Intravital Fluorescence Microscopy: Impact of Light-induced Phototoxicity on Adhesion of Fluorescently Labeled Leukocytes

Abstract

Alterations in leukocyte/endothelium interaction due to phototoxic effects of the fluorescent dyes acridine orange (AO) and rhodamine 6G (Rh6G) were studied by intravital microscopy using the dorsal skinfold model in awake Syrian golden hamsters. AO (0.5 mg/kg/min; constant IV infusion) and Rh6G (0.1 μmol/kg; bolus IV) were administered via an indwelling venous catheter. Five to seven arterioles (35–55 μm) and postcapillary venules (30–65 μm) were investigated in each animal. Vessels were exposed four times for 30 sec to continuous light of the appropriate excitation wavelength with a 10–15-min time interval between exposures. Animals were randomly assigned to five experimental groups (five distinct light energy levels). AO and Rh6G induced leukocyte rolling/sticking in postcapillary venules and arterioles when exposed to high light energy levels. AO, but not Rh6G, induced arteriolar vasospasm when exposed to high light energies. The potential phototoxic effect of AO and Rh6G is demonstrated, as assessed by the stimulation of leukocyte–endothelium interaction and arteriolar vasospasm in vivo. This study underscores the necessity to optimize microscopic set-ups for intravital microscopy, to reduce the excitation light energy level significantly, and to perform stringent control experiments, ruling out an artificial phototoxicity-induced stimulation of leukocyte adhesion.

Keywords

intravital fluorescence microscopy leukocyte–endothelium interaction acridine orange rhodamine 6G phototoxicity microcirculation

Leukocyte–endothelium interaction is a key feature of inflammation (Granger and Kubes 1994; Menger and Lehr 1993; von Adrian 1993), wound healing (Erban 1993), and ischemia–reperfusion, as well as diverse acute and chronic inflammatory diseases (Lehr and Arfors 1994; Lehr et al. 1993b; Ross 1986). Intravital fluorescence videomicroscopy has been established as a versatile technique for the study of cell–cell interaction and blood flow at the level of the microcirculatory unit during these processes by using fluorescent markers such as acridine orange (AO) and rhodamine 6G (Rh6G) for direct visualization of leukocytes (Lehr et al. 1993a,c; Menger et al. 1992a,b).

That the illumination of fluorescently labeled cells or tissues with high energetic light of the appropriate excitation wavelength can cause structural changes or even cell death was suggested several years ago in in vitro cell culture experiments (Zdolsek 1993; Zdolsek et al. 1990; Olsson et al., 1989). This phenomenon has been ascribed to the phototoxic effect that depends on the energy level of the excitation light, the duration of light exposure, and the concentration of the fluorescent dye.

On the basis of these considerations, we have investigated whether these phototoxic effects can lead to in vivo activation of leukocytes labeled with AO or Rh6G and thereby influence their adhesion properties during intravital microscopic investigations in the dorsal skin-fold chamber preparation in hamsters. A better understanding of the direct effects of photoexcitation during in vivo studies of leukocyte–endothelium interaction might also help to explain discrepancies among different laboratories investigating leukocyte adhesion to microvascular endothelium.

Materials and Methods

Animal Model and Preparation

The experiments were performed in compliance with guidelines of the National Institute of Health for the use of laboratory animals. Forty female Syrian golden hamsters weighing 55–65 g were used for this study. The animals had free access to food (Purina lab chow 8604: vitamin E content 50.58 IU/kg) and water. This concentration of vitamin E in the laboratory chow is unlikely to exert inhibitory effects on leukocyte rolling and adhesion in response to fluorescent dyes (Willy et al. 1995).

The surgical technique has previously been described in more detail (Endrich et al. 1980). Briefly, under anesthesia with sodium pentobarbital (50 mg/kg ip) (Abbott; Chicago, IL), a dorsal skinfold on the back of the hamsters was stretched out perpendicular to the spine and sandwiched between two specially manufactured titanium frames. With an operation microscope and microsurgical instruments, one layer of the skinfold was carefully excised. The remaining layer, consisting of epidermis, subcutaneous tissue, and thin striated skin muscle was covered with a 12-mm circular coverglass (Schubert & Weiss; Labortechnik, Munich, Germany) incorporated into the metal frames. After a recovery period of 5 days, venous catheters (PE50) were inserted into a jugular vein. The catheters were tunneled subcutaneously to the dorsal side of the neck and exteriorized through the skin at a site close to the metal frame of the titanium chamber. Microcirculatory studies were performed on awake animals 1–2 days after catheter implantation.

Intravital Microscopy: Technical Set-up

An epi-illuminiscence fluorescence microscope (Laborlux D 513 591; Leitz, Wetzlar, Germany) was used for the intravital microcirculatory investigations. A 100-W/2 HBO mercury lamp (Osram; Berlin, Germany) connected to a short arc lamp power supply (model 1230; Leitz, Rockleigh, NJ) served as a light source and emitted light with a relative intensity peak at 490 nm wavelength. The light leaving the lamp housing was filtered through a heat absorption filter (514-396 heat, thickness 2 mm; Leitz) and various neutrodensity filters (ND), i.e., ND6, ND12, ND25, ND50, to reduce light energy. The number specification of each of these neutrodensity filters (∗∗∗ 45 mm) indicates the amount of light transmitted through the filter given as percent of the total incident light. By applying and combining different neutrodensity filters, we were able to expose the tissue repeatedly to distinct light energy levels. A heat absorption filter was installed in all experiments to protect the neutrodensity filters from temperature damage. The light was further passed through excitation filters for AO (D480/X 30, 4039; Chroma, Brattleboro, VT or Rh6G (TRITC 31002, 506; D605/55m; Chroma) and then reflected by a dichroic mirror which reflected wavelengths >400 nm to the skin muscle tissue of the hamster, and passed emitted wavelengths >500 nm to the camera. The dichroic filter and the excitation and emission filters were parts of the microscope filterblock (H3 513673 and TRITC 31002, 506; Leitz). For the intravital fluorescence microscopic investigations, a X20 water-immersion objective (WI 20, 0.4; Olympus, Tokyo, Japan) was used. The final magnification was 660-fold. The output of the CCD camera was then displayed on a monitor (12 VM 968; Audiotronics, North Hollywood, CA). The images were captured with a videocassette recorder (Video Hi8, EV-C100, NTSC; Sony, Tokyo, Japan) at a video frame rate of 30 frames/sec. A video timer (VTG33; Fora Company, Tokyo, Japan) was connected to the video recorder.

Non-intensified Camera System

A non-intensified CCD camera [CCD72, horizontal 570 TVL (television lines), vertical 350 TVL, pickup area 8.8 × 6.6 mm; Dage-MTI, Michigan City, IN∗∗∗J was used for visualization of fluorescently labeled leukocytes at higher light energy levels because it required stronger fluorescence signals for detection of the cells. The light sensitivity of this camera 0.001–0.002 fc (footcandles), which was equivalent to the sensitivity of other camera systems such as COHU 4400, FK6990 (COHU; Prospective Measurements, San Diego, CA) employed in other intravital microscopic studies using the identical animal model (Vollmer et al. 1994; Menger et al. 1992a,b; Nolte et al. 1992; Lehr et al. 1991).

Intensified Camera System

To detect weaker fluorescent signals at very low light levels, the CCD 72 camera was connected to an image intensifier; (GENIISYS Image intensifier; Dage–MTI, Michigan City, IN). This system enabled us to reduce the incident light energy level by a factor of 40-fold. The GENIISYS device uses an 18-mm GENII proximity-focused image intensifier as the light amplification device, with three basic components: a photocathode, a microchannel plate (MCP), and a phosphor screen. When light reaches the photocathode surface (P20), electrons are accelerated and are proximity-focused onto the micro-channel plate (MCP). The MCP multiplies the number of electrons through secondary emission and the output of the MCP is focused onto a phosphor screen. This electron bombardment of the phosphor screen creates an image that is optically coupled to the camera system (CCD 72). The photocathode (S20) has a spectral emittance (blue and green portion of the visible spectrum) similar to the spectral response of the dark adapted-human eye. The maximal light sensitivity of the intensified camera system was 10^-6 fc (lumen/feet2) which corresponds to 10^-5 lx (lumen/m2) (lx stands for lux). This camera system is approximately 100–1000-fold more light sensitive compared to the CCD 72 camera alone.

Measurement of Light Energy at the Tissue Level

Before the experiments a photodiode (UV-100BQ; EG&G Electro-Optics, Salem, MA) was positioned horizontally beneath the 20-fold objective (20WI 20, NA 0.4; Tokyo, Japan). As in the intravital microscopic studies, a drop of water was placed beneath the objective. The diode was adjusted until the light beam was centered and focused in the middle of the diode's window. We determined experimentally that the light did not overfill the diode sensitive surface which was a 2.5-mm-diameter circle. The outlet of the diode was connected to an ammeter (Beckman Industrial 310; Brea, CA). The photodiode's readout, given in milli-amperes (mA), was multiplied by a constant factor K = [(3.333 mW × 60 sec) / 1 mA] to obtain the correct value of light energy in mJ/min, as stipulated by the diode specifications. The diameter of the field (d) that an objective illuminates is approximately the microscope tube diameter divided by the objective magnification. In our microscopic setup this was d = (16 mm / 20) = 0.8 mm, which corresponded to an illuminated area (A) of A = 5 × 10^-3 cm². This represented the area to which the previously computed energy was applied. The energy per unit area (mJ/min/cm²) was E = K / (5 × 10^-3 cm²) = [(3.333 mW/mA) × 60 sec)] / 5 × 10^-3 cm² = (3.333 × 60) / 5 × 10^-3 mJ/min/cm². Our measurements revealed that the coverglass of the titanium chamber did not absorb any light, so that the above described measurements in fact represented the light energy at the tissue level.

Experimental Protocol

As described in previous publications, we exposed the skinfold microcirculation four times for 30 sec to the excitation light (Menger et al. 1992a,b; Nolte et al. 1992). A time interval of 10–15 min was allowed between each exposure. Five distinct light energy levels were chosen: 77.6 mJ/min/cm², 192.1 mJ/min/cm², 698.8 mJ/min/cm², and 1514.2 mJ/min/cm² and 3106.3 mJ/min/cm2 to which the vessel segments had been exposed. Approximately five to seven arterioles (diameter 30–55 μm) and five to seven venules (diameter 30–65 μm) were selected in each animal. AO (excitation wavelength 470 nm, emission wavelength 530–560 nm; Sigma, St Louis, MO) or purified AO A-1301; Molecular Probes, Eugene, OR) was given as a constant infusion via an indwelling jugular vein catheter at a concentration of 0.5 mg/kg/min. Rh6G (excitation wavelength 527 nm, emission wavelength 551 nm; Sigma) was given iv (via an indwelling catheter) as a bolus of 0.1 μmol/kg 5 min before the first observation. Both fluorescent dyes were administered at concentrations that have been used in previous investigations (Nolte et al. 1994; Vollmer et al. 1994; Lehr et al. 1992; Menger et al. 1992a,b).

Leukocyte rolling and leukocyte sticking in postcapillary venules and arterioles were evaluated and quantified off-line. In addition, the occurrence of arteriolar vasospasm was assessed. In accordance with previous studies, sticking leukocytes were defined as the total number of leukocytes that were firmly attached to the microvascular endothelium and did not change their location throughout the entire 30-sec light exposure period. Sticking leukocytes were given as number of cells/per mm² of vessel surface, calculated from the diameter and standardized length (200 μm) of the vessel segment under investigation.

Rolling leukocytes were defined as the total number of leukocytes rolling along the endothelial cell lining at a substantially slower velocity compared to the midstream blood cell velocity. Rolling leukocytes were given as number of cells passing an imaginary line perpendicular to the vessel's longitudinal axis over a period of 30 sec.

Vasospasm was defined as a complete closure of the vessel lumen with no signs of microvascular flow.

Statistical Analysis

The data in the figures are given as mean ± SD at each light energy level. Five animals were included in each of the investigated light energy level. p<0.05 was considered statistically significant and was calculated by single-factor, repeated-measures ANOVA.

Results

Figure 1 demonstrates that the photodiode output (mAmp) responded in a linear fashion to the reduction of incident light energy level (%). Reduction of light energy was achieved by using various neutrodensity filters (ND).

Acridine Orange

To obtain adequate images at the lower light energy levels (77.6 mJ/cm²/min, 192.1 mJ/cm²/min, 698.8 mJ/cm²/min), the intensified camera system had to be used because the fluorescence signal was too weak to be detected by the non-intensified camera alone (CCD 72). At higher light energy levels (1514.2 mJ/cm²/min, 3106.3 mJ/cm²/min), the experiments were executed with the CCD 72 without the intensifying system.

Figures 2 and 3 demonstrate the effect of AO-induced phototoxicity on leukocyte rolling (Figure 2) and sticking to the endothelium in arterioles and postcapillary venules (Figure 3). In postcapillary venules, we observed a baseline leukocyte rolling value of 6–8 leukocytes/30 sec observation period. Repeated exposures at the lowest light energy level (77.6 mJ/cm²/min) caused no changes in leukocyte rolling (Figure 2). Leukocyte rolling increased at light energy levels equal to or greater than 192.1 mJ/cm²/min. Slightly less pronounced leukocyte rolling was observed in arterioles. At the lower light levels, ranging between 77.6 mJ/cm²/min and 192.1 mJ/cm²/min, arteriolar leukocyte rolling could not be evoked by repeated light exposures (Figure 2). Reproducible data for leukocyte–endothelium interaction could not be obtained at light levels equal to or greater than 698.8 mJ/cm²/min because of significant reductions in arteriolar diameters as well as complete vasospasm.

Figure 1

This graph demonstrates the linear response of the photodiode according to the incident light energy level applied during the experiments. A 50 (75) % reduction of light energy by inserting the neutrodensity filter ND 50 (ND 25) led to a 50 (75) % reduction of the photodiode output from 0.078 to 0.039 mAmp (0.018 mAmp), respectively.

Figure 2

Effect of AO on leukocyte rolling in arterioles and postcapillary venules according to the light energy level applied during the experiments. Data are depicted as mean ± SD of n = 5 animals per light energy level. ∗p<0.05, single-factor repeated-measures ANOVA.

No sticking leukocytes during our baseline measurements (first light exposure) were found in arterioles and postcapillary venules (Figure 3). No leukocyte adhesion could be detected at light energy levels at and below 192.1 mJ/cm²/min. At higher light levels, repeated light exposures of the same vessel segment resulted in a steady, almost linear increase in leukocyte sticking in postcapillary venules (Figure 3).

In a series of control experiments, no leukocyte–endothelium interaction or arteriolar diameter changes were observed when the tissue was repeatedly exposed to light of various energy levels but without previous injection of the photosensitizing dye (data not shown).

Acridine Orange-induced Vasospasm

At light energy levels of 77.6 and 192.1 mJ/cm²/min, no arteriole that was exposed to the incident light showed complete vasospasm (0%). At light energy levels of 698.8, 1514.2, and 3106.3 mJ/cm²/min, 66%, 100%, and 100% of all investigated arterioles, respectively, went into complete vasospasm. At light energy levels of 1514.2 mJ/cm²/min and 3106.3 mJ/cm²/min virtually all arterioles went into spasm after the second light exposure. Upstream and downstream from the site of light exposure, the afflicted arteriole exhibited normal vessel diameters. Observation of the identical vessels 24 hr after light exposures revealed in most cases that the arteriolar vasospasm was still present at the site of light epi-illumination. Vasospasms occurred after 1.9 ± 0.3, 2.1 ± 0.3, and 2.9 ± 0.2 exposures at light levels or 3106.3, 1514.2, and 698.8 mJ/cm²/min, respectively.

Rhodamine 6G

Because of the much weaker fluorescence signal from Rh6G- labeled leukocytes, we could not obtain satisfactory images at light levels of 77.6 mJ/cm²/min and 192.1 mJ/cm²/min, even using the intensified system. All the images were captured with the image-intensifying camera system.

At light levels beyond 698.8 mJ/cm²/min, leukocyte rolling increased slightly after each exposure in postcapillary venules, but not in arterioles (Figure 4). Similar results were obtained for sticking leukocytes in postcapillary venules and arterioles (Figure 5). No sticking leukocytes were evident during the baseline measurements (first exposure). In postcapillary venules, the number of sticking leukocytes increased slightly after each subsequent exposure to light levels of 1514.2 mJ/cm²/min and 3106.3 mJ/cm²/min, but not at 698.8 mJ/cm²/min.

Figure 3

The effect of acridine orange on leukocyte sticking in arterioles and postcapillary venules according to the light energy level used during the experiments. Data are depicted as mean ± SD of n = 5 animals per light energy level. ∗p<0.05, single-factor repeated-measures ANOVA.

We did not observe any arteriolar vasospasm or arteriolar diameter changes at any of the light levels investigated when Rh6G was used as the fluorescent dye.

Discussion

The purpose of this investigation was to test the hypothesis that the energy level of the incident light used for illumination of fluorescently labeled leukocytes influences leukocyte–endothelial interaction in vivo and to determine the most appropriate experimental methodology for direct in vivo microcirculatory studies to avoid this artifact. The principal findings of this investigation are as follows: Leukocyte rolling and adhesion to endothelial cells can be provoked by photo-excitation of these fluorescent dye molecules. The influence of these photochemical processes on cell activation is directly related to the amount of light energy used for illumination of these cells. In contrast to Rh6G, AO caused arteriolar vasospasm and diameter changes at high light energy levels. Finally, visualization of fluorescently labeled leukocytes by an intensified camera system allows significant reduction of incident light energy to levels at which phototoxic interference can be excluded.

Intravital Microscopic Set-up

The basic set-up for intravital fluorescence microscopy requires a high-energy light source (usually a mercury or xenon lamp), a microscope suited for epi- or transillumination, and a camera. The quality of the camera (light sensitivity) is the most important determinant of the amount of incident light energy that must be used during the experiments. In the present study, the light sensitivity of our non-intensified camera system (CCD72) was comparable to those used by us in previous studies, as well as by other investigators (Nolte et al. 1994; Vollmer et al. 1994; Menger et al. 1992a,b; Nolte et al. 1992; Lehr et al., 1991). This camera allowed a twofold reduction (from 3106 mJ/min/cm² to 1514) of the incident light energy level in our experimental set-up. This light energy level was still too high to reliably prevent AO-induced phototoxicity under the experimental conditions of this study. The incident light levels could not be sufficiently lowered to avoid these effects and still provide enough light for visualization without the use of a light-intensified camera system. Upgrading the non-intensified camera system by combining it with an image intensifier allowed a significant reduction of the incident light energy by a factor of 40-fold (from 3106 mJ/min/cm² to 77 mJ/min/cm²). At this very low light level, no AO-induced phototoxic artifacts were observed (Figures 2 and 3).

Figure 4

Effect of rhodamine 6G on leukocyte rolling in arterioles and postcapillary venules according to the light energy level applied during the experiments. No satisfactory images of the fluorescently labeled leukocytes within the microcirculation could be obtained at light levels of 77.6 mJ/cm²/min and 192.1 mJ/cm²/min. Data are depicted as mean ± SD of n = 5 animals per light energy level. ∗p<0.05, single-factor repeated-measures ANOVA.

Cell Activation Through Photoexcited Acridine Orange

AO (mw 669.7) is a weak acidophilic dye that can readily diffuse through cell membranes. Because of its weak acidotropic properties, AO accumulates in acidic cell compartments on protonation, e.g., lysosomes, endosomes, and the Golgi apparatus (Zdolsek, 1993; Allison et al., 1966). It also accumulates in cell nuclei and intercalates with DNA and RNA (Ito 1978). The mechanism of action responsible for cell activation or even cell destruction in vitro by the photosensitizing fluorescent AO has been defined (Zdolsek 1993; Zdolsek et al., 1990). It was demonstrated in studies with cell cultures that the AO-mediated phototoxic effect was predominantly the result of a singlet oxygen mediated reaction (Type II reaction) and a radical-mediated (Type I) reaction (Zdolsek, 1993; Zdolsek et al. 1990; Olsson et al. 1989; Martin and Logsdon, 1987). These authors speculated that lysosomal membranes of AO loaded and then light-exposed cells are the primary site for attack by these reactive oxygen intermediates. As a consequence, peroxidative damage to lysosomal membranes would allow leakage of hydrolytic enzymes into the cytosol, resulting in uncontrolled cell activation or, in extreme cases, self-digestion (Olsson et al. 1989). These processes are more pronounced in a hyperoxic environment, whereas hypoxia significantly protected cells against photodamage (Zdolsek 1993; Zdolsek et al. 1990; Olson et al. 1989; Martin and Logsdon, 1987), emphasizing the involvement of lipid peroxidation. Even though these processes have thus far been noted only in cell cultures, we can speculate that similar photochemical processes might occur in vivo. In fact, Povlishock et al. (1983) showed in vivo that excited intravascular sodium fluorescein (whose excitation and emission wavelengths are identical to those of acridine orange) induced microvascular endothelial cell injury, resulting in subsequent platelet aggregation. Electron microscopy of the injury site demonstrated that the luminal cell membrane damage, the swelling of the nuclear membrane, and the cellular vacuolization closely mimicked the pattern of cell damage seen after free radical-mediated lipid peroxidation (Farber et al. 1990; Povlishock et al. 1983). The occurrence of arteriolar constriction during the first light exposures might be due to similar radical mediated events. Inhibition of endothelial cell-mediated vasodilatation has been proposed (Rosenblum 1986,1988), resulting in an unbalance between vascular smooth muscle contracting and dilating mechanisms (Olsson et al. 1989; Gryglewski et al. 1986; Rubanyi and Vanhoutte 1986).

Figure 5

Effect of Rh6G on leukocyte sticking in arterioles and postcapillary venules according to the light energy level applied during the experiments. No satisfactory images of the fluorescently labeled leukocytes within the microcirculation could be obtained at light levels of 77.6 mJ/cm²/min and 192.1 mJ/cm²/min. Data are depicted as mean ± SD of n = 5 animals per light level. ∗p<0.05, single-factor repeated-measures ANOV.

Figure 6

(a) Arteriole (diameter 50 μm) in striated muscle at the end of the first light exposure period. A postcapillary venule crosses the arteriole. The vessel borders of the arteriole are clearly demarcated by AO-labeled endothelial cells. (b) The entire segment of the same arteriole (diameter 15 μm) that was exposed to light (a) is almost completely constricted. This image was taken during the second light exposure period. The postcapillary venule does not show signs of vascular narrowing.

There are a number of potential mechanisms through which radical-mediated cell activation could elicit leukocyte adhesion. Patel et al. (1991) showed in in vitro experiments that intra- and extracellular reactive oxygen species induce prolonged upregulation of adhesion molecules such as P-selectin, which mediate the rapid induction of leukocyte–endothelium interaction. In addition, reactive oxygen species induce the generation of platelet activating factor-like lipids that recognize specific receptors for PAF on the leukocyte surface and elaborate firm leukocyte adhesion to endothelial cells (Lehr et al. 1993c; Zimmerman et al. 1992). Previous studies using the same animal model have shown that leukocyte activation in response to pro-oxidant stimuli can be effectively blocked by PAF receptor antagonists or either by superoxide dismutase or by anti-P-selectin antibodies, suggesting that these mechanisms of leukocyte–endothelial interactions are operative under the conditions of our experiments (Lehr et al. 1992a,b,1993c,1994). Interestingly, it has been demonstrated that, in contrast to excited AO, non-excited AO inhibits leukocyte metabolism, thereby reducing the ability of leukucytes to adhere to bovine serum albumin matrix through CD11/CD18 and to produce superoxide anions (Hansell et al. 1994).

Cell Activation Through Photoexcited Rhodamine 6G

Rh6G is a cationic fluorescent dye (mw 479) that selectively accumulates in nuclei and mitochondria of living cells (Horobin and Rashid 1990; Ranganathan et al. 1989). Rhodamines are positively charged at physiological pH, which does not interfere with their lipophilic nature. Once the Rh6G enters the mitochondria, it becomes trapped owing to the high negative electrical potential across the mitochondrial membrane (Rashid and Horobin 1990; Ranganathan et al. 1989). Excited Rh6G can inhibit oxidative phosphorylation and mitochondrial respiration (Ranganathan et al. 1989). It has been proposed that dye excitation leads to reduction of the dye molecule, thereby releasing dye and solvent radicals (Soper et al. 1993). Oxygen may play a key role because it is the major dissolved component in aqueous solutions and is consumed during the photochemical reaction of Rh6G (Kato and Sugimura 1974). At present it is not clear how these processes might lead to cell activation and hence cell adhesion. It appears that oxygen radicals and intra-and extracellular radical formation might play an important role in this process, leading to lipid peroxidation and upregulation of adhesion receptors as previously described for AO (Olsson et al. 1989).

Phototoxicity-induced alterations of microvascular perfusion, vascular diameter, and cell function are common phenomena inherent not only to AO or Rh6G but also to other fluorescent dyes when excited at the appropriate wavelengths (Kerger et al. 1995; Friesenecker et al 1994; Gawlowski et. 1989; Herrmann 1983; Rosenblum and El-Sabban 1977).

The results of the present study emphasize that intravital fluorescence microscopic approaches to study cell–cell interactions are susceptible to phototoxic artifacts. The amount of light needed to visualize leukocytes in vivo is determined by the quality of the camera system. Non-intensified cameras with limited light sensitivity options require significantly stronger fluorescence signals to obtain images with an adequate signal-to-noise ratio. To generate stronger signals, fluorescently labeled cells and tissues must be exposed to higher incident light energy levels, thus substantially increasing the risk for phototoxic effects. In contrast, intensified cameras, which are 100–1000-fold more light sensitive than non-intensified systems, can register much weaker fluorescent signals. When the amount of excitation light energy is reduced considerably, by use of an intensified camera system, the risk of phototoxicity is abolished.

Our study therefore emphasizes the need to optimize the microscopic set-up to obtain adequate imaging of the microcirculation with a minimum of light energy. This can be achieved (a) by using various optical filters and image intensified camera systems, (b) by applying the smallest amount of the fluorescent dye in a standardized fashion (constant infusion or single bolus), (c) by reducing observation time periods and, most importantly, (d) by performing stringent control experiments to exclude phototoxic activation of leukocytes. When these conditions are established, acridine orange and rhodamine 6G can continue to be of great help in the investigation of leukocyte–endothelial cell interaction by means of intravital microscopy.

Footnotes

Acknowledgements

Supported by NIH grant no. HL-46022. The results were presented in part during the 43rd Annual Conference of the Microcirculatory Society, April 13–14, 1996, in Washington, DC.

References

Allison

Magnus

Young

(1966) Role of lysosomes and of cell membranes in photosensitization. Nature 209:874–878

Endrich

Asaishi

Götz

Me∗∗∗mer

(1980) Technical report—a new chamber technique for microvascular studies in unanesthetized hamster. Res Exp Med 177:125–134

Erban

(1993) P-selectin and wound healing. Behring Inst Mitt 92:248–257

Farber

Kyle

Coleman

(1990) Biology of disease: mechanisms of cell injury by activated oxygen species. Lab Invest 62:670–679

Friesenecker

Tsai

Intaglietta

(1994) Capillary perfusion during ischemia-reperfusion in subcutaneous connective tissue and skin muscle. Am J Physiol 267:H2204–H2212

Gawlowski

Harding

Granger

(1989) Leukocyte phagocytosis and alterations in microvascular integrity elicited by FITC-dextran 150 and epi-illumination in the microcirculation of the hamster cheek pouch. Microvasc Res 37:1–15

Granger

Kubes

(1994) The microcirculation and inflammation: modulation of leukocyte/endothelial cell adhesion. J Leukocyte Biol 55:662–675

Gryglewski

Palmer

RMJ

Moncada

(1986) Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320:454–455

Hansell

Berger

Chambers

Arfors

(1994) Influence of acridine orange tracer dyes on neutrophil function. J Leukocyte Biol 56:464–468

10.

Herrmann

(1983) Platelet aggregation induced in the hamster cheek pouch by a photochemical process with excited fluorescein isothiocyanate-dextran. Microvasc Res 26:238–249

11.

Horobin

Rashid

(1990) Interactions of molecular probes with living cells and tissues. Part 1. Some general mechanistic proposals, making use of a simplistic Chinese box model. Histochemistry 94:205–209

12.

Ito

(1978) Cellular and subcellular mechanisms of photodynamic action: the 1O2 hypothesis as a driving force in recent research. Photochem Photobiol 28:493–508

13.

Kato

Sugimura

(1974) Deterioration of rhodamine 6G dye solution in methanol. Opt Commun 10:327–339

14.

Kerger

Torres Filho

Rivas

Winslow

Intaglietta

(1995) Systemic and subcutaneous microvascular oxygen tension in conscious Syrian golden hamsters. Am J Physiol 268:H802–810

15.

Lehr

H-A

Arfors

(1994) Mechanisms of tissue damage by leukocytes. Curr Opin Hematol 1:92–99

16.

Lehr

H-A

Becker

Marklund

Hübner

Arfors

K-E

Kohlschutter

Messmer

(1992a) Superoxide-dependent stimulation of leukocyte adhesion by oxidatively modified LDL in vivo. Arterioscler Thromb 12:824–829

17.

Lehr

H-A

Hübner

Finckh

Angermüller

Nolte

Beisiegel

Kohlschdütter

Messmer

(1991) Role of leukotrienes in leukocyte adhesion following systemic administration of oxidatively modified human low density lipoprotein in hamsters. J Clin Invest 88:9–14

18.

Lehr

H-A

Hübner

Menger

Messmer

(1993b) Mechanisms and mediators of leukocyte/endothelium interaction during atherogenesis. Atheroscler Rev 25:49–57

19.

Lehr

H-A

Kress

Menger

(1993a) Involvement of 5-lipoxygenase products in cigarette smoke-induced leukocyte/endothelium interaction in hamsters. Int J Microcirc Clin Exp 12:61–73

20.

Lehr

H-A

Kress

Menger

Friedl

Hübner

Arfors

K-E

Messmer

(1992b) Cigarette smoke elicits leukocyte adhesion to endothelium in hamsters: inhibition of CuZn-SOD. Free Radical Biol Med 14:573–581

21.

Lehr

H-A

Olofsson

Carew

Vajkoczy

von Andrian

Hübner

Berndt

Steinberg

Messmer

Arfors

(1994) P-Selectin mediates the interaction of circulating leukocytes with platelets and microvascular endothelium in response to oxidized lipoprotein in vivo. Lab Invest 71:380–386

22.

Lehr

H-A

Seemüller

Hübner

Menger

Messmer

(1993c) Oxidized LDL-induced leukocyte/endothelium interaction in vivo involves the receptor for platelet-activating factor. Arterioscler Thromb 13:1013–1018

23.

Martin

Logsdon

(1987) Oxygen radicals mediate cell inactivation by acridine dyes, fluorescein, and Lucifer yellow CH. Photochem Photobiol 46:45–53

24.

Menger

Lehr

(1993) Scope and perspective of intravital microscopy—bridge over from in vitro to in vivo. Immunol Today 14:519–522

25.

Menger

Pelikan

Steiner

Messmer

(1992b) Microvascular ischemia-reperfusion injury in striated muscle: significance of “reflow paradox.” Am J Physiol 263:H1901–H1906

26.

Menger

Steiner

Messmer

(1992a) Microvascular ischemia-reperfusion injury in straited muscle: significance of “no reflow.” Am J Physiol 263:H1892–H1900

27.

Nolte

Bayer

Lehr

Becker

Krombach

Kreimeier

Messmer

(1992) Attenuation of postischemic microvascular disturbances in striated muscle by hyperosmolar saline dextran. Am J Physiol 263:H1411–H1416

28.

Nolte

Schmid

Jäger

Botzlar

Roesken

Hecht

Uhl

Messmer

Vestweber

(1994) Leukocyte rolling in venules of striated muscle and skin is mediated by P-selectin, not by L-selectin. Am J Physiol 267:H1637–H1642

29.

Olsson

Brunmark

Brunk

(1989) AO-mediated photo-damage of microsomal- and lysosomal fractions. Virchows Arch [B] 56:247–257

30.

Patel

Zimmerman

Prescott

McEver

McIntyre

(1991) Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils. J Cell Biol 112:749–759

31.

Povlishock

Rosenblum

Sholley

Wei

(1983) An ultrastructural analysis of endothelial change paralleling platelet aggregation in a light/dye model of microvascular insult. Am J Pathol 110:148–160

32.

Ranganathan

Churchill

Hood

(1989) Inhibition of mitochondrial respiration by cationic rhodamines as a possible teratogenicity mechanism. Toxicol Appl Pharmacol 99:81–89

33.

Rashid

Horobin

(1990) Interaction of molecular probes with living cells and tissues. Part 2. Histochemistry 94:303–308

34.

Rosenblum

El-Sabban

(1977) Platelet aggregation in the cerebral microcirculation. Circ Res 40:320–328

35.

Rosenblum

(1986) Endothelial dependent relaxation demonstrated in vivo by cerebral arterioles. Stroke 17:494–497

36.

Rosenblum

(1988) Loss of endothelium-dependent relaxation in mouse cerebral microvessels may be rapidly reversible. Microvasc Res 35:132–138

37.

Ross

(1986) The pathogenesis of atherosclerosis—an update. N Engl J Med 314:488–500

38.

Rubanyi

Vanhoutte

(1986) Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol 250:H822–H827

39.

Soper

Nutter

Keller

Davis

Shera

(1993) The photophysical constants of several fluorescent dyes pertaining to ultrasensitive fluorescence spectroscopy. Photochem Photobiol 57:972–977

40.

Vollmer

Lang

Menger

Messmer

(1994) Hypertonic hydroxyethyl starch restores hepatic microvascular perfusion in hemorrhagic shock. Am J Physiol 266:H1927–H1934

41.

Von Andrian

Chambers

Berg

Michie

Brown

Karolak

Ramezani

Berger

Arfors

Butcher

(1993) L-selectin mediates neutrophil rolling in inflamed venules through sialyl LewisX-dependent and -independent recognition. Blood 82:182–191

42.

Willy

Thiery

Menger

Messmer

Arfors

K-E

Lehr

(1995) Impact of vitamin E supplement in laboratory animal diet on ischemia-reperfusion injury. Free Radical Biol Med 19:919–926

43.

Zdolsek

(1993) AO-mediated photodamage to cultured cells. APMIS 101:127–132

44.

Zdolsek

Olsson

Brunk

(1990) Photooxidative damage to lysosomes of cultured macrophages by acridine orange. Photochem Photobiol 51:67–76

45.

Zimmerman

Prescott

McIntyre

(1992) Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol Today 13:93–100