A. Imaging Phosphorescence Intensity from the Vasculature of the Retina
The camera was focused on the retina, the brightness adjusted, and a sequence of images collected. The sequence of images used delays ranging from 0° to 360° relative to the excitation. A sequence of phosphorescence intensity images, collected by use of 1030 Hz modulation, is shown in . There is a network of small vessels between the major arterioles and veins in the retina that appear to be quite uniformly ~10 µm in diameter. We refer to this network of small vessels as capillaries, although this is likely to be an oversimplification. Observing these small vessels requires that there be little movement of the mouse during acquisition of the image.
As shown in , the individual images of phosphorescence intensity show with good resolution the veins and the arteries that supply blood to the retina. The images are similar to those obtained during fluorescein angiography. In both cases, the luminophors are dissolved in the blood plasma and, therefore, the intensity images show vascular structure. Leaks of either fluorescein or the phosphor through the vessel walls lead to the appearance of small local regions of increased intensity spots in the images, and both the intensity and the size increase with time after phosphor injection. The permeability of fluorescein to the vessel walls is much higher than that of Oxyphor G2 (charge, 1 compared with −16; molecular weight, 332 compared with 2,642). The change in the image that is due to dye leakage is greater for the phosphor than for fluorescein. As phosphor leaks out of the vessel, the phosphorescence intensity increases not only because of the increase in the amount of dye near the locus of the leak but also because the oxygen pressure is lower in the extravascular space than in the vessels.
The phosphor is present only in the blood plasma, so the phosphorescence intensity in the images is related to both the blood volume and the oxygen pressures. Thus the lower oxygen pressures in the veins relative to the arterioles result in the veins’ being brighter than the arterioles. Larger vessels are brighter than small vessels, and the capillary bed areas, which lack vessels larger than ~10 µm diameter, show much weaker phosphorescence than the larger vessels. Individual capillaries can be seen in the phosphorescence images but are not resolved in the lifetime and oxygen maps unless they are blocked or otherwise depleted in oxygen relative to the surrounding capillaries.
B. Maps of Phosphorescence Lifetime and Oxygen Pressure
Typically, a set of phosphorescence intensity images (see above) with phase delays of 0° to 360° and at 30° intervals was taken. The in-phase correction was obtained by imaging with frequency 36 kHz at 0° and 180° (see above). Subtracting the 180° image from the 0° image yields the intensity image that corresponds to lifetimes of less than 1 µs, i.e., reflected light and fluorescence. This in-phase signal was subtracted from all images in the set to give a set of corrected images for calculation of the phosphorescence lifetimes. shows a plot of the phosphorescence intensity in three regions of interest, corresponding to two veins and an arteriole, as a function of the phase delay used to collect the intensity images. The sets were fitted to sinusoids in each pixel, and the phase shift was determined for each fit. The phosphorescence-lifetime image was calculated from Eq. (3)
and converted to the oxygen image by use of Eq. (8)
Fig. 2 Phosphorescence intensity versus phase delay (in degrees) for three regions of interest in the image, two veins and one arteriole. Least-squares fits to a sinusoid yielded phase shifts of 43.6° and 40.4° for the veins and 35.1° (more ...)
The phase delay image, the phosphorescence-lifetime image and, the oxygen pressure image are displayed in . Visualizing the phase delay image is important because the conversion of the lifetime image into the oxygen image can be done most reliably if the lifetimes are determined at frequencies at which the delays are 25° to 35°. Each phosphor is characterized by a distribution of lifetimes and, therefore, changing the phase delay in a frequency-domain measurement can lead to differences in the apparent lifetimes, τ. The narrower the distribution in lifetimes, the less effect the difference in phase delays has on the calculated lifetime. When there were regions in the map with phase shifts that differed substantially (> 10°) from the phase (28°) used for phosphor calibration, they were imaged again at a frequency for which the phase shift was nearer 28°.
Fig. 3 Maps of (a) the phosphorescence intensity at 30° phase shift, (b) the phase shift between excitation and phosphorescence, (c) the phosphorescence lifetime, and (d) the oxygen pressure calculated from the set of images in . The maps correspond (more ...)
In the region of the optic nerve’s head, the images show relatively large veins and arterioles radiating from a small central region (). As you progress around the disk, the vessels alternate between arterioles and veins, and this is clearly seen in both lifetime and oxygen images. The vessels alternate between low and high oxygen pressures and long and short phosphorescence lifetimes. The oxygen pressure in the veins is typically 30–45 mm Hg, whereas that in the arterioles is 60–80 mm Hg. In regions farther from the optic nerve’s head there are substantial areas of capillary bed that exhibit higher oxygen pressures, usually 50–100 mm Hg. This result is consistent with some of the blue (450 nm) excitation light passing through the retina and exciting a small amount of diffuse phosphorescence from the underlying choriocapillaris where the oxygen pressure is high (short lifetime). When this choroidal signal is added to the weak phosphorescence from the retinal capillaries, the resultant mixed signal gives calculated oxygen pressures between those of the retina and of the choroid. The total intensity is very low, such that the oxygen values also become noisy. Where the phosphorescence signal from the retinal vessels is higher, such as for the veins and arterioles, the oxygen pressures in the individual vessels can be accurately determined.
C. Dependence of the Retinal Oxygen Maps on Time after Injection of the Anesthetic
When mice are given anesthetics, the animals often go through a transient period of decreased blood pressure, abnormal breathing patterns, or both. The duration and severity of this period are quite variable among animals owing to individual differences in biochemical and physiological responses to the anesthetic. In the experiment shown in and , oxygen maps were repetitively measured at 1–2 min intervals from the earliest time attainable (3 min) until the mouse began to recover from anesthesia. Representative data are shown in as the phosphorescence intensity images and oxygen pressure maps at 5 and 35 min after anesthesia, shortly before the mouse awakened. In this animal, the venous oxygen pressures increased with time after anesthesia from ~15 mm Hg at 5 min to ~50 mm Hg at 35 min. In contrast, the arteriolar values were essentially constant and near 75 mm Hg. The time course of the process is shown in , where the oxygen pressures in selected regions of two arterioles and two veins are plotted against the time after anesthesia.
Fig. 4 Dependence of the retinal oxygen pressures on time after induction of anesthesia. The mouse was given an intraperitoneal injection of anesthetic, the pupil was dilated, and retinal imaging of the phosphorescence lifetimes began ~3 min later. The measurements (more ...)
Fig. 5 Time course of the oxygen pressures in the retinal veins and arterioles following anesthesia. Oxygen pressure maps of the retina were repetitively measured; a total of 19 measurements was made over the period of 3–35 minutes following anesthesia. (more ...)
The time course of the oxygen measurements after induction of anesthesia is highly variable among mice, ranging from almost no change over time to failure to attain stable levels before recovery (usually because of an erratic breathing pattern). In most cases, however, there is a transient lowering of the oxygen pressure in the veins, which returns to a nearly stable condition within 5–10 min. In the period when the values are stable, the arteriolar values are 65–85 mm Hg and the venous values 35–55 mm Hg. Oxygen extraction is 25–30 mm Hg. Similar time dependence has been observed for microvascular oxygen levels in normal muscle tissue in mice used as controls for tumor oxygen measurements.24
D. Identifying Vascular Lesions Induced by Alterations in Tissue Oxygenation
In initial experiments, a laser was used to block one of the larger retinal blood vessels and the phosphorescence images were collected 48 h later. The measured phosphorescence was well defined, with no evidence of leakage from the vessels (spreading of intensity, increase in intensity with time, or both). The phosphorescence from the large damaged area was high owing to the low oxygen levels, and this obscured the emission from the surrounding normal tissue. As a result, the oxygen maps showed only large areas with oxygen pressures near zero, confirming the presence of a massive injury. This model was not further pursued because such large injuries can be readily detected by simple ophthalmoscopy. It was more interesting to test for the ability to detect injury that is due to the pathology involving microvessel failure, where blockage of capillaries would result only in small hypoxic regions. To this end, a model of local vascular failure was generated. Two or three 75 µm focal spots of laser photocoagulation with indocyanine green as an intravascular sensitizer were made in regions that spared observable major blood vessels. This procedure restricted injury to the capillary bed within the focal area. Oxygen measurements were made 48 h later to limit the contribution of immediate local edema and vascular leakage. An oxygen pressure map of a region with two laser-induced lesions is shown in .
Fig. 6 Oxygen pressure maps of (a) the region containing two laser photocoagulation spots and (b) a region centered on one of the two spots. The measurements were made with modulation frequencies of 3030 and 800 Hz, respectively. The area of retina covered by (more ...)
The lesions appear in phosphorescence intensity images as bright spots (not shown). In the oxygen map they appear as nearly round areas with central core oxygen values well below those of the surrounding tissue. The absence of significant leakage of the phosphor from the vessels was confirmed by measurements made over periods of 1 h following phosphor injection, during which time the size and the oxygen profile of the region of hypoxia remained constant. presents an oxygen map of one of the two lesions reimaged at a lower frequency (800 Hz) to yield a better estimate of the core oxygen pressure. The laser-induced lesion consists of a central region of acute hypoxia surrounded by a region of graded oxygen deficit that extends outward to approximately 150–200 µm from the center. The mean oxygen pressure in the core was less than 7 mm Hg.
E. Naturally Occurring Vascular Anomalies
Images of the retina in mice, particularly old obese mice, occasionally show anomalous hypoxic vessels within the capillary bed of the retina. An example of such vessels is shown in . The phosphorescence intensity image  does not show any obvious vascular anomalies, whereas when the oxygen pressure map is calculated there are vessels in three regions with retinal capillaries that resemble, on a microscale, varicose veins. The oxygen pressures in these vessels are well below that in the surrounding capillaries. It is likely that they have greatly diminished blood flow and that, as a result, much more oxygen has been extracted from the blood within these vessels.
Phosphorescence intensity image (left) and oxygen pressure map (right) of the eye of a 2-year-old mouse. The measurements were made at 1000 Hz. The retinal area imaged was approximately 0.92 mm high by 1.2 mm wide.