High-Resolution Wide-Field Optical Imaging of Microvascular Characteristics: From the Neocortex to the Eye

Abstract

Measuring microvascular characteristics in cortical tissue and individual microvessels has important applications for functional imaging, biomedical research, and clinical diagnostics. Multiphoton fluorescence microscopy approaches are most effective and allow to reliably record red blood cell (RBC) velocity in individual vessels, but require injecting fluorescent tracers. Moreover, only one or few vessels in a small area can be imaged at a time. Wide-field CCD/CMOS-based optical imaging of intrinsic absorption or reflection changes in macroscopic vascular networks allows to overcome these shortcomings, by recording RBCs’ trajectories over several mm² of cortical surface. The RBC velocity can then be extracted from these wide-field data using specialized algorithms. Here, we describe two of those, which provide robust RBC velocity estimations that are independent and can thus be used as a control one for another. Although this approach can be used in any part of the body with optically accessible blood vessels, here we show its application in two cases: first the cerebral cortex and then the eye. In this latter application, we go into some more detail in describing the retinal function imager (RFI): a unique, noninvasive multiparameter functional imaging instrument that directly measures hemodynamic parameters such as retinal RBC velocity, oximetric state, and metabolic responses to photic activation. In addition, it allows capillary perfusion mapping without any contrast agent. These parameters of retinal function are degraded by retinal abnormalities. Here, we thus focus on the characterization of microvessels properties. Indeed, clinical studies suggest that knowing these properties should yield multiple clinical applications for early diagnosis of retinal diseases, possible critical guidance of their treatment, as well as implications for vascular diseases of cortex and eye.

Appendix: Surgery, Optical Imaging Data Acquisition Stimulation Parameters

1.1 Surgery

Details for the surgeries in all three preparations (rat, cat, monkey) can be found elsewhere [29, 30, 85]. Briefly: in all three cases, a recording chamber was implanted on the skull—either in an acute procedure (rat, cat) or chronically (monkey). A cranial window was opened above the cortical area of interest and the dura was either left in place (rat) or removed (cat, monkey), and in the awake monkey case, replaced by an artificial transparent dural substitute [86]. The chamber was filled with saline (rat), silicon oil (cat), or agar (monkey) to stabilize the cortex against movements (heartbeat, respiration…) and closed with a cover-glass to provide a flat optical interface and, in the case of the monkey, a sterile seal. Anesthesia was urethane in the rat, sodium pentobarbital for the cat and isofluorane for the monkey (in this case for surgery only). All surgical and maintenance procedures were in agreement with NIH guidelines.

1.2 Stimulation Protocol

In the rat case, a spreading depression was elicited by carefully touching the cortex with a needle about 2 mm away from the border of the imaged area (for details, see [29]). In the alert monkey case, the experimental paradigm was a simple fixation task. A trial started when the monkey began fixating on a fixation point (0.1°), displayed on a CRT screen. After 200 ms, a high luminance contrast drifting square grating (0.125–0.25 cycles/deg, 8–32°/s) appeared for 3 s, except in the “blank” conditions (no grating). The stimulus was then turned off, and the monkey had to continue to fixate until the fixation point disappeared, for a total fixation period of 6.5 s. An isoluminant, uniformly gray-screen inter-trial interval of ~10 s followed. Data from trials where the monkey broke fixation were rejected.

1.3 Optical Imaging

The light source was a tungsten-halogen lamp (100 or 150 W, Zeiss, Germany) from which specific wavelengths can be selected using adequate interference filters (Omega Optical, Brattleboro, USA). The cortex was illuminated using light guides. To maximize RBC contrast and thus blood vessel contrast in general, we used green light, since, in this part of the spectrum, hemoglobin (Hb) absorption is large.

To reach a satisfactory illumination level despite small exposure times corresponding to high frame-rate collection (100 Hz in the rat, 200 Hz in the monkey) and high magnifications, we used an interference filter with a rather large transmission window (30 nm), centered at 540 nm in the case of the rat, and a more narrow one (10 nm) centered at the isosbestic point of 570 nm in the case of the monkey. Images were acquired using a commercial CCD-based imaging system (Imager 3001, Optical Imaging Inc., Germantown, USA). A macroscope [87] was mounted onto the camera. It was composed of a 50 mm lens and an inverted 135 mm lens, yielding a magnification of 2.7, such that our spatial resolution was 9 × 9 μm/pixel, in the case of the rat. In the case of the monkey, we used an upper lens of 100 mm, yielding a magnification of 2 and a spatial resolution of ~12 × 12 μm/pixel. To obtain an optimal identification of vascular activity, the optical system was focused onto the cortical vasculature.

In the case of the monkey, data acquisition lasted the time of a trial (~6 s). Data were written onto the hard disk after each trial. In the rat case, data acquisition lasted 7.5 min and was split into 1 s long periods of continuous 100 Hz acquisition (100 frames), interleaved with 20 ms pauses necessary to store the data onto the hard disk, as required by the data acquisition software we used for this purpose (Optical Imaging Inc., Germantown, USA).

Veins and arteries were first identified by visual inspection (morphology, pulsation, color of vessels…) through a microscope and through the camera. This classification was then integrated with oximetric information obtained from an “oximetric image” of the vasculature [30]: Two images of the cortical surface were taken, one at 560 and the other at 540 nm illumination (peaks of deoxy- and oxyhemoglobin absorption, respectively). One image was then divided by the other and the gray level scale of the resulting “oximetric image” was shifted, such that the parenchyma, which contains mainly capillaries, appeared to be in the middle of it (gray). With this procedure, the various vessel types in the image were thus automatically shifted to the bright or the dark half of the grayscale according to their different degree of oxygenation, allowing for a further identification of veins and venules (dark), as well as of arteries and arterioles (bright).