Measurements of microcirculation were initiated upon the ethical consent was approved. 15 healthy volunteer were included in the study. All volunteers were asked for the existence of any symptoms for the on-going disease like flu, infectious disease, and chronic disease (Diabetes, hypertension, rheumatologic disorders, epileptic disorders, etc.). Once the volunteer met inclusion criteria, he/she became eligible for study. Volunteers were advised to beware from food and drink for at least two hours before measurement. Also, they were advised to come at least two hours after big meals like lunch or dinner. All the volunteers were evaluated at the same position (sitting and hand at heart level), room and temperature to provide equal standards. The volunteer had rest for at least 30 minutes in a semi-recumbent and extending leg position. The room temperature was 24-26C and checked with a thermometer intermittently. The door was closed to prevent air movement. Lights were switched off, and the curtains were closed to prevent the effect of light on devices. Once all the environmental conditions met, two investigators evaluated the volunteer. One investigator checked the volunteer's blood pressure, temperature, and heart rate.

A vacuum pillow was used to hold the arm stable during the procedure. Two different devices (laser speckle contrast imaging (LSCI) and laser doppler imaging (LDI)) were ready next to the volunteer. An appropriate intact skin area, inner side of the forearm were used for the investigation and both electrodes were attached. The skin surface was cleaned with alcohol or deionized water to remove remnants of cosmetics and creams with low conductivity. This helped to prevent spots with high current density that may result in local burns and non-specific vascular reactions. After peeling off the protective tape layer on the bottom of the Drug Delivery Electrode, the electrode was firmly attached to the skin. The Dispersive Electrode was placed approximately 10-15 cm from the Drug Delivery Electrode. Power supply was connected by attaching the electrodes to the leads. After lifting the lid of the Drug Delivery Electrode using the pointed tab, tip of the syringe was slided under the lid of the Drug Delivery Electrode and the Drug Delivery Chamber was filled with approximately 0.5 mL of a 10 mg/ml SNP solution. The drug was then delivered using cathodal iontophoresis for 1 minute with a current strength of 200 µA. The LDI and LSCI instruments are positioned so they can alternately measure the delivery site. Basal perfusion was measured with both instruments before starting iontophoresis. Subsequently, iontophoresis was started and measurements are made alternating between the two instruments. Therefore, we would be able to compare both devices in terms of supra-baseline flux values. When all measurements were finished, the electrodes removed from the patient and the Drug Delivery Electrode and the Dispersive Electrode was removed.

The second part of the study was performed as step-wise occlusion. The arm which was not used for iontophoresis test was used for step-wise occlusion. The baseline blood flux was followed for 2 minutes by LDI and LSCI. After 2 minutes, the upper arm was occluded for 30 seconds with a pneumatic cuff up to 50 mmHg, 80 mmHg, 110 mmHg, 140 mmHg, and 170 mmHg. Between each part, the volunteer had rest for 5 minutes to let the blood flux to turn the baseline. Once reaching to the planned pressure, LSCI and LDI devices were alternately be used to record blood flux. Therefore, we were able to compare both devices in terms of sub-baseline flux values.

All data were recorded in 2 external electronic storage, and the names of the volunteers were coded with numbers. Only main investigator was aware of who corresponds to which number. Results of Perimed and Moore Devices were analyzed with the software PIMsoft 1.5 (Perimed AB, Järfälla, Sweden), MoorLDI2-BI Burn's Software Version 4.0, respectively. Two investigators performed the analysis. If one investigator made the analysis, the other checked the results, as well.