result, the transparent light image of a cell within a well can be recorded and correlated with its fluorescence signal. Each cell being held by a well, biological manipulations on the cell are possible (introduction of staining solutions, drugs, etc.) while preserving its identity. The excitation beam individually and sequentially illuminates the retained cells, and FLT and FAD of an individual fluorescent cell are measured.

2.7.

Data Analysis

The model function was represented by a sum of exponentials as shown in Eq. (2), where I(t) is the fluorescence intensity at time t and α_i is the amplitude of the i’th lifetime τ_i, such that ∑_iα_i=1.

I(t)=I_∥(t)+2MI₊(t), and M is the microscope polarization correction factor which compensates for any optical and/or electrical distortion of the polarization components.

FAD was analyzed by the global fitting of I_∥(t) and I₊(t) to the following equations:

The time resolved measurements were compared to steady state anisotropy (average over time) by utilizing both equalities in Eq. (2):

The quality of fits was evaluated by chi-square (χ²) values based on weighted residuals.⁶ A fit was considered to be within acceptable limits when χ²<2.

Decay curves of FI were analyzed by the standard multi-exponential MATLAB (MathWorks version 5.3 R11 Natick, MA, USA) fitting software and compared to ISS data analysis as well as to the software of the Center for Fluorescence Spectroscopy (CFS, University Maryland, College Park, MD, USA).

3.1.

System Performance Validation

All measurements were conducted at room temperature. Checkups and calibration runs of the entire system were performed daily, and included calculation of the relative modulation depth, phase shift, and the correction factor M=I_∥/I₊. The latter ratio was determined by measuring the FI of a nonviscose fluorescein solution. For all practical purposes, the measurements yield the M value of unity.

Calibration and validation runs were performed by comparing two types of measurements by two different systems (ISS-K2 and FDTRM) for the same sample: two steady-state FA measurements, and two TRM measurements. The TRM results were compared to the steady-state results using Eq. (5). For that purpose, 10 μM fluorescein-glycerol solutions with varying viscosities were measured. The results and their standard deviations (based on 10–50 repetitions) are listed in Table 1. They indicate a high level of correlation and thus the fitness of FDTRM.

Table 1

3.2.

Repeatability Test

The repeatability of the FDTRM performance was checked on fluorescent beads having 2000–50 000 MESF. One lifetime component equal to 4±0.1 ns was found. The FLT value of 4 ns for fluorescein is known to be as that in water solution (see Table 5, row 1, and Refs. 7 and 8), i.e., the dye labeling the beads experiences the same environmental conditions as in water solution. In addition, in FAD measurements (more than 50 measurements were conducted), only one component of τ_R equal to 3.1±0.85 ns was observed.

Computation of the steady state FA via Eq. (5), utilizing the measured values of τ_F and τ_R, yielded r=0.18±0.02, in agreement with the steady state FA measured by both systems: the ISS-K2 and the FDTRM in a steady state mode.

3.3.

Measurements of FLT and FAD in Cellular Models

Two cellular models were considered: the mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cells (JTC). Previous studies^{9 10 11} have indicated that in mitogenic activation the steady state FA decreases, whereas in apoptosis the FA increases. These two findings were the main reason for choosing these models for the present study.

3.4.

Complementary Experiments

Generally, cellular events involve several chemo-physical alterations that may affect the steady-state FA. Among these are pH, viscosity, polarity, concentrations of various intracellular ions, etc.^{8 12} In the following experiments we investigated the possibility of a direct influence of some of the above variables upon the FLT and FAD of a hosted probe.

3.4.3.

FLT dependence on H₂O₂

One of the best-known collisional quenchers of fluorescence is molecular oxygen, a product of H ₂ O ₂. Hence, in the presence of H ₂ O ₂, FLT might be changed, and consequently FA may change as well.⁶ In order to negate the possibility that during H ₂ O ₂ apoptosis induction the measured fluorescence characteristics were directly influenced by the presence of H ₂ O ₂ and/or its products, the following experiment was preformed in cell free solutions with known viscosity. Fluorescein (10 μM) glycerol (80%) solutions, in the presence of different concentrations of H ₂ O ₂ (0, 50, 1000, and 16 000 μM) were prepared and FA and FLT were measured.

The results are shown in Fig. 3. The FLT did not change for the H ₂ O ₂ range of 0–16 000 μM, and remained at the value of 3.6±0.15 ns. However, FA increased by more than 10% from 0.28±0.05 at 0 μM, to 0.312±0.06 at 16 000 μM H ₂ O ₂, which indicates that FA is not influenced by FLT.

Figure 3

FLT and FA of 10 μM fluorescein in 80% glycerol solution with different H ₂ O ₂ concentrations. The full line indicates the FA (right scale) and the dashed line indicates the FLT (left scale). The abscissa scale indicating the H ₂ O ₂ concentrations is drawn logarithmically.