In the last decade several approaches have been developed, assessing more sensitive and quantitative alterations of retinal microvascular changes. Although protocols may differ in some minor points, the principles are similar. According to standardized protocols, one (e.g., 45°) nonstereoscopic color retinal photograph centered between the optic disk and the macula and approximately two disk diameters nasal to the optic disk has to be done in a darkened room. Hence, due to dark adaption, mydriatic agents are no longer necessary. However, in some studies (e.g., Rotterdam Study), pharmacological mydriasis was routinely done. For quantitative assessment of retinal vessels, the photographs have to be converted to digital pictures and analyzed by specific imaging software, e.g., the “Interactive Vessels Analysis” (IVAN) (University of Wisconsin, Madison, WI, USA). This software analysis provides semiautomated measurement of retinal arterioles and venules. Using formulas (e.g., Parr and Spears [8] or Knudtson et al. [9]), a single “central retinal artery equivalent (CRAE)” and a “central retinal vein equivalent (CRVE)” are calculated. Subsequently, arteriole-to-venule ratio (AVR) can be computed; for details see Hubbard et al. [10]. However, by this method, it is not possible to evaluate the retinal vascular wall thickness or vessel diameter directly.

SLDF, introduced by our study group about 10 years ago, allows the dynamic assessment of both functional (i.e., vascular tone) and structural parameters (i.e., wall and lumen diameter). In brief, SLDF is performed in the juxtapapillary area of the right eye, 2–3 mm temporal superior of the optic nerve at 670 nm (Heidelberg Retina Flowmeter, Heidelberg Engineering, Germany). A retinal sample of 2.56 × 0.64 × 0.30 nm is scanned within 2 s (at least one full systolic and one diastolic phase) and measured every 10 μm of this specific length of the retinal arteriole (80–140 μm). The confocal technique of the device ensures that only capillary flow of the superficial layer of 300 μm is measured. No pupil dilation is necessary (i.e., no constriction of patient daily routine) [11].

For assessment of functional parameters, mean retinal capillary flow (RCF) is assessed in the area of interest, and for further dynamic analysis, non pharmacological and pharmacological tools can be applied. Flicker light increases RCF at least in part via a nitric oxide (NO)-dependent mechanism and represents a non pharmacological tool to investigate vasodilatory capacity of retinal arterioles. It is noteworthy to mention that flicker light exposure has no effects on systemic blood pressure (BP), thereby minimizing potential systemic hemodynamic influences on RCF. Moreover, basal NO activity can be assessed by administration of the NO synthase inhibitor N^G-monomethyl-l-arginine (l-NMMA).

For assessment of structural parameters, the outer arteriole diameter (AD) is measured by reflection images, and the lumen diameter (LD) is measured by perfusion images. From the raw parameters, wall thickness (WT, [AD−LD]/2), WLR ([AD−LD]/LD) (Fig. 11.2), and wall cross-sectional area (WCSA, п/4 × [AD²–LD²]) can be calculated.

Importantly, also individual pulsatile pattern of functional (RCF) and structural (e.g., WT) parameters of retinal arterioles in systole and diastole can be reliably assessed (Fig. 11.2).

All analyses are performed offline with automatic full-field perfusion imaging analysis (AFFPIA) (SLDF Version 4.0 by Welzenbach with improved resolution) [11].