The study was approved by the ethics committee board of the University of Miami and conducted according to the tenets of the Declaration of Helsinki. All subjects were recruited and informed about the purposes and methods of the study and each volunteer signed a consent form.
Study subjects
A total of 58 normal healthy subjects were recruited at the Bascom Palmer Eye Institute of the University of Miami. All subjects with a refractive error of no more than − 6.00 D and no greater than + 3.00 D were recruited. Only one eye of each subject was selected to be imaged. All subjects were imaged within 1 hour at the same visit during office hours (from 9 AM to 5 PM) [7]. The exclusion criteria included a history of contact lens wear (within 6 months), ocular surgery and trauma, systemic diseases, and the use of medications. Subjects with hypertension, diabetes, sickle cell anemia, cerebral small vessel disease, history of stroke, cardiovascular diseases, and other vascular diseases were also excluded.
Study examinations
All subjects underwent complete ophthalmologic examinations, including the slit-lamp biomicroscope, best-corrected visual acuity (BCVA) measurement, fundus examination, and intraocular pressure. The best-corrected visual acuity of all subjects was 20/20 or above. Their blood pressures, including systolic pressure and diastolic pressure, and heart rates were also measured, and personal medical histories were obtained from all subjects.
Functional slit-lamp biomicroscope (FSLB) for measuring conjunctival microcirculation
FSLB settings and image procedures have been well described in previous studies [2, 8], and its repeatability had been validated [7]. In brief, the FSLB was modified from a traditional slit-lamp by adding a digital camera which has a special function called Movie Crop Function (MCF). The MCF enables the addition of a 7× magnification, which can be combined with the slit-lamp magnification (30×), resulting in extremely high magnification (~ 210×) for imaging the motion of the cluster of red blood cells (Fig. 1). In this study, the field of view of the FSLB was 0.9 × 0.7 mm. The pixel interval on the acquired video was 1.4 μm. The other settings of the camera were the same as in previous studies [7, 9]. To measure the mean BFV and blood flow rate (BFR), six different locations approximately 1 mm away from the limbus were recorded. The measurement was taken on conjunctival venules because the majority of the conjunctival vessels are venules. Custom software was used to obtain BFV and BFR and the detailed image processing procedures have been reported previously [7, 9].
Retinal function imager (RFI) for measuring retinal microcirculation
RFI is an advanced ophthalmic imaging modality based on a fundus camera. The system and its applications have been recently reviewed [10] and the system has a high reproducibility [11]. A high-speed camera is attached to the fundus camera with a green illumination system to acquire a series of fundus photos. The image processing software processed the sequential images to obtain the motion of a cluster of red blood cells in the 2nd and 3rd branches of pre-capillary arterioles and post-capillary venules for the measurement of the mean retinal BFV (Fig. 1) [4, 5]. To measure the mean retinal BFR, a circle (diameter = 2.5 mm) was drawn to outline the vessels crossing the circle. The diameter of these vessels was measured. Using both the measured BFV and vessel diameter, BFR was calculated. In the present study, the field of view was 4.3 × 4.3 mm. The pixel interval of the captured image was 4.2 μm. Before imaging, 1% tropicamide was used to dilate the pupil [12].
Statistical analysis
All data are presented in the format of mean ± standard deviation (SD) and analyzed using Excel (version 2010; Microsoft, Redmond, WA, USA). The sample size was calculated by a software program (G*Power, version 3.1.9.4) recommended by Faul et al. [13] and Bonett and Wright [14]. A sample size of 14 subjects would be enough to detect the difference of the microcirculation in the retina and conjunctiva with a detection power of 0.99. Paired student t-test was used to compare the microvascular parameters in the conjunctiva and retina. Pearson correlation coefficients were used to determine the relationships between the microvascular parameters in the conjunctiva and the retina. All P < 0.05 were regarded as statistically significant.