In the present study we developed, evaluated, and validated an IAS for the SDF device. The IAS was based on creating adherence of the SDF probe to the sublingual tissue by applying negative pressure to the periphery of the microscopic field of view. The main findings were that: 1) the IAS did not affect microcirculatory perfusion in the SDF imaging field of view; 2) the IAS prevented pressure artifacts up to a significantly greater force applied by the SDF probe onto the tissue; 3) the time required to obtain a stable image sequence was similar with and without the IAS; and 4) the duration of maintaining that stable image sequence was significantly increased with the IAS. Ultimately, to demonstrate the clinical applicability of the SDF setup with the IAS, simultaneous bilateral sublingual SDF measurements were conducted in intensive care patients undergoing a standard lung recruitment maneuver with one handheld SDF device and one SDF device mounted in a mechanical arm and equipped with the IAS. It was shown that the IAS significantly reduced image drifting and enabled the acquisition of significantly longer image sequences. A final and important finding is also that we showed, in proof of concept, that with the IAS it is possible to perform a measurement without the need for an operator by mounting the device on a mechanical arm, leaving the operator free to perform a clinical maneuver.
The design of the IAS presented here is based on an IAS developed by Lindert et al. for OPS imaging, including the negative pressure level of ≈100 mmHg . To show that application of peripheral negative pressure did not affect microcirculatory perfusion in the SDF imaging field of view Lindert et al. measured blood flow velocities in venules and arterioles. They found that the velocities did not change after switching the negative pressure source on. In the present study, for validation purposes, we investigated the effects on blood flow velocities in small, medium, and large microvessels in five healthy volunteers and provided evidence that indeed microcirculatory perfusion is not affected by application of negative pressure though the IAS. These experiments demonstrated that the IAS is a valid method for SDF image stabilization, not affecting microcirculatory perfusion in the microscopic field of view.
It has been well established that pressure artifacts are easily induced and diminish the reliability of SDF measurements of microcirculatory perfusion [6, 11]. This appreciation known from daily application of SDF imaging is confirmed and highlighted by the low force level required to induce pressure artifacts found in the present study. The SDF imaging device has a mass of approximately 360 g. The critical force onto the sublingual tissue without the IAS, at which pressure artifacts are induced, was found to amount approximately 1/6 of the mass of the SDF device. Hence, physical feedback is impossible for SDF operators and visual feedback in the microcirculatory images is necessary to avoid excessive pressure. In fact, most SDF operators use visual feedback to gauge the pressure exerted by the SDF probe on the imaged microcirculation as exemplified in a recent publication . De Backer et al., defined the critical pressure inducing perfusion artifacts at the point where venular flow either stopped or significantly slowed down . Using a similar cut-off in the present study we were able to show that the larger surface contact area created by the presence of the IAS resulted in an approximately five times greater force required for the induction of pressure artifacts. This significantly improved SDF image acquisition.
Another important advantage of using an IAS for SDF imaging is that it allows acquisition of longer and more stable SDF image sequences. Previous studies reported that SDF measurements have low intra- and inter-observer variability  and that microcirculatory density and perfusion vary highly per site and in time . Hence, studying the microcirculation under pathophysiological conditions requires multiple measurements per time point in order to eliminate this site- and time-dependency of the obtained results. The current microcirculatory image acquisition guidelines dictate that microcirculatory density and perfusion should be measured in 3-5 sites per time point to allow adequate interpretation of the results . Furthermore, according to these guidelines, the length of each SDF image sequence should be > 20 s. This was proven to be rather difficult without the IAS and fairly easy with the IAS. An alternative for multiple measurements to determine the microcirculatory state at a certain time point, continuous measurements of microcirculatory perfusion and density during a clinical maneuver or intervention (e.g., nitroglycerin administration) would allow direct assessment of their effects on the microcirculation. The presented IAS would potentially enable such studies.
Non-invasive intravital imaging modalities, such as OPS and SDF imaging, have been used in studies for monitoring the severity of shock and efficacy of resuscitation and alterations in sublingual microcirculatory density and perfusion have been associated with patient morbidity and mortality [3, 6, 16]. 'Normalizing' microcirculatory density and perfusion has become focus of new clinical studies and microcirculatory images are gaining a more prominent role in clinical monitoring. Adequate interpretation of microcirculatory images is essential and relies heavily on the quality of the images, in terms of axial and lateral stability. In the present study we showed that the IAS improves both axial and lateral stability of the acquired microcirculatory images and significantly reduced pressure artifacts and image drifting.