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Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136–144 www.elsevier.com / locate / jphotobiol Interaction of oxygen-sensitive luminescent probes Ru(phen) 21 and 3 21 Ru(bipy) 3 with animal and plant cells in vitro Mechanism of phototoxicity and conditions for non-invasive oxygen measurements Jurek W. Dobrucki* Laboratory of Confocal Microscopy and Image Analysis, Department of Biophysics, Institute of Molecular Biology, Jagiellonian University, ul. Gronostajowa 7, 30 -387 Kracow, Poland Accepted 15 October 2001 Abstract Understanding the role of oxygen in the physiology, pathophysiology and radio- and chemosensitivity of animal cells requires accurate and non-invasive measurements of oxygen concentrations in the range of 0–2310 24 M, in cells in vitro or in vivo. High resolution 3D imaging techniques could be particularly useful in investigating tissue oxygenation in vivo and in model tissues (multicellular spheroids) in vitro. The goals of this work were to develop microscopy techniques and (i) to define conditions under which two oxygen-sensitive luminescent dyes, Ru(bipy) 21 (tris(2,29-bipyridyl)ruthenium(II) chloride hexahydrate) and Ru(phen) 21 (tris(1,10-phenanthro3 3 line)ruthenium(II) chloride hydrate) can be used to probe oxygen concentrations within viable cells in vitro, when no phototoxic effects are evident, and (ii) to investigate the mechanism of phototoxicity once cell damage occurs. This report demonstrates that Ru(bipy) 21 and 3 Ru(phen) 321 do not pass through intact biological membranes, do not cause measurable photodamage to plasma membranes at a concentration of 0.2 mM and, when loaded into endosomes, yield a strong luminescent signal. However, at an extracellular concentration of 1 mM, in the presence of 457-nm light, detectable amounts of both complexes accumulate at the plasma membrane and cause a loss of membrane integrity via a mechanism which may involve the generation of singlet oxygen. 2001 Elsevier Science B.V. All rights reserved. Keywords: Ruthenium bipyridyl; Ruthenium phenantroline; Oxygen; Cells; Phototoxicity; Confocal microscopy 1. Introduction Oxygen is a known modifier of radio- and chemosensitivity of animal cells. Survival of mammalian cells irradiated with low LET radiation in the absence of oxygen is approximately three times higher than in the presence of oxygen [1]. Oxygen may also modify the interaction of drugs with cells [2]. Understanding the role of oxygen in physiology and pathophysiology as well as in radio- and chemosensitivity of animal cells requires accurate and non-invasive measurements of oxygen concentrations in the range of 0–2310 24 M, in cells in vitro or in vivo. As hypoxic cells and tissue areas undergoing reoxygenation may be very small, high resolution 3D imaging techniques could be particularly useful in investigating these phenom*Tel.: 148-12-252-6382; fax.: 148-12-252-6902. E-mail address: [email protected] (J.W. Dobrucki). ena in vivo and in model tissues (multicellular spheroids) in vitro. Measurements in single cells, at low partial pressures of oxygen, seem particularly interesting as they might aid studies of the role of hypoxic cells in tumor regrowth and investigations of tissue reoxygenation. A number of methods have been designed to measure oxygen in solution. These rely on oxygen-sensitive electrodes, luminescence [3], EPR [4] and NMR [5]. Some of these methods appear to be amenable for use in cell cultures and tissues, even though several shortcomings restrict their versatility. For instance, small oxygen electrodes consume oxygen themselves and the measurements are invasive. EPR approaches use nitroxides or particulate probes, like lithium phthalocyanine (LiPc), coal, soot or even India ink [6]. While some of these agents exhibit no apparent cytotoxicity (LiPc), others, like nitroxides, may inflict damage on cells. EPR probes exhibit a spectrum of dynamic ranges and sensitivities to dissolved oxygen, from 1011-1344 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S1011-1344( 01 )00257-3 J.W. Dobrucki / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136 – 144 relatively low for nitroxides like CAT1, to very high, for crystals of LiPc. The spatial resolution of EPR oxygen images obtained in phantoms and tissues is too low for imaging individual cells [4]. NMR approaches appear to be non-invasive but also offer limited resolution [7]. In contrast, approaches based on the use of luminescent probes like ruthenium(II) complexes may allow the sensitive, high resolution and non-invasive analysis of oxygen in living tissues and even individual cells [8]. Several factors need to be considered in order to select the right luminescent probe for physiologically relevant measurements of oxygen in viable cells. These factors include intracellular concentration, localization and distribution of a probe, toxicity and phototoxicity, probe stability, pH dependence of luminescence, and influence of soluble proteins on luminescence properties. As demonstrated in this report, ruthenium(II) complexes cannot freely pass through intact biological membranes. Therefore, they could be deliberately confined to the extracellular space or to endosomes. Moreover, luminescent signals should be sufficiently strong to perform measurements of phase shift and further calculations of luminescence lifetimes [8]. Measurements in primary endosomes seem particularly interesting due to the rapid consumption of oxygen by active NADPH oxidase. Moreover, luminescence of ruthenium(II) complexes appears to be independent of pH [9,10] within the range found naturally in endocytic vesicles (i.e. pH 5.0–7.2). Ru(II) complexes do not appear to be destroyed in lysosomes, at least not at a rate detectable under the experimental conditions we used. Obviously, due to vesicle fusion and formation of secondary lysosomes concentrations of Ru(phen) 21 complex3 es may vary between vesicles in the same cell and between cells. Hence, fluorescence lifetime imaging microscopy (FLIM) [8,11] provides the preferred technique for oxygen determination in cells and cell organelles such as endosomes. Measurements of fluorescence lifetime are independent of probe concentration—within the range we can expect in cells—and thus immune to probe redistribution, destruction by metabolism or photobleaching and removal from cells. Unfortunately, potential obstacles to intracellular FLIM measurements also exist, since oxygen sensitivity of Ru(II) complexes bound to proteins or nucleic acids is lower than in solution and may be difficult calibrate in live cells. While luminescence lifetimes of Ru(bipy) 21 and 3 Ru(phen) 321 complexes determined in aerated solutions were apparently only negligibly influenced by the presence of serum proteins [8], the luminescence lifetimes of a new oxygen probe [Ru(dpp(SO 3 Na) 2 ) 3 ]Cl 2 were shown to be strongly influenced by proteins [12]. Luminescence of several ruthenium(II) complexes dissolved in water has been shown to depend on oxygen concentration [13]. Luminescence lifetimes in aerated solutions are shorter than in deoxygenated environments by 36 and 51% for Ru(bipy) 21 and Ru(phen) 321 , respec3 tively [14]. New ruthenium complexes are also being 137 developed as better oxygen probes [15,16]. Ruthenium complexes embedded in plastic were used for constructing oxygen sensors [17–22]. Dissolved ruthenium(II) complexes were also applied in oxygen measurements in solution, for studies of enzymatic reactions, and in cells grown in vitro [8,23–25]. Most, if not all, experiments involving fluorescent probes in contact with live cells are limited by the phototoxicity of the dyes used [26]. This problem was also encountered in our preliminary experiments to measure oxygen in individual intact cells, using Ru(phen) 21 and 3 Ru(bipy) 21 as luminescent probes [8]. Although the results 3 were promising, it became clear that knowledge of the interactions between these dyes and viable cells in culture would be required to further develop this technique. With this goal in mind, the current report describes interaction of two complexes of ruthenium, Ru(bipy) 21 and Ru(phen) 21 3 3 , with viable animal and plant cells maintained in vitro. It attempts to define conditions under which these Ru(II) complexes can be used as oxygen probes in microscopy studies, with no measurable phototoxic effects and investigate the mechanism of phototoxicity under the conditions when it does occur. 2. Materials and methods 2.1. Chemicals Tris(2,29-bipyridyl)ruthenium(II) chloride hexahydrate (Ru(bipy) 21 [27] and tris(1,10-phenanthroline)3 ) ruthenium(II) chloride hydrate (Ru(phen) 21 3 ) were obtained from Aldrich. 2.2. Cell cultures J144 mouse macrophages were obtained from American Tissue Collection and cultured in Dulbecco’s MEM (Sigma) supplemented with 10% fetal bovine serum (Sigma) and antibiotics. Cells were subcultured by washing off substratum with a stream of culture medium. Normal human fibroblasts were cultured as described previously [28]. Logarithmically growing cell cultures were used for experiments (|48 h following seeding). For imaging experiments, cell cultures were seeded onto round ¨ coverslips (22 mm diameter, Menzel-Glaser), placed in Petri dishes and maintained in a CO 2 incubator (Jouan) for 2 days. Subsequently, coverslips were mounted in a microscope stage microincubator (Life Science Resources, Cambridge, UK). Prior to seeding cells, coverslips were selected for thickness of 0.1760.005 mm, degreased, washed, wrapped individually in aluminum foil and sterilized by autoclaving. Integrity of plasma membranes was tested by exclusion of propidium iodide or by hydrolysis of fluorescein diacetate. 138 J.W. Dobrucki / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136 – 144 2.3. Incubation of cells with Ru( II) complexes For incubation of cells with ruthenium complexes, culture medium was supplemented with the drug immediately prior to experiments. Prior to adding to cell cultures, solutions of Ru(II) complexes were sterilized using a 0.22-mm Millipore filter. 2.4. Fluorescence confocal microscopy and image analysis Images of macrophages exposed to Ru(II) complexes were collected using a Bio-Rad MRC1024 laser scanning confocal system (with an inverted microscope Nikon Diaphot 300), using a 603 PlanApo NA1.4 oil immersion lens. Excitation was 457 nm (100-mW Ar ion laser, ILT) with a 3% neutral density filter in the excitation light path. The dichroic splitter was 510 DLRP (VHS block); emission was collected using a 580 LP emission filter. Cells growing on round glass coverslips were maintained in a microincubator resting on a microscope stage, at 37 8C in Dulbecco’s MEM supplemented with HEPES (pH adjusted to 7.2), without Phenol Red. Microincubator facilitated replacement of culture medium, adding dye solutions and changing gas atmosphere during illumination and imaging, without altering the position of the imaged area of cell culture. Sequences of images (5123512 pixels, 256 gray levels) were collected and analyzed using LaserSharp TimeCourse software (Bio-Rad). Black level of the photomultiplier was adjusted so that noise values were within 1–10; gain was adjusted so that the values of red autofluorescence of intact cells were within a range of 5–50. For timecourse recordings, horizontal cross-sections of single cells (six to ten in one series) were selected and followed over time. An equivalent area of dye-containing culture medium was recorded as a control. Traces of a mean of luminescence intensity in each selected cell and in surrounding medium as well as time-series of images were displayed live during the experiment and recorded simultaneously. Further analysis of images and traces were performed using LaserSharp, Origin or SigmaPlot software. 2.5. Illumination of cells and simultaneous observation of damage to plasma membrane Preliminary measurements of phototoxicity of Ru(II) complexes were conducted by illuminating cell cultures on a stage of an inverted microscope (Nikon Diaphot 300), with 450–490-nm light emitted by a high pressure mercury arc lamp (Nikon). Cell cultures were maintained on plastic Petri dishes at room temperature or on coverslips mounted in a microincubator, at 37 8C. Following illumination, accumulation of Ru(II) complexes in the regions of plasma membranes was observed. Subsequently, entry of the dyes into damaged cells occurred. Following different periods of illumination, the dye was removed, standard medium was introduced and cultures were maintained in a CO 2 incubator for 3–6 days. Subsequently, cells were counted once a day and growth curves of individual cultures were constructed in order to estimate cell damage (data not shown). In further experiments described below, illumination of cells was achieved during confocal imaging, i.e. by scanning a focused laser light beam in a raster pattern over a selected area of a cell culture maintained in a microincubator. One scan over the whole imaged area of 1103110 mm took 1 s. Thus, one pixel was illuminated for less than 4 ms in 1-s intervals. The beam delivered |0.1 mW (unless indicated otherwise) of 457-nm light focused onto a diffraction limited spot in the sample. The fluence rate was 50 kW/ cm 2 ; fluence was 0.16 J / cm 2 for one scan over one pixel. Thus, the total light dose delivered to cells was proportional to the number of illuminations (confocal frames). The setting of a black level and gain of the detector (see above) allowed accurate detection of the onset of the penetration of the dye into damaged cells. At these gain settings the luminescence of the dye accumulated in the nuclei of damaged cells eventually exceeded the dynamic range of the detector. Where indicated, during illuminations cells were submerged in physiological saline made in D 2 O (Institute of Nuclear Physics, Swierk, Poland). Anoxia was achieved by purging the saline solution with nitrogen. 3. Results 3.1. Penetration of Ru( II) complexes through biological membranes Penetration of Ru(II) complexes through cell plasma membranes was investigated by incubating fibroblasts with Ru(bipy) 21 (data not shown) or macrophages with 3 21 Ru(phen) 3 (0.2 mM; Fig. 1), under standard growth conditions. As shown in Fig. 1B, following an 8-min long incubation, luminescence of Ru(II) complexes was detected only in culture medium, outside cells. Following a 70-min incubation (in the dark) a growing number of cytoplasmic vesicles loaded with Ru(phen) 21 was ob3 served (Fig. 1C). Apparently, in a process of endocytosis Ru(phen) 21 was sequestered into membrane vesicles and 3 transported into the cell interior. The data presented in Fig. 1 indicate that Ru(phen) 21 has no capacity to cross intact 3 outer cellular membranes or membranes of endocytic vesicles. Similarly, when algae (Clostridium) and a fern leaf (Adiantum capillus veneris) were incubated in water supplemented with Ru(bipy) 21 3 , the fluorophore did not penetrate into cells [9]. 3.2. Affinity to plasma membranes When cells were incubated for 5 min with Ru(phen) 21 3 J.W. Dobrucki / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136 – 144 139 es, and the dyes subsequently removed, red luminescence remained in the region of outer cell membrane (Fig. 2; see also Fig. 1C). This observation indicates that, although Ru(II) complexes themselves had no affinity for membranes, illumination with light did result in a measurable accumulation of the dye in the region of plasma membrane. In this experiment, plasma membrane integrity was not compromised as demonstrated by fluorescein diacetate assay (data not shown) and the absence of Ru(II) complexes from cell nuclei (Discussion section). Thus, it is likely that the dye accumulated at the outer surface of plasma membrane. 3.3. Affinity to nucleic acids Affinity of Ru(II) complexes to intracellular structures exposed by fixation of cells with formaldehyde (1% at room temperature for 10 min) was investigated. A dye solution (2 mM) was added to fixed cells for 5 min and the unbound dye was removed by rinsing cell preparations three times with PBS. Microscopy observation revealed that the dye accumulated primarily in cell nuclei and some residual luminescence of the dye remained in cytoplasmic regions (data not shown). Following photodamage to plasma membranes a similar pattern of Ru(phen) 21 ac3 cumulation in cells was seen (see below). These observations were consistent with the reported affinity of Ru(phen) 21 for nucleic acids [14,29–31]. 3 3.4. Phototoxicity of Ru( II) complexes Fig. 1. Excluding Ru(phen) 21 by viable, intact macrophages and entry of 3 the dye into cells by means of endocytosis. Images represent red luminescence in a culture of macrophages. (A) Prior to adding Ru(phen) 21 (0.2 mM). Some granular autofluorescence in cytoplasm can 3 be detected. Bar represents 10 mm. (B) Following 8 min of incubation with Ru(phen) 21 3 . A confocal section through cells and the surrounding medium showing that Ru(phen) 321 does not enter cells and remains extracellular. (C) Following 70 min of incubation with Ru(phen) 21 and 3 removal of the dye-containing medium. Luminescence of Ru(phen) 21 is 3 detected exclusively in endocytic vesicles in the cytoplasm, and in plasma membranes (arrowhead); no Ru(phen) 21 can be detected in nuclei. 3 or Ru(bipy) 21 and the dyes subsequently removed, no 3 measurable luminescence of Ru(II) complexes remained associated with cells. However, if cells were illuminated with 457-nm light during incubation with Ru(II) complex- Although Ru(phen) 21 is excluded by cells during short 3 incubations in the dark, exposure to light may result in phototoxicity of this fluorophore. The phototoxic effects exerted by Ru(phen) 21 were measured in order to establish 3 the concentrations of the dye and the number of confocal sections that could be collected if Ru(phen) 21 was to be 3 used as a fluorescent oxygen probe in direct contact with live cells. Viable macrophages were maintained under optimal growth conditions, in the microincubator resting on the microscope stage. Immediately after adding Ru(phen) 21 (0.2 mM) to culture medium a selected area of 3 the cell culture was exposed to 457-nm light. Under these conditions no measurable endocytosis of Ru(phen) 21 took 3 place. When the number of illuminations exceeded 120 some deterioration of plasma membrane integrity became evident and Ru(phen) 21 started to slowly enter cells (Fig. 3 3). This implies that illumination-induced phototoxic damage compromises membrane integrity. Initially, Ru(phen) 21 which passed through plasma membranes 3 accumulated in cell nuclei (a representative sequence of images can be viewed at http: / / helios.mol.uj.edu.pl). At this stage the cytoplasm exhibited no measurable luminescence of the dye. Subsequent doses of light resulted in a sudden collapse of plasma membrane integrity and a massive entry of the dye followed by Ru(phen) 21 accumu3 140 J.W. Dobrucki / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136 – 144 Fig. 2. Accumulation of Ru(phen) 21 on plasma membranes of macrophages. A cross-section of a confocal image of one cell, recorded prior (A) and 3 following (B) 20 illuminations (457 nm, 0.4-mW laser beam) in the presence of 2 mM Ru(phen) 321 . Arrows depict position of plasma membranes. lation in cell nuclei and cytoplasm. The intensity of intracellular Ru(phen) 21 luminescence exceeded that of 3 culture medium. These high luminescence values resulted partly from an increase of luminescence of Ru(phen) 21 3 upon binding to DNA [14] and partly from an accumulation of the dye bound to DNA [14], and possibly RNA, in damaged cells. As a control, at the end of each experiment the area of the culture, which had not been exposed to laser illumination, was examined. Typically, no accumulation of the dye was detected confirming that damage to plasma membranes was a result of phototoxicity rather than any inherent toxicity of the Ru(II) complex. Fig. 3. Phototoxicity of Ru(II) complexes manifested by a loss of plasma membrane integrity and entry of the dye into cytoplasm. The traces depict intensity of luminescence of Ru(phen) 21 determined in culture medium 3 (a) and in the interiors (cytoplasm and nucleus) of six selected cells (b), during illumination with 457-nm light. For clarity, in each trace only every tenth reading of luminescence is plotted. Luminescence of the dye accumulated in nuclei eventually exceeded the dynamic range of the photomultiplier. A summary of a series of similar experiments, depicting phototoxic effects occurring in the presence of three different concentrations of Ru(phen) 321 , is shown in Fig. 4. The number of confocal sections that could be collected without inflicting damage on plasma membranes was |20, 80, and over 120, at concentrations of 2, 1 and 0.2 mM of Ru(phen) 21 3 , respectively. Membranes of endocytic vesicles were also subject to photodamage. In a similar experiment, macrophages were exposed to Ru(phen) 21 for 90 min prior to illumination 3 and allowed to internalize the dye. Following this incubation the cytoplasm contained a large number of endosomes loaded with Ru(phen) 21 (Fig. 5B). The subsequent illumi3 nation of cells resulted in breakage of the membranes of Fig. 4. Photodamage exerted on plasma membranes by Ru(phen) 21 at 3 three different concentrations. Each point is a mean of intensities of luminescence of Ru(phen) 21 entering eight selected cells. The values of 3 standard deviation were within the 10–60 range. The traces for individual cells were collected as in Fig. 3. The curves were normalized to a level of red autofluorescence of cytoplasm. J.W. Dobrucki / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136 – 144 141 endosomes and release of Ru(phen) 21 3 . Consequently, intact luminescing vesicles disappeared from images and the liberated dye accumulated in cell nuclei (Fig. 5C). When only selected regions of large cells (spindle-shape human fibroblasts and some plant cells) were illuminated in the presence of Ru(phen) 21 and Ru(bipy) 21 3 3 , the dyes entered cells and accumulated exclusively in the regions exposed to light. An example of this phenomenon is shown in Fig. 6. This could be explained by the fact that plasma membranes, and subsequently internal membranes, were damaged exclusively in the illuminated areas. Subsequently, Ru(phen) 21 became bound to membranes in these 3 damaged regions. 3.5. Mechanism of phototoxicity of Ru( phen) 21 3 In order to understand the mechanism of phototoxicity induced by Ru(phen) 21 3 , damage to plasma membranes was measured in the presence and absence of oxygen. Measurements were also carried out in heavy water, which is known to lengthen the lifetime of singlet oxygen [32], and in the presence of histidine (1 mM), a photoprotector that acts by scavenging singlet oxygen [33]. Fig. 7 illustrates representative experiments in which luminescence of Ru(phen) 21 was followed in individual cells. 3 Under standard conditions, |100–150 illuminations could be performed before loss of membrane integrity allowed the dye to enter cells (Fig. 3). In the absence of oxygen over 500 illuminations delivered to cells caused no damage to plasma membranes (Fig. 7). In the presence of heavy water |20–30 frames were sufficient to compromise plasma membrane integrity. When histidine was added to physiological saline made in D 2 O, the number of frames resulting in damage to plasma membranes was again over 100. Accumulation of the dye in cells was significantly slower (Fig. 7) and cell swelling was observed. Cells in unilluminated areas did not accumulate the dye. These observations indicate that singlet oxygen is involved in the photodynamic damage exerted by Ru(phen) 21 3 . 4. Discussion 4.1. Interaction of Ru( II) complexes with cells 21 3 Fig. 5. Photodamage to endosomes loaded with Ru(phen) (2 mM). (A) A transmitted light image of a group of macrophages (prior to incubation with the dye). Bar represents 10 mm. (B) A confocal image of red luminescence, collected after 90-min incubation with Ru(phen) 21 in the 3 dark and removal of the dye-containing medium. Endosomes are loaded with Ru(phen) 21 3 . (C) Red luminescence of the dye following 110 illuminations. This illumination caused rupture of endosomes, release of the dye into cytoplasm and subsequent accumulation in nuclei. The photomultiplier gain was three times higher in C than in B. This report demonstrates that selected Ru(II) complexes are unable to cross intact biological membranes. This behavior could be anticipated as they carry a positive charge and are insoluble in lipids. In this respect Ru(II) complexes behave similarly to a known ‘vital dye’, propidium iodide (PI) [34]. PI is also a water soluble, positively charged molecule and is known to remain extracellular with respect to live cells. The analogy can be taken further, since Ru(phen) 21 3 , just as PI, enters cells 142 J.W. Dobrucki / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136 – 144 Fig. 7. Photodamage exerted on plasma membranes by Ru(phen) 21 (0.2 3 mM), in the environment in equilibrium with air, in the absence of oxygen, in the presence of D 2 O, and D 2 O with histidine. Each curve depicts changes of intensity of luminescence of Ru(phen) 21 in interior of 3 six to eight selected cells. Data for individual cells were collected as in Fig. 3. Fig. 6. Photodamage in a selected region of a cell. (A) A group of macrophages incubated with Ru(bipy) 21 (0.2 mM). The dye does not 3 penetrate into cells. Bar represents 10 mm. (B) Following illumination with 457-nm light photodamage occurs, the dye enters cells, and accumulates in nuclei and on internal membranes. (C) Following removal of extracellular Ru(bipy) 21 3 , the dye remains in cell nuclei and membranes. (D) An image of an area adjacent to the region illuminated previously. The border between the illuminated and unilluminated areas is marked with a white line. In some cells extending across this border, Ru(phen) 21 accumulated exclusively in the area illuminated previously, 3 while parts of the same cells that had not been illuminated remained devoid of the dye. with damaged plasma membrane and stains nucleic acids. In other words, Ru(phen) 21 can be used as a ‘vital dye’ in 3 vitro. However, the luminescence of free PI is very weak and increases 20–30-fold upon binding to DNA, while the intensity of luminescence of unbound Ru(phen) 21 is 3 relatively strong and increases by less than 50% upon binding to DNA [14,35]. Extracellular PI is usually not detectable while unbound Ru(phen) 21 in culture medium is 3 strongly luminescent and can be used to visualize intracellular space between intact cells in multicellular spheroids (J. Dobrucki, unpublished). In experiments involving Ru(phen) 21 and live cells, penetration of the dye through 3 plasma membranes of intact cells can serve as a convenient ‘internal control’ for photodynamic damage to cells. However, in an interesting report describing an attempt to measure intracellular oxygen concentrations using Ru(phen) 21 (1 mM) [24] potential phototoxic effects and 3 release of the dye from endosomes were not seen. If Ru(phen) 21 enters cells by endocytosis it will become 3 trapped in endocytic vesicles. However, when it is free to enter cells and diffuse throughout the cell interior, it appears to accumulate in cell nuclei and, to a lesser extent, in the cytoplasm. Since it is known that Ru(phen) 21 has 3 affinity for nucleic acids it appears most likely that it binds to nuclear DNA and cytoplasmic RNA. A pattern of binding in nuclei, which resembles the typical distribution of heterochromatin and nucleoli, is consistent with this hypothesis. This binding may also explain a higher local concentration of Ru(phen) 21 in nuclei than in medium 3 surrounding damaged cells. J.W. Dobrucki / Journal of Photochemistry and Photobiology B: Biology 65 (2001) 136 – 144 4.2. Potential use of Ru( II) complexes as cellular oxygen probes Potential use of Ru(II) complexes as oxygen probes in direct contact with living cells will require a compromise between the relatively high probe concentrations needed to record a strong luminescence signal and the need to minimize the dye concentration and light dose to limit phototoxic effects. Under the experimental conditions used here, phototoxic effects as expressed by plasma membrane integrity were undetectable when Ru(phen) 21 concentra3 tion was 2310 24 M. Under these conditions cells could tolerate a dose of light required to collect |100 confocal frames, using |0.1-mW beam of excitation light used in a confocal microscope. Preliminary measurements of luminescence lifetimes of Ru(II) complexes in single cells [8] and data presented here indicate that it may be possible to optimize conditions to non-invasively detect luminescence of Ru(II) complexes in extracellular space or in endosomes of single cells, and calculate extra- or intracellular concentrations of oxygen. 4.3. Mechanism of phototoxicity Photosensitizing action of Ru(II) complexes bound to DNA has been studied extensively. However, in the viable cell system used here, the dyes remained extracellular and had no direct contact with DNA. The first observable phototoxic effect was damage to plasma membranes. The influence of anoxia, heavy water and histidine suggests that singlet oxygen was involved in the damage inflicted on plasma membrane in the presence of Ru(phen) 21 3 . Indeed, generation of singlet oxygen by illuminated Ru(II) complexes has been reported [13,36]. Thus, one may put forward the following hypothesis: upon illumination extracellular and membrane-bound Ru(II) complex generates singlet oxygen. A local high concentration of singlet oxygen causes a sequence of events that eventually lead to plasma membrane damage, which is, in turn, manifested by a loss of membrane integrity and entry of the dye into cells. 5. Conclusions The data presented here and preliminary results reported previously [8] indicate that Ru(bipy) 21 and Ru(phen) 21 3 3 may be suitable for measurements of extracellular or endosomal concentrations of oxygen, using FLIM. 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