1.

Introduction

Eukaryotic cells depend on dioxygen, ${\mathrm{O}}_2$ for production of cellular energy in the form of ATP via aerobic respiration. Variation of extracellular oxygen concentrations influences cellular metabolism and is key to tissue development and function. Methods that have been used to examine oxygen gradients in tissue or three-dimensional constructs include computational modeling,¹ photo-acoustic microscopy,² electron paramagnetic resonance combined with magnetic resonance imaging,^3,4 invasive probes, and non-invasive luminescence.^5,6 Modeling approaches rely heavily on experimental data using the Michaelis-Menten equation to describe the maximum oxygen consumption rate of cells ⁷ combined with Fick’s 2nd law governing diffusion to estimate macro-gradients in tissues or constructs.¹ However, estimation of macro-environmental conditions may not be representative of the pericellular micro-environment that is influenced by microgradients directly around individual cells. Obtaining information in close proximity to the cell periphery requires high spatial resolution, $<1\mathrm{\mu m}$, and the flexibility of detection in different solutions unattainable through photo-acoustic microscopy, electron paramagnetic resonance or magnetic resonance imaging. The only remaining methods are invasive probes and non-invasive luminescence.

Invasive probes, such as the Clark’s electrodes ^5,8 or optrodes,^6,9,10 are able to derive oxygen measurements from within a three-dimensional cell system. The invasive nature of the probe causes damage to the surrounding site and, in the case of the electrode, directly consumes oxygen at a rate of $0.4–5\mathrm{pmol}/\mathrm{h}$ thereby influencing the measured oxygen environment.¹¹ For both probes, the spatial resolution is limited by the size and surface area of the probe head with measurements restricted to a single point.¹² These probes also lack the ability of registering the measurement location. Alternatively, luminescence utilizes fluorescence or phosphorescence to measure the intensity or lifetime of a fluorophore.^12,13 Luminescence applied non-invasively, and used with lifetime rather than intensity, results in measurements that are independent of probe concentration, photobleaching and excitation drifts.¹⁴

Lifetime measurements are conducted using time resolved techniques based on the time or frequency domain, although commercially available equipment tends to focus on the former.^15–17 The two methods generally used for acquisition of lifetime data are time-gate or time-correlated single photon counting (TCSPC) methods. TCSPC is generally applied to confocal laser scanning microscopes,¹⁸ whereas, time-gate is predominately used on widefield set-ups.¹⁹ The advantage of TCSPC over time-gate relates to the low-photon counting noise during decay acquisition and the superior time-resolution for photon count registration.²⁰ In these studies, a two-photon excitation confocal system was used to aid excitation within 3D tissue constructs. This takes advantage of tissues natural transparency at IR wavelengths and provides optical sectioning that minimizes tissue exposure and fluorophore photobleaching. TCSPCs time-to-analogue converter has a time range limit of 2 μs restricting the maximum lifetime of any dye.

All fluorophores experience some form of quenching effects from oxygen,²¹ but certain dyes exhibit a greater sensitivity to oxygen. In particular, polycyclic aromatic hydrocarbons, metalloporphyrin and transition metal polypyridyls such as, pyrene, platinum, palladium, and ruthenium have been used extensively. Pyrenebyutic acid has previously been used for intracellular measurements of oxygen concentration in rat liver cells.²² However, there are number of factors that make these derivative unsuitable as an oxygen sensitive dye. For example, emission overlap with autofluorescence, poor chemical- and photostability,²³ limited sensitivity in the physiological range²⁴ and quenching caused by reactive oxygen species.²⁵ Platinum and palladium based probes have phosphorescent decays allowing encapsulation which protects it from the applied environment. However, only single point measurements are collated due to the restriction in acquisition speed derived from the long lifetime decay.^26,27 Ruthenium(II) complexes have been used for the detection and quantification of oxygen in-vitro²⁸ and in-vivo^27,29 and have lifetimes in the order of 600 ns making them suitable for TCSPC detection. The other advantages of ruthenium(II) complexes in homogeneous solution are the linear response to oxygen concentration,³⁰ large Stokes shift which provides high photo-stability,³¹ and a lifetime longer than endogenous fluorophores and mono-exponential decay. Tris(2,2’-bipyridyl)ruthenium(II) chloride hexahydrate, ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$, is the least likely to bind to protein and DNA,³² is hydrophilic, and inexpensive.

Dobrucki³³ investigated the impact of laser excitation on ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ and ${[\mathrm{Ru}{(\mathrm{phen})}_3]}^{2+}$ incubated with macrophage cells. His research suggested that the creation of reactive oxygen species from the illumination of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ and ${[\mathrm{Ru}{(\mathrm{phen})}_3]}^{2+}$ lead to cellular membrane permeability, collapse, and eventually cell death. The concentration level at which phototoxicity occurs is disputed among researchers and can be between 0.2 mM to 1.34 mM.^28,33 This variation in concentration may be due to differences in the excitation source which may be pulsed or continuous, method of excitation which may be widefield or confocal, number of illuminations and illumination time period. Reactive oxygen species can also induce damage to the dye causing increased photobleaching. This has been shown in systems where ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ was embedded in silica.³⁴ In this case, the restriction to diffusion prevents radical oxygen dissipating away from the dye and has a higher likelihood of reacting with the immediate environment causing photobleaching. The dependence of phototoxicity on excitation variables highlights the importance of conducting independent assessments on each system.

The present study investigates the suitability of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{+2}$ using commercially available equipment for accurately measuring spatial oxygen gradients at $<400\mathrm{nm}$ spatial resolution. Concentration dependence was previously tested using a range of concentrations between 0.104 to 3.34 mM and showed a linear relationship with intensity, but no effect on lifetime.³⁵ This techniques utilizes a fluorescence lifetime imaging microscopy (FLIM) TCSPC system with multi-photon laser and pulse-picker, which will reduce the 80 MHz laser repetition rate to accommodate the long lifetime of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{+2}$ which is ($<600\mathrm{ns}$ at 21 °C). The oxygen consumption of primary chondrocytes, as used in this study, is of particular interest given their specialist metabolism associated with the avascular nature of articular cartilage. Furthermore, there is increasing interest in the use of tissue engineered techniques for articular cartilage repair that require the culture of chondrocytes in monolayer or 3D systems. The agarose model has been well characterized in numerous previous studies and has demonstrated the ability to maintain chondrocytic phenotype with the production of cartilaginous extracellular matrix. However, the oxygen consumption and spatial gradients are unclear, hence the requirement for a non-invasive technique that is capable of providing high spatial resolution. Using this technique, spatial oxygen gradients were quantified for the first time around individual viable cartilage cells or chondrocytes which have been seeded in three-dimensional agarose constructs.

2.

Instrumentation

The excitation was provided from a tuneable 720 to 990 nm multi-photon 80 MHz Mai Tai laser which was manufactured by Sapphire, Newport Spectra-Physics in the United Kingdom, and was subsequently delivered to a pulse-picker which was a Model 3980 which was manufactured by Newport Spectra-Physics in the United Kingdom. The laser excitation rate was controlled via a pulse selector Model 3986 which was manufactured by Newport Spectra-Physics in the United Kingdom, that provided a selection of repetition rates between 8 MHz down to single shot. After exiting the pulse-picker, a beam splitter created a reference beam to log excitation pulses using a photon counter PHD-400N manufactured by Becker &amp; Hickl GmbH in Germany. Prior to the laser beam entering the Leica SP2 confocal microscope DM IRE2 which was manufactured by Leica-Microsystems Ltd. in the United Kingdom, the laser power was controlled via an electro-optical modulator LIV20 to 60 produced in Linos, Germany, as represented by the schematic in Fig. 1. Photon emission was collected with a photo-multiplier tube detector PMH-100, manufactured by Becker & Hickl GmbH in Germany is based on Hamamatsu H5773P module, attached to the side port of the confocal microscope and was coupled to the Time-Correlated Single Photon Counting module SP830, enabling lifetime measurements.

Fig. 1

Schematic layout of the MP-FLIM system. Abbreviations: PM—periscope mirror, FM—Flip mirror, M—Mirror, BS—Beam splitter, PC—Photon counter, PH—Pinhole, EOM—electro-optical modulator, TCSPC—Time correlate single photon counting module, EC—Environmental chamber, HP—Heating plate.

The method of photon collection using TCSPC is fully described in the Becker & Hickl textbook.³⁶ To summarize, photon detection is discriminated from noise using a constant fractional discriminator. The signal is converted from analog to digital counts that are assigned by arrival time to the correct time channel. This is represented by a histogram depicting the decaying fluorophores after excitation and reflects the fluorophore’s environment. Collection rates were maintained at $\le 1\%$ of the repetition rate to avoid systematic pile inducing artificial lifetime components in the decay during collection.³⁷

During experimentations, a repetition rate of 500 kHz at 780 nm was delivered through $\times 63$ (NA 1.4) oil immersion objective lens 506192, HCX PL APO, Leica-Microsystems Ltd., GmbH, and produced a maximum power of 164 μW at the sample. The sample was measured using a Model 1830 power meter and a 818 IR/CW detector which were both manufactured by Newport Physics in the United Kingdom. All imaging was performed with the $\times 63$ objective and using TCSPC settings of ADC 1024, unless otherwise stated.

3.

Materials & Methodology

3.1.

Chemical Reagents and Preparation of Cell-Seeded Constructs

Cell culture media consisted of standard Dulbecco’s Modified Eagle Medium supplemented with $3.8\mathrm{g}/\mathrm{l}$ HEPES, $80.6\mathrm{U}/\mathrm{ml}$ penicillin and $80.6\mathrm{mg}/\mathrm{l}$ streptomycin, $0.12\mathrm{g}/\mathrm{l}$ L-ascorbic acid (A0278), 1% L-glutamine and 16% ($v/v$) fetal bovine serum. Calcein AM and Ethdium homodimer-2 were used as live and dead cell stains, respectively, at concentration of $5\mathrm{\mu l}/\mathrm{ml}$. Constructs were created with agarose powder which was dissolved in Hanks balance salt solution, autoclaved for 45 min and incubated in a 60 °C oven for 1 h, to create a sterilized solution which was cooled to 37 °C prior to mixing with cells. The oxygen sensitive dye used was Tris(2,2’-bipyridyl)ruthenium(II) chloride hexahydrate ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$. A filtered stock solution of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ was prepared with de-ionized water at a concentration of 13.34 mM.

3.1.1.

Cell preparation

Primary chondrocyte-agarose constructs were prepared following the well established model and protocols described in numerous previous studies.^38–40 Briefly, cartilage was dissected under sterile conditions from the proximal surface of adult bovine metacarpal-phangel joints. Tissue from each joint, $n=3$, was sequentially digested at 37 °C, with pronase and collagenase for one and 14 h, respectively. The digested cell suspension was passed through a 70 μm nylon cell strainer, washed three times with $\mathrm{DMEM}+16\%\mathrm{FBS}$, pooled together and the isolated cells were resuspended in 30 ml fresh $\mathrm{DMEM}+16\%\mathrm{FBS}$. Cells were seeded in 3% ($w/v$) agarose at $4\times {10}^6\text{cell}/\mathrm{ml}$ and $20\times {10}^6\text{cell}/\mathrm{ml}$ for pericellular measurements and viability tests, respectively. The cell-agarose mixture was gelled at 4 °C for 20 mins in specially designed air-tight chambers, as shown in Fig. 2. For pericellular measurements, quarter sections were removed using a sterile scalpel blade to provide even and rapid access for nutrients, oxygen and fluorophore. Each chamber was filled with 1 ml of media, loosely capped and left overnight in a 5% ${\mathrm{CO}}_2$ incubator at 37 °C. Media was replaced 1.5 h prior to the experiment with a working concentration of ${[\mathrm{Ru}{(\mathrm{bpy})}_3]}^{2+}$ media and incubation was continued.

Fig. 2

Schematic of the sample chamber containing agarose construct filled with media.

3.3.

Cellular Application of Ruthenium(II)

3.3.1.

Phototoxic effects on cell viability

Coverslips, no. 1.5, $\varnothing 22\mathrm{mm}$, within each chamber were marked with permanent marker pen to aid re-location of the specific cells exposed to laser excitation. Chambers were individually mounted into the heating plate on the inverted microscope stage and maintained at 37 °C within an environmental chamber set to 35 °C. The perpendicular lines were located using the eye piece and orientated so that the intersection was positioned in the top left corner of the field of view.

A single brightfield image was taken using the transmitted PMT and $\times 20$ objective lens with a $2048\times 2048$ format, two line averages and two frame average. Excitation was provided at 633 nm, via a helium-neon laser, to avoid direct excitation of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$. This image was used to relocate the specific cells and FOV 24 h after excitation of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$. A $\times 63$ objective lens ($\mathrm{NA}=1.4$) was used with an image format, speed and laser repetition rate of $1024\times 1024$, 400 Hz and MP 4 MHz, respectively, to select two cells for illumination per field of view. The image format was adjusted to $256\times 256$ and a zoom in of the selected cell produced an image voxel size of $130\times 130\mathrm{nm}$, half the Rayleigh criteria for maximum resolution, which will maximize possible photodamage. The selected cell was located centrally for maximum exposure and the detector switched to TCSPC PMT. The laser repetition rate was dropped to 500 kHz and EOM gain adjusted to provide a count rate $<5\times {10}^3\text{counts}/\sec$. The TCSPC parameters were also set to $256\times 256$ image format and 256 time channels with a collection time of 1800 s.

After the laser exposure, agarose constructs were washed three times in media and continued incubation with 1 ml $\mathrm{DMEM}+16\%\mathrm{FBS}$ for a further 24 h at 37 °C. Two cells were imaged per chamber and the procedure was repeated for a total of 11 cells. The control cells were treated in an identical manner and mounted on the microscope for the same length of time with the ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$, but without excitation.

Cellular viability was determined 24 h after excitation of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ including the corresponding control studies. The chambers were removed from the incubator, $5\mathrm{\mu l}/\mathrm{ml}$ of Calcein AM and Ethdium homodimer-2 were added, and incubation continued for an additional 1 h. Brightfield images from the previous day were used to locate each exposed cell by realigning the perpendicular lines in the FOV. The FITC and TRITC programs were run on the Leica using an argon laser calibrated to an ex 488 nm and an em 500 to 535 nm and a He-Neon laser calibrated to/ex 543 nm and an em 555 to 700 nm, respectively. The pre-adjusted programs were placed into a sequence file and the selected cells imaged using two line and frame averages per image.

3.3.2.

Pericellular spatial oxygen gradient

Chambers seeded at $4\times {10}^6\text{cells}/\mathrm{ml}$ were mounted onto the stage of the inverted mircscope and maintained at 37 °C. Using the pulse-picked 4 MHz MP laser and an image format of $512\times 512$, an individual cell was identified and centrally located in the $z$-axis at ex 780 nm, and em $600\pm 50\mathrm{nm}$. The repetition rate was then adjusted to 500 kHz, image format readjusted to $64\times 33$, voxel size changed to $350\times 350\mathrm{nm}$ and field of view positioned to the right of the cell. In the Single Photon Counting Module, the ADC was set to 1024, image format was matched to the Leica and set to $64\times 33$, photon collection rate was maintained at $<1\%$ of the repetition rate which is $<5000\text{counts}/\mathrm{s}$ and the collection time was set to 900 s for each image. This procedure was repeated for each of the 52 cells. Control experiments were performed on agarose constructs prepared in an identical manner, but without cells.

Lifetime images were processed in Time Resolved Imaging software TRI2, Version 2.3.9.1.⁴² Analysis of preliminary lifetime maps showed considerable high frequency spatial heterogeneity. Normal processing methods allow for binning to occur at increasing square sizes to increase pixel counts, however, they bare no relation to the subject of interest. Alternatively, TRI2 allows a masking feature where any pattern can be designed. Therefore, in order to determine the existence of any spatial oxygen gradients, as might be produced by cellular oxygen consumption, a circumferential binning mask was created and moved radially in incremental steps of one pixel creating a set of 59 masks. This method loses circumferential spatial information, but retains high spatial resolution in the radial $x$-axis.

Each drawn mask was loaded individually on to each FLIM intensity image where the counts in the mask were transferred for time resolved analysis and binned to produce a lifetime decay, as shown in Figs. 3(a) through 3(d). Individual pixels, without binning, had accumulated between 450 and $800\text{counts}/\text{pixel}$ and after masked binning contained between 16,000 and $\text{25000\hspace{0.17em}\hspace{0.17em}}\text{counts}/\text{decay}$. Each decay was individually analyzed using a mono-exponential model following a Levenberg-Marquardt fitting to determine $\tau$ where, $Z$ is the offset, $A$ is the amplitude and $t$ is the time, as shown in Eq. (2).

Eq. (2)

$$f(t)=Z+A{\exp }^{(-t/\tau )}.$$

Repeated iterations of Chi-square, ${\chi }_r^2$, were performed until ${\chi }_r^2<1.25$ with an offset fixed to zero. The derived lifetime of each mask represented single points at discrete distances from the cell as shown in Fig. 3(e). The lifetime values of each mask and cell were extracted from excel files, using Matlab, and imported into Origin. The mask lifetimes of each cell were plotted against distance from the cell edge and data points analyzed. Confirming whether a significant change of lifetime was seen with distance, a linear fit was applied at close proximity to the cell, although this would not be assumed for cellular consumpution over larger distances. Conversion into oxygen concentration was performed as described by Gnaiger et al.⁴³ and units follow the recommended standard stated in the International Units of Physics and Chemistry (IUPAC) of $\mathrm{mol}/\mathrm{kg}$ for gas concentrations in liquid.

Fig. 3

Diagrammatic representation of the lifetime data analysis process. (a) A user user defined mask is illustrated in green; (b) the mask is applied to an intensity image of an unresolved test cell; (c) only the selected pixels in the mask are transferred for time resolved analysis; (d) pixels are binned to produce a single lifetime decay and mono-exponential fit applied with weighted residual below and (e) calculated lifetime is transferred to graph and positioned at mask distance represented by green circle. Other separate masks are created along the entire image to produce a complete data set of lifetime values versus distance from the cell periphery.

4.

Results

4.1.

Ruthenium Characterization

4.1.1.

${[\mathrm{Ru}{(\mathrm{Bipy})}_3]}^{2+}$ Shows temperature dependence

Figure 4 shows the influence of temperature on the lifetime response of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$. Representative semi-log lifetime plot, represented in Fig. 4(a), show a reduction in gradient and lifetime with increasing temperature. The standard deviation for the fitting of each lifetime is $<0.2\%$. The mean lifetime values from Fig. 4(a) at each temperature are plotted in Fig. 4(b) and demonstrate a statistically significant linear correlation over this temperature range. The error bars represent the standard deviation in lifetime and temperature from the five repeated measurements. The data lies within the upper and lower confidence limits of the relationship reported by Morris.⁴⁴

Fig. 4

Response of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ in deionzied water to a change in temperature. (a) Semi-log plot showing decay of photon counts with time and the effect induced by temperature. Inset: the central section of the decay between 500 to 1500 s displaying the shift in gradient when ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{+2}$ is exposed to 32 °C is represented by a red line, 34 °C is represented by a green line, and 39 °C is represented by a dark blue line. (b) The corresponding relationship between lifetime and temperature is shown as the lifetime measurements which are represented by red squares with error bars in $x$-axis representing temperature drift and error bars $y$-axis representing standard deviation of fit. Measured data is compared with Morris⁴⁴ which is indicated by a black line shown with upper and lower confidence ($>95\%$) limits which are indicated by a purple dot dash line and a blue dot dash line, respectively.

4.1.2.

No correlation between ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{+2}$ lifetime and pH

The stock solution of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ was dissolved in phosphate buffer between pH 5 and 7.5 to produce a final ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ concentration of 0.412 mM. Repeated lifetime measurements at room temperature were taken of each pH concentration. There was no correlation between lifetime and pH concentration between 5 and 7.5 pH. The mean and standard deviation of the measured lifetimes were calculated as $388.4\pm 0.76\mathrm{ns}$ ($\pm 0.19\%$), respectively.

4.1.3.

Fetal bovine serum (FBS) shields ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ from quenching

The emission wavelength of increasing concentration of FBS diluted in deionised water with ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ are presented in Fig. 5(a). All emission spectra show identical profiles regardless of percentage FBS concentration with asymmetric responses. An asymmetric double sigmoidal peak function was used to determine the peak emission and FWHM as $608.1\pm 0.64\mathrm{ns}$ and $60.1\pm 0.64\mathrm{ns}$, respectively.

Fig. 5

Response of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ to concentrations of FBS diluted in de-ionized water. (a) Normalised intensity emission wavelength at an excitation of 780 nm and repetition rate of 500 kHz across an emission range of 540 to 700 nm. (b) Mean intensity and lifetime decays of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ with increment concentrations of FBS. Error bars represent standard deviations. Left and right $y$-axes correspond to the lifetime and intensity which are represented by black squares and red circles, respectively. Data points are fitted with a dose response curve using an adjusted $R^2>0.96$. The percentage of FBS in the cell culture media is represented by the purple dashed line which corresponds to a lifetime of 388 ns. The maximum response is represented by the dotted green line with a lifetime of 389 ns and intensity of 56702.

The mean lifetime and intensity of the samples in Fig. 5(a) were plotted against increasing percentage of FBS in solution at ambient temperatures, as shown in Fig. 5(b). The standard deviations for the data of both lifetime and intensity were between 1% and 0.29% of the measurement at a given FBS concentration. Measurements for lifetime and intensity were acquired using the same sample. Both display an identical response to the increase in FBS concentration with an initial change in lifetime at increasing concentration that, subsequently, plateaus at the higher concentrations. Both sets of data have been fitted with a dose response curve, with adjusted $R^2>0.96$, reaching a maximum for the lifetime and intensity at 389.1 ns and 56702, respectively, represented by the green dotted line. The percentage of FBS used in the cell culture media was 16%, represented by the purple dashed line, reaches a point on the curve that corresponds to a lifetime of 387.9 ns. The difference between the lifetime at the cell culture concentration and the maximum response of the fitted lifetime was 0.28%.

4.1.4.

Oxygen has linear relationship with ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{+2}$

Lifetime measurements of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ dissolved in water and media at a temperature of 37 °C have been acquired at incrementing concentrations of dissolved oxygen, as shown in Fig. 6(a). A linear correlation can be seen between lifetime and oxygen molality this is characterized by the following negative linear models with an adjusted $R^2>0.98$:

Eq. (3)

$${\tau }_{\text{water}}=464.9\times {10}^{-9}–689.1\times {10}^{-6}·[{\mathrm{O}}_2](\mathrm{mol}/\mathrm{kg});$$

Eq. (4)

$${\tau }_{\text{media}}=460.4\times {10}^{-9}–616.9\times {10}^{-6}·[{\mathrm{O}}_2](\mathrm{mol}/\mathrm{kg}).$$

The measured ${\tau }_0$ for de-ionized water and media was 470.9 ns and 463.5 ns, respectively. By rearranging Eqs. (3) and (4), the oxygen molality can be calculated from the measured lifetime by applying Eq. (1), which produces Fig. 6(b).

Fig. 6

Establishing a relationship lifetime and oxygen ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ in solutions of de-ionized water, indicated by black squares and culture media, indicated by red circles, at 37 °C. (a) The mean lifetime values for water and culture are fitted with linear models. Vertical error bars represent an oxygen concentration accuracy of $\pm 0.1\%$ of the Xvivo system and horizontal error bars the standard deviation for the sample of lifetime measurements at each oxygen concentration. (b) The relationship between oxygen concentration and ${\tau }_0/\tau$ was fitted by linear models to determine $k_{\mathrm{sv}}$ water and media as indicated by black squares and red circles, respectively.

Figure 6(b) depicts the ratio of ${\tau }_0/\tau$ plotted against oxygen molality for ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ in water and in media. Linear fits of both sets of data show a positive linear relationship between the ratio ${\tau }_0/\tau$ and oxygen molality. The gradient for water is steeper than for media, but both intercept at approximately 1. The determined Stern-Volmer constant can now be used to calculate the bimolecular rate for ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{}$ in water and media at 37 °C using Eq. (1) $k_{{\mathrm{sv}}_{\text{water}}}=2108.8\mathrm{mol}/\mathrm{kg}$, $k_{{\mathrm{sv}}_{\text{media}}}=1819.8\mathrm{mol}/\mathrm{kg}$, $k_{2_{.\text{water}}}=4.478\times {10}^9\mathrm{mol}/\mathrm{kg}/\mathrm{s}$, $k_{2_{.\text{media}}}=3.926\times {10}^9\mathrm{mol}/\mathrm{kg}/\mathrm{s}$, respectively.

4.2.

Interaction of ${[\mathit{Ru}{(\mathit{bipy})}_3]}^{2+}$ with Cells

4.2.1.

Phototoxicity of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$

Using the transmitted images, it was possible to relocate and identify the same cell 24 h after excitation of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ and to assess viability, as illustrated in Figs. 7(a)–7(c). The percentage viability 24 h after exposure was 90% with an identical 90% viability for control cells.

Fig. 7

Process of selecting and identification of cell 1 prior to after exposure to assess viability. (a) Brightfield image of identified cells before exposure, $\text{scale bar}=50\mathrm{\mu m}$. (b) Lifetime image cells exposed to the imaging protocol and then chamber returned to the culture incubator for 24 h. (c) Same cells relocated using Brightfield and imaged using 488 nm and 564 nm excitation to reveal cells labelled with calcien AM and EthD-2. Live cells are illustrated in green, whereas, dead cells are illustrated in red with a $\text{scale bar}=50\mathrm{\mu m}$.

4.2.2.

A spatial oxygen gradient exists around a sub-population of chondrocytes

The lifetime values acquired from each mask were converted to oxygen concentrations using a rearranged form of Eq. (1) and derived values from section 4.1.4 correlated with the central voxel distance from the cell periphery. In Figs. 8(a) and 8(b), two cells are displayed with oxygen concentration at an incrementing distance from the cell periphery, represented as zero μm. Figure 8(a) represents a cell displaying no statistically significant gradient or slope originating at the cell periphery, whereas, Fig. 8(b) displays a statistically significant positive gradient or slope that has developed from the cell periphery, reflecting a lower oxygen concentration close to the cell which would be compatible and expected with cellular consumption.

Fig. 8

Converted lifetime data of representative cells showing spatial variation in oxygen concentration with distance from the periphery and the oxygen spatial gradients of cells in a population. All cells have been fitted with linear models assumed at close proximity to the cell to provide the spatial oxygen gradient. (a) For cell 2, the linear model shows no statistically significant correlation. (b) For cell 3, there is a significant positive correlation with a gradient of $0.5\mathrm{\mu mol}/\mathrm{kg}/\mathrm{\mu m}$ and pericellular oxygen concentration of $174\mathrm{\mu mol}/\mathrm{kg}$. (c) Box and whiskers plot of spatial oxygen gradients derived from assumed linear models at close proximity to the cell which have been fitted to data sets of change in oxygen concentration versus distance from the cell periphery. Whiskers and box represent outliers and 25&75 quartiles respectively.

Performing this conversion for all the measured cells revealed a sub-population of 54% of cells in agarose gel, had no statistically significant correlation between oxygen concentration and distance from the cell periphery, as shown in Fig. 8(c). For the other 46% of cells, there was a statistically significant positive correlation. Only 4 percent of cells (52) showed a negative gradient, however, neither of these were statistically significant and matched that seen for agarose with no cells. The average gradient for the significant, non-significant and no cells was 0.53 $(\pm 0.11)$, 0.21 $(\pm 0.16)$ and 0.02 $(\pm 0.11)\mathrm{\mu mol}/\mathrm{kg}/\mathrm{\mu m}$, respectively. The cells associated with statistically significant oxygen gradients were larger than those with no statistical significant gradient with median values of 12.5 and 10.9 μm, respectively. This difference in cell size was statistically significant based upon a Mann-Whitney U-test ($p<0.05$).

5.

Discussion

The development of a pulse-picked TP confocal system required the characterisation of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ within its intended environment in order to understand the restrictions, limitations and the sensitivity to its application for pericellular spatial oxygen quantification. This involved investigating possible effect from temperature, pH, cell culture media and characterizing the relationship between lifetime and oxygen concentration.

There was a negative correlation between temperature and lifetime measurements, in accordance with findings by other researchers.^44–46 In Fig. 4 the relationship between lifetime and temperature appears to be linear. However, over a larger temperature range, previous studies have shown that the relationship is best represented by a polynomial.⁴⁴ An increase in the temperature of water reduces dissolved oxygen and, therefore, should reduce quenching and increase the lifetime. At the same time, there is an increase in the rate of diffusion and therefore collisional quenching. Studies of the temperature effect on ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ without oxygen present have shown that this relationship is due to the thermal activation of a non-radative pathway that operates via the non-luminescent d-d energy state.^44–46 For example, a change of 1 °C in ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ in aqueous solution can induce a 1% change in the lifetime. This highlights the vital importance of maintaining stable and reliable temperature control system to obtain accurate results when using ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$.

Altering pH to mimic the physiological range, 5 to 7.5 pH, did not affect the measured ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ lifetime. This finding was consistent with previous studies.³³ The variability between the measurements across all pH levels shows a standard deviation of $<0.19\%$.

The emission spectra showed identical profiles and no spectral shift for increasing concentrations of FBS in $\text{water}+{[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$. No spectral shift suggests that there is no physical binding to ruthenium(II) and that only one population of fluorophores are emitting.²¹

Examining the lifetime and intensity measurements of ${[\mathrm{Ru}{(\mathrm{bipy})}_{}]}^{2+}$, with increasing concentrations of FBS, reveals similar responses with a positive correlation that gradually decreases to disassociation as illustrated in Fig. 5(b). Since both the lifetime and intensity increase, this indicates that there is less oxygen quenching the fluorophore. However, as the overall oxygen concentration has not been changed, it suggests that the FBS must be abating the oxygen surrounding the ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$. This is supported by previous studies by Vaughan and Weber, ²² who looked at pyrenebutyric acid as a dynamic probe for microenvironments. They showed that if pyrenebutyric acid was conjugated to bovine serum albumin, there was no response to changes in oxygen. However, when free in solution, there was a positive linear relationship. Lakowicz and Weber⁴⁷ stated that this effect is caused by the protein structure that prevents the diffusion of oxygen reaching the fluorophore. However, the fluorophores used in these previous studies were conjugated to bovine serum albumin and showed a slightly different response between the intensity and lifetime measurements. This was a combination of static and dynamic quenching, where the intensity measurements were unaffected by bovine serum albumin concentration and produced a linear response. The lifetime measurements only produced a partial response and then became static, as found in the current study.⁴⁷ This suggests that ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ is not conjugated to FBS or its constituents. This was further supported by the fact that there was no shift in the emission spectra. Similar effects are also seen when ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ comes into contact with DNA. In this case, it can become electrostatically bound or can exhibit partial intercalation depending on the ratio of dye and DNA.⁴⁸ It has been shown by Jain et al.³² that ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ has the weakest binding of all ruthenium(II) complexes to DNA, proteins and serum, due to the presence of hydrophobic organic ligands. This suggests that FBS is partially shielding ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ from oxygen until the FBS increase in concentration has shielded all ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$. At this point, no further response is seen with increasing FBS concentrations. In theory, it would then be expected to change with increasing concentrations of the dye.

Experiments have confirmed that there is a negative linear relationship between lifetime and oxygen molality. As the oxygen partial pressure increases, so does the molality leading to fluorescence quenching and a decrease in lifetime. The ${\tau }_0$ for water and culture media containing FBS are different, which indicates that some form of electrostatic binding could be affecting the radiative rates, as was observed for the interaction between water and FBS in section 4.1.3. The linear fits that establish the relationship between ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ in water and media display smaller standard deviations for media than for water, as illustrated in Fig. 6(a). This indicates that the dye is less sensitive to the environmental change, such as oxygen concentration or temperature fluctuations, when in media. The Stern-Volmer plots indicate dynamic quenching is involved due to the linear lifetime measurement.²¹ This relationship has also been confirmed by other researchers.^49,50 The difference in the Stern-Volmer gradients between water and media reflects different bimolecular quenching interactions. The $K_{\text{SV}}$ values are reflective of the fluorophores environment, such as the influence of temperature, viscosity and binding. A large effect to $K_{\text{SV}}$ was reported by Maheswari et al. ⁵¹, where ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ was measured in the presence of DNA due to electrostatic intercalation with DNA base pairs. The effect of binding is apparent in Maheswari et al. ⁵¹ data with an 83% greater $K_{\text{SV}}$ value in comparison to the current study for ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ in media. This confirms that ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ in media is not bound, but rather has a restrictive quenching relationship. Instead of using the Smoluchowki’s equation to calculate $k_0$, Demas et al. ⁴¹ and Rusak et al.⁵⁰ used Debye’s approximate equation⁵² which assumed that the size and the specific molecules were unimportant, and only the temperature and viscosity of the solvent need to be considered. Equation 5 shows their applied equation⁵⁰:

Cells maintained $>90\%$ viability after 1800 s of laser exposure to the surrounding ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$, at a concentration of 0.412 mM. This is in contradiction to the phototoxicity reported by Dobrucki, when macrophages were exposed to confocal imaging in the presence of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ at concentrations of 0.2 mM, and with 112 illuminations.³³ However, other researchers have used concentrations up to 1.34 mM with no reported loss of viability.⁴⁹ The difference stems from the power of the excitation, which governs the generation of reactive oxygen species created by oxygen quenching ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$, and confirms the variability in phototoxicity between different imaging protocols and excitation methods. Dobrucki states, that the high pressure mercury arc lamp used in their experiments delivered a light beam with 0.1 mW of light focused onto a diffraction limited spot delivering $0.16\mathrm{J}/{\mathrm{cm}}^2$ for one scan/pixel. The current systems excitation wavelength was 780 nm at 500 kHz repetition rate and had a 160 μW of average power at the sample. The energy per pulse $E$, is calculated as $3.2\times {10}^{-10}\mathrm{J}$ using Eq. (6):

Analyzing the relationship between oxygen concentration and distance from the cell periphery has shown that 98% of cells show a positive oxygen gradient such that oxygen concentration reduces close to the cell. There was some variability in the magnitude of this gradient with a sub-population of 46% of cells for which correlation between oxygen and distance was statistically significant, as well as, having a statistically larger cell diameter. None of the gradients measured from the population displayed any statistically significant negative spatial oxygen gradients. This is consistent with the fact that all cells are consuming oxygen, although some do so at a faster rate. This variability in apparent oxygen consumption may reflect the known heterogeneity of the chondrocyte population,^53,54 with larger, more metabolically active cells from the deep zone of articular cartilage being those cells with a greater statistically significant oxygen gradient.

6.

Conclusion

A TCSPC based TP confocal, with added pulse-picker, was used for the detection of ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ lifetime response to changes in oxygen concentration. This was specifically applied to extract high spatial resolution at close proximity to the cell periphery. It was established that ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ was independent of pH concentration, however, it was susceptible to variations in serum concentration and temperature. We suggest that ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ temperature dependence can be linearly described over short temperature ranges, 32 to 39 °C, and that the addition of FBS creates a shielding effect from oxygen quenching forming a concentration dependence and slightly reducing ${[\mathrm{Ru}{(\mathrm{bipy})}_3]}^{2+}$ sensitivity to fluctuations in its environment.

The optimized protocol maintained chondrocyte viability at $>90\%$ and was effective in quantifying pericellular spatial oxygen gradients with a high degree of accuracy and reproducibility. This demonstrated that living cells in-vitro establish a self-induced pericellular oxygen gradient, which was successfully quantified using the implemented TCSPC FLIM system. The extrapolation of this technique to in-vivo studies is complex due to environmental effects, such as protein binding, which cannot be so easily controlled. However, the present study provides a powerful technique for in-vitro investigation of cellular oxygen consumption and its associated spatial gradients. Such experimental studies will support further in silico modeling approaches for understanding oxygen consumption and micro-environment both in-vitro and in-vivo.