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Phillip A Bedggood, Angelina Duan, Bang V Bui, Andrew B Metha; Phase and absorption contrast for functional analysis of retinal microvessels. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5191.
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© ARVO (1962-2015); The Authors (2016-present)
The intensity of small retinal vessels in adaptive optics images is influenced by hemoglobin absorption and interference effects. We aimed to determine where each of these effects dominates to inform future endeavors in functional and phase contrast imaging.
The foveal microvascular network was imaged in 3 healthy subjects with adaptive optics at 200 fps. Phase effects were assessed by altering defocus in 0.05 D (14 µm) steps. Hemoglobin absorption was assessed by local flicker stimulation to assess those retinal venules that showed a stimulus-induced drop in intensity. We believe this drop to result from increased hemoglobin absorption following increases in vessel diameter and deoxygenated blood fraction. The stimulus was the imaging light (593 ± 25 nm) set to low power (5 µW), flickering at 8 Hz for 30s over the 1.2° imaged field. This was compared to a baseline of 30s fixation on a dimly lit target. Vessel diameter was measured by finding peaks in the local image intensity gradient. Vessel intensity was measured halfway between the center and the edge of each vessel.
In all capillary segments <8 µm diameter (n=40), the sign of contrast between individual red cells and background tissue was invertible by altering defocus, and ranged from +15% (bright cells) to -20% (dark cells). Conversely, vessel intensity was always darker than background in vessel segments >13 µm diameter (n=37). Vessels of intermediate diameter (8-13 µm) took on a range of appearances. Functional stimulation produced a detectable change in intensity of venules as small as 13 µm. In some cases a large enough vessel length was imaged to demonstrate a diminished response in downstream portions of the same venule - e.g. the response in one venule transitioned from -11.3 ± 0.9%, to -5.8 ± 1.1%, to -1.9 ± 1.3% over a distance of 130 µm.
Intensity of the blood column in vessels < 8-13 µm was dominated by phase contrast, which requires a small amount of defocus and a narrowband source. Imaging methods sensitive to changes in optical path length may particularly enhance the study of vessels of this caliber. Venules as small as 13 µm yielded detectable changes in intensity on flicker stimulation. The diminished downstream response suggests either dilution with blood from neighboring tissue and/or local changes in the efficacy of oxygen extraction.
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