2.1.

Optical Setup

The custom-built 2PM system (Fig. 1)³³ was used for both 3-D retinal microvasculature angiography and measurements of ${\mathrm{pO}}_2$. All measurements were performed in the dark.

Fig. 1

Optical setup for 2PM imaging of ${\mathrm{pO}}_2$ in mouse retinal microvasculature. EOM, electro-optic modulator; SH, shutter; GS, $xy$ galvanometer scanner; DM, dichroic mirror; OBJ, microscope objective lens; PMTs, four-channel detector unit. The inset illustrates the coupling of the long-working-distance microscope objective with the mouse eye and eye anatomical features considered, when modeling the propagation of the optical rays. $P_{r1}$ and $P_{r2}$ are top and bottom retina planes, respectively. $P_{r1}^{\prime }$ and $P_{r2}^{\prime }$ are virtual images of $P_{r1}$ and $P_{r2}$, respectively, created by the compound optical system of the mouse eye.

Two-photon excitation was performed using a tunable femtosecond laser (InSight, Spectra-Physics, 680 to 1300 nm, 80 MHz rep. rate). The power level and temporal gating of the excitation beam were controlled by an electro-optic modulator (EOM) (ConOptics, 350 to 160). The beam was steered in the lateral plane ($X$ and $Y$) by two galvanometer mirrors (Cambridge Technologies, 6215HB) and focused onto a desired retina layer by an objective lens (Olympus, XLPLN10XSVMP, $10\times$, 0.6 NA, water immersion), which was separated from the mouse cornea by transparent ultrasound gel, a glass cover slip, and a thin layer of eye lubricant (see Fig. 1 inset). The working distance ($\mathrm{WD}=8\mathrm{mm}$) of the objective lens was chosen to be significantly longer than the physical cornea-to-retina distance ($\sim 3.2\mathrm{mm}$),^34–36 since the apparent optical depth of the retina position is higher due to the optical properties of the eye. The emission originating from the retinal microvasculature was collected by the objective lens, reflected by an epidichroic mirror (Semrock Inc., FF875-Di01-25x36), passed through a short-pass filter (Semrock Inc., FF01-890/SP-50), and split into two channels. One was directed to a photon-counting photomultiplier tube (PMT) (PMT, Hamamatsu, H10770PA-50) through a bandpass filter (Semrock Inc., FF01-795/150-25) and used for recording the phosphorescence of the probe. Another was passed through a band-pass filter (Semrock Inc., FF01-525/50-25) and directed into analog PMT (Hamamatsu, R3896), for recording the fluorescence of vascular markers [e.g., fluorescein isothiocyanate (FITC)-dextran] in order to construct microvascular angiograms of the regions of interest.

2.2.

Animal Preparation

All surgeries and experimental procedures were performed in accordance with the guidelines established by the MGH Institutional Animal Care and Use Committee (IACUC). We used three female C57BL/six mice (Charles River Laboratories, 3-months old, 22 to 23 g). Mice were anesthetized during surgery using 1.5% to 2% isoflurane in a mixture of air and oxygen, as they underwent tracheotomy for ventilation and femoral artery cannulation for measuring the blood pressure and heart rate, injection of the contrast agents (probes), and blood gas measurements. The body temperature was maintained at 37°C using a heating blanket and monitored by a rectal thermometer.

Prior to imaging, the pupils were dilated using 1% tropicamide eye drops, and an eye lubricant gel (Goniovisc Hypromellose Ophthalmic solution USP 25 mg, 2.5%) was applied over the cornea. Animals were placed in a prone position; the head was slightly tilted and restrained using a metal bar previously attached to the skull with dental cement to minimize motion artifacts induced by respiration and heartbeat. A microscope coverslip was placed over the right cornea. The coverslip was in contact with the eye lubricant gel, providing a flat surface, and it was optically coupled with the objective lens by using ultrasound gel. A mixture of the phosphorescent probe [molecular weight (MW) $\sim 75\mathrm{kDa}$, 0.1 mL, $34\mu \mathrm{M}$] and dextran-conjugated fluorescein (FITC, 0.1 mL, $89\mu \mathrm{M}$) was administered into the blood plasma via a femoral artery cannula. The mice were kept anesthetized during imaging using 1% isoflurane in a mixture of air and oxygen. The average power limit for ocular safety (based on ANSI 2000)³⁷ was calculated to be 61 mW in our imaging configuration ($10\text{-}\mu \mathrm{s}$-long excitation gate at 950 nm). The average laser power after the objective during application of the excitation gate (high-repetition-rate pulse train) was 1.3 to 58 mW. We induced hyperoxic and normoxic conditions by manipulating the ${\mathrm{O}}_2$ concentration in the inhaled mixture of ${\mathrm{O}}_2$ and air (${\mathrm{O}}_2$: 21% during normoxia and 100% during hyperoxia) and confirmed the results through systemic arterial blood gas measurements. The animal blood pressure (BP), heart rate, body temperature, and expired ${\mathrm{CO}}_2$ were monitored continuously during the experiments. The range of BP and systemic ${\mathrm{pCO}}_2$ during the experiments were 67 to 90 mmHg and 30 to 37.5 mmHg, respectively. Surgical preparations took $\sim 30$ to 45 min and moving the animal to the microscope, positioning of the animal, injection of contrast agent, and testing blood gasses took additional 30 to 45 min.

2.3.

Depth-Resolved Imaging of Retinal Microvasculature Using Two-Photon Fluorescence Microscopy

In order to visualize retinal microvasculature, high-resolution volumetric angiograms were recorded by means of 2PM fluorescence imaging of the FITC-labeled blood plasma. We started our measurements by selecting a region of interest (ROI) using an air-coupled, low-resolution objective (Olympus, XLFLUOR4x/340, $4\times$, 0.28 NA, 30-mm WD) with a large field-of-view. Then we switched to a high-resolution water-immersion objective (Olympus XLPLN10XSVMP, $10\times$, 0.6 NA, 8-mm WD) to record the angiograms. The excitation beam (840 nm) was raster scanned with a dwell time of $4\mu \mathrm{s}/\text{pixel}$. The FITC fluorescence was recorded at multiple planes through the retinal depth by translating the imaging objective along the optical $Z$ axis.

2.4.

Depth-Resolved Measurement of Absolute ${\mathrm{pO}}_2$ in Retinal Microvasculature Using Two-Photon Phosphorescence Lifetime Microscopy

Intravascular ${\mathrm{pO}}_2$ was quantified using oxygen-dependent quenching of phosphorescence and a new probe based on a new highly two-photon absorbing Pt(II) tetraphthalimidoporphyrin.³² The probe does not cross the blood brain barrier and/or cellular membranes and can be used for both intravascular and extravascular oxygen imaging. The new probe belongs to the class of dendritically protected phosphorescent probes³⁸ and is characterized by high two-photon absorption cross-section ($\sim 600\mathrm{GM}$ at 950 nm) and high phosphorescence quantum yield ($\sim 0.23$ in aqueous solution at 22°C). Before recording phosphorescence decays in selected locations, the excitation beam (950 nm) was raster scanned in the corresponding axial plane, and a two-dimensional phosphorescence intensity image (survey image) of the ROI was recorded. This survey scan helped to position the beam at the desired location(s) for collection of the time-resolved phosphorescence data. We used $300\text{-}\mu \mathrm{s}$-long interexcitation cycles, during which the EOM was in the opened state during the initial $10\mu \mathrm{s}$ to provide an excitation gate, followed by a $290\text{-}\mu \mathrm{s}$-long phosphorescence collection period. We averaged 500 interexcitation cycles during $\sim 150\mathrm{ms}$ to collect the decay at each measurement location. In each imaging plane, we selected 30 to 60 locations inside the microvascular segments. Measurement of ${\mathrm{pO}}_2$ at selected locations took 5 to 10 s. The digital acquisition card (National Instruments, PCIe-6537) was used at a $50\mathrm{MS}/\mathrm{s}$ acquisition rate to collect PMT photon counts after the discriminator (Hamamatsu, C9744). The photon counts were binned into $2\text{-}\mu \mathrm{s}$ bins. The first $5\mu \mathrm{s}$ of the data after the end of the excitation gate were rejected to match the probe calibration conditions and the remaining $285\mu \mathrm{s}$ of phosphorescence decays were fitted with a single exponential function to calculate the decay time. The phosphorescence decay times (lifetimes) were converted to absolute ${\mathrm{pO}}_2$ values using a Stern–Volmer-like expression obtained in independent calibration experiments, which were performed using solutions of the probe in a physiological buffer (phosphate, 20 mM) at 36.6°C. Throughout the range assessed by our calibrations (${\mathrm{pO}}_2=0$ to 160 mmHg), the probe displayed nearly ideally linear Stern–Volmer behavior, i.e., the quenching rate constant changed linearly with ${\mathrm{pO}}_2$. In the hyperoxia experiments, we assumed that this linearity is retained at ${\mathrm{pO}}_2$ values above 160 mmHg, and thus the hyperoxia values shown in Fig. 5 were obtained by extrapolation of the oxygen titration curve to the higher ${\mathrm{pO}}_2$ region. This extrapolation was justified by the linearity of the Stern–Volmer plot throughout the physiological range (0 to 160 mmHg) and the fact that the Stern–Volmer approximation, i.e., the assumption about zero-order of the quenching reaction with respect to quencher (${\mathrm{O}}_2$), should become even more justified physically at higher oxygen concentrations. The objective was translated along the optical $Z$ axis as ${\mathrm{pO}}_2$ measurements were proceeded to different axial planes (typically six to seven per experiment). The measurement results were classified according to three retinal depth ranges. The survey intensity images in each group were merged by maximum intensity projection (MIP), i.e., selecting the maximum intensity value per pixel. The resulting MIP image was contrast enhanced for better visualization and segmentation of the microvascular branches. The resulting MIP images and ${\mathrm{pO}}_2$ measurements were overlaid. Within each vessel segment, ${\mathrm{pO}}_2$ was typically measured in one to five locations (as shown in Fig. 4), then the average ${\mathrm{pO}}_2$ values were computed per vessel segment if more than one point was measured. Finally, the colors (using MATLAB colormap “jet”) were assigned to the entire vessel segments showing average ${\mathrm{pO}}_2$ in mmHg (Fig. 5).

2.5.

Statistics

The individual ${\mathrm{pO}}_2$ measurements with standard error higher than 15% were rejected. Reported ${\mathrm{pO}}_2$ values within major vessels were presented as $\text{mean}\pm \text{standard deviation}$ (SD), where mean ${\mathrm{pO}}_2$ and its standard deviation were calculated across multiple ${\mathrm{pO}}_2$ measurements within a vascular segment.

3.1.

Calibration of the Compound Eye-Objective Optical System

The compound optical system, which consisted of the imaging objective and the intrinsic optical system of the eye, was calibrated in order to quantify the distances within the measured tissue volume in both lateral and axial dimensions. The optical system of the mouse eye, i.e., cornea, aqueous chamber, lens, vitreous chamber, and retina, can be represented by volumes with different refractive indices separated by curved interfaces (Fig. 1, inset).

The dimensions of the mouse eye and hence its optical properties vary with age and strain. We used the previously published data^34–36 to estimate the optical properties of the eye of the 3-month-old C57BL/6 mice that were used in our experiments (see Table 1) at the excitation wavelength of 950 nm. We constructed the compound ray transfer matrix of the mouse eye, using the previously published approach,³⁶ taking into account the propagation of light through the cornea, lens, aqueous, and vitreous chambers, and the diffraction at the interfaces between them. The whole eye was modeled as a single optical element (i.e., a thick lens). Marginal rays were traced from both directions (cornea-to-retina and retina-to-cornea) in order to calculate front and back focal points, principal planes, and effective focal length (3.1 mm). We then used the lens equation to estimate the distances [Fig. 2(a)] and magnification [Fig. 2(b)] in our measurements. To determine the lateral dimensions, we focused the excitation beam on the inner-retina surface, translated the animal in $X$- and $Y$-directions at known distances and recorded fluorescence images of the vasculature (using FITC-dextran as vascular marker) at each position. The shifts between the images in $X$- and $Y$-dimensions were quantified and scaled with the depth-dependent magnification factor [Fig. 2(b)].

Table 1

Optical parameters used to calculate the depth of the imaging plane within the retina and corresponding lateral magnification.34–36

Fig. 2

Calibration of the compound eye-objective optical system. (a) Calibrated retina depth as a function of the distance between the objective lens and the cornea surface. (b) Calibrated lateral magnification introduced by mouse eye optics as a function of imaging plane depth inside the retina.

The working distance of the objective lens was quickly depleted as deeper layers of retina were imaged [Fig. 2(a)]. The entire retinal depth ($\sim 220\mu \mathrm{m}$) was accessible by adjusting the gap between the surfaces of the objective lens and the cornea from 2.7 to 1.6 mm. Upon lowering the focal plane deeper into the retina, the magnification factor induced by the eye optics changed from 2.2 to 2.7. These calibration experiments revealed that our optical system had sufficient working distance and could be used to image through the entire retinal microvascular plexus.

3.2.

Depth-Resolved Imaging of Retinal Microvasculature

After the optical system was calibrated, we demonstrated the capability of our imaging setup to generate high-resolution volumetric angiograms by two-photon excited fluorescence of FITC-labeled blood plasma.

Mouse retina is holangiotic, i.e., it does not have an avascular central region, unlike human retina.³⁹ Major retinal vessels on the superficial layer are arranged in a radiating star-like pattern consisting of arteries and veins in alternating order [Fig. 3(a)]. The total retinal thickness is $\sim 220\mu \mathrm{m}$,³⁴ and the outer retinal layers ($\sim 100\text{-}\mu \mathrm{m}$ thick) are avascular and positioned close to the choroid plexus.^15,40 The inner retinal layers ($\sim 100\text{-}\mu \mathrm{m}$ thick) comprise three microvascular layers.⁴¹ Starting from the retinal surface, these layers are: (1) the superficial layer, which is rich in arterioles and located in the ganglion cell layer; (2) the intermediate layer, located in the inner plexiform layer; and (3) the deep layer, which is rich in venules and located in the outer plexiform layer.^40–43

Fig. 3

Two-photon depth-resolved retinal microvasculature imaging. (a) Microvascular structures, as imaged using a low-magnification objective, of the three main microvascular layers in the mouse retina: superficial, intermediate, and the deep. The superficial microvascular layer shows a radiating pattern of major arteries and veins falsely colored in red and blue, respectively. Yellow arrows indicate the direction of the blood flow. ROI is shown by white-dashed square. (b) and (c) Retinal microvasculature layers of two mice, imaged using high-magnification objective. Three imaging depths ($z$) in (b) and (c) show microvascular structure in three retinal microvascular layers. The imaging depths ($z$) shown in the graphs represent calculated values based on the calibrations (see the main text and Fig. 2 for details).

Figures 3(b) and 3(c) show images of the retinal microvasculature in two mice. The microvascular structures shown in three panels in Figs. 3(b) and 3(c) represent mostly microvascular segments from the three individual retinal microvascular layers, with little overlap with the vasculature from the adjacent microvascular layers. This confirms that our setup is capable of high-resolution 3-D imaging in the eye and able to distinguish between individual arterioles, venules, and capillaries from different microvascular layers as well as between major arteries and veins.

3.3.

Depth-Resolved Measurement of ${\mathrm{pO}}_2$ in Retinal Microvasculature

We quantified absolute ${\mathrm{pO}}_2$ in the blood-plasma using the phosphorescence quenching method.

A representative phosphorescence intensity image and the locations of the local microvascular ${\mathrm{pO}}_2$ measurements in the superficial layer are shown in Fig. 4(a). ${\mathrm{pO}}_2$ was measured in a large set of microvascular segments within $438\times 438\mu {\mathrm{m}}^2$ field-of-view (FOV), including in arterioles, venules, and capillaries. Representative phosphorescence decays (average photon counts during interexcitation interval of $300\mu \mathrm{s}$) corresponding to a major artery and a vein under normoxia (systemic arterial ${\mathrm{pO}}_2=95.2\mathrm{mmHg}$ according to blood gas analyzer) are shown in Fig. 4(b). These decays were fitted with single exponentials to derive the phosphorescence lifetimes and subsequently the ${\mathrm{pO}}_2$ values.

Fig. 4

Two-photon imaging of retinal microvascular ${\mathrm{pO}}_2$. (a) Intravascular ${\mathrm{pO}}_2$ (color coded, color bar in mmHg) at various locations within retinal microvasculature. ${\mathrm{pO}}_2$ measurement locations are marked by colored dots on the gray-scale survey intensity image. (b) Examples of phosphorescence decays measured within the artery (red) and vein (blue). The measurement locations corresponding to the decays shown were marked on image (a) with a red star and a blue circle, respectively. $10\text{-}\mu \mathrm{s}$-long laser excitation gate was marked with the shaded area at the beginning of the interexcitation interval. Fitted phosphorescence lifetimes in arteriole and venule (${\tau }_A$ and ${\tau }_V$, respectively) and their corresponding ${\mathrm{pO}}_2$ values are provided inside the panel. Values are presented as $\text{mean}\pm \text{standard}$ error of mean (SEM).

Depth-resolved ${\mathrm{pO}}_2$ measurements in the retinal microvasculature, performed in the same mouse during normoxia and hyperoxia, are shown in Fig. 5. We measured ${\mathrm{pO}}_2$ in a major artery, a major vein, a number of arterioles, venules, and capillaries within the superficial, intermediate, and deep retinal microvascular plexus. During normoxia, the mean ${\mathrm{pO}}_2$ values in the major artery and vein were $93\pm 3\mathrm{mmHg}$ ($\text{mean}\pm \mathrm{SD}$) and $63\pm 2\mathrm{mmHg}$ ($\mathrm{mean}\pm \mathrm{SD}$), respectively. The arterial value was consistent with the systemic arterial ${\mathrm{pO}}_2=112\mathrm{mmHg}$, measured using a blood gas analyzer. Longitudinal ${\mathrm{pO}}_2$ gradients, starting from high ${\mathrm{pO}}_2$ in the major artery at the superficial retinal layer, could be visualized along the precapillary arterioles as approaching lower ${\mathrm{pO}}_2$ values in the capillaries close to the postcapillary venules, and finally to the major vein.

Fig. 5

${\mathrm{pO}}_2$ distribution in the retinal microvasculature during normoxia and hyperoxia. Survey images (top) and ${\mathrm{pO}}_2$ measurements superimposed on these images during normoxia (middle row) and hyperoxia (bottom row). Columns represent different microvascular layers: superficial (first column), intermediate (middle column), and deep layer (third column). Vascular segment colors in second and third rows represent average of all ${\mathrm{pO}}_2$ point measurements within the vascular segment (colorbar in mmHg). Color-coded masks were overlaid on the gray-scaled MIP survey scan images. Both the imaging depth ranges ($z$) and the dimensions of the FOV were estimated using the calibration curves presented in Fig. 2. Note that the ${\mathrm{pO}}_2$ ranges during normoxia and hyperoxia are far apart, so we used separate ${\mathrm{pO}}_2$ color scales to aid visualization of ${\mathrm{pO}}_2$ distributions in the retinal microvasculature at these two conditions.

We subsequently perturbed the microvascular oxygenation in the retina by inducing hyperoxia, whereby the systemic arteriolar ${\mathrm{pO}}_2$ was increased to 334 mmHg. Similar to the measurements under normoxia, the longitudinal gradients of intravascular ${\mathrm{pO}}_2$ could be visualized between the major artery ($327\pm 2\mathrm{mmHg}$, $\mathrm{mean}\pm \mathrm{SD}$) and vein ($224\pm 4\mathrm{mmHg}$, $\text{mean}\pm \mathrm{SD}$) in the superficial retinal layer, i.e., the small vascular segments close to the major artery were more oxygenated than those close to the major vein.

The ${\mathrm{pO}}_2$ values in the major artery and vein, as well as in the combined superficial, intermediate, and deep microvascular plexus (microvascular bed) under normoxia and hyperoxia are shown in Fig. 6. During normoxia, the median ${\mathrm{pO}}_2$ values in the major artery, vein, and in the microvascular bed were 93, 62, and 87 mmHg, respectively, with the microvascular bed ${\mathrm{pO}}_2$ distribution being highly heterogeneous, spanning the range of ${\mathrm{pO}}_2$ values from arteriolar to venular ${\mathrm{pO}}_2$.

Fig. 6

${\mathrm{pO}}_2$ distribution across different retinal compartments: artery, vein, and microvascular bed in between, under (a) normoxic and (b) hyperoxic conditions (same data as in Fig. 5). The top and bottom edges of the boxes (shown in blue) mark the 75th and 25th percentiles; the error bars show the extreme ends of the data spread (red + symbols), excluding outliers, and the red-center lines mark the medians.

Using the Hill equation with coefficients characteristic of C57BL/6 mice ($h=2.59$ and $P_{50}=40.2$),⁴⁴ we calculated the hemoglobin oxygen saturation (${\mathrm{sO}}_2$) in the major artery and vein during normoxia. The corresponding values were 90% and 75%, respectively. These results suggest that the oxygen extraction fraction was 16%. During hyperoxia, the median ${\mathrm{pO}}_2$ in the artery, vein, and the microvascular bed were 323, 226, and 277 mmHg, respectively, with the microvascular bed ${\mathrm{pO}}_2$ distribution, as in the normoxic case, being highly heterogeneous and spanning the range of ${\mathrm{pO}}_2$ values from arteriolar to venular ${\mathrm{pO}}_2$.