SIDESTREAM DARK FIELD IMAGING: A NOVEL MICROSCOPIC MODALITY FOR CLINICAL ASSESSMENT OF THE MICROCIRCULATION
Adapted from P.T. Goedhart*, M. Khalilzada*, R. Bezemer*, J. Merza, C. Ince Optics Express 2007;15(23):15101-14 *Equal contribution
and R. Bezemer, M. Khalilzada, C. Ince Yearbook of intensive care medicine 2007; J.L. Vincent (ed.), Springer-Verlag, Berlin, Heidelberg, Germany

aBstract
The introduction of Orthogonal Polarization Spectral (OPS) imaging and its implementation into a clinically-applicable hand-held microscope opened the field of studying the human microcirculation in exposed organ and tissue surfaces. Driven by the success of OPS imaging and the drawbacks it has, we developed a novel imaging modality for the microcirculation, which we have termed (sidestream dark field (SDF) imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation). In this study, we first validated SDF imaging by comparison of SDF-mediated measurements of capillary diameters and red blood cell velocities in the human nailfold microcirculation to OPS¬mediated measurements. Secondly, we compared OPS and SDF image quality, in terms of contrast and sharpness. For this purpose, OPS and SDF images of exactly the same microcirculatory areas were obtained sublingually. We found that SDF imaging provides superior venular and capillary contrast compared to OPS imaging. Thirdly, we explored the SDF imaging (Renal microcirculation, kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) capabilities with respect to imaging individual red and white blood cells and the glycocalyx in the sublingual microcirculation. In conclusion, the present study has introduced SDF imaging as a novel imaging modality, incorporated in a hand-held clinically-applicable device and validated it by quantitative comparison to OPS imaging. It is anticipated that SDF imaging (Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation)will serve as a novel and improved imaging modality to contribute to the clinical assessment of the microcirculation in various clinical scenarios and, additionally, allow more reliable application of computer-aided image processing and analysis software for quantification of microcirculatory alterations associated with disease and therapy.
intrOductiOn
Up to one decade ago, direct intravital observation of the microcirculation in humans was limited to the use of bulky capillary microscopes, mainly applied to the nailfold capillary bed, thus severely limiting microcirculatory investigation under clinical conditions. The introduction of Orthogonal Polarization Spectral (OPS) imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) by Slaaf et al. and its implementation into a clinically-applicable hand-held microscope opened the field of studying the human microcirculation in exposed organ and tissue surfaces [Slaaf et al., 1987; Groner et al., 1999]. Since then, numerous studies have been undertaken in various clinical scenarios where cardiovascular function is at risk [Mathura et al., 2001a;Cerný et al., 2007]. Studies have focused on the microcirculation during disease and therapy in surgery, emergency medicine, and intensive care medicine [Spronk et al., 2002; Sakr et al., 2004, 2007; Ince, 2005] as well as during such diverse conditions as cancer [Mathura et al., 2001b], wound healing [Lindeboom et al., 2007], and infectious diseases [Dondorp et al., 2007]. OPS imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) has had an important clinical impact by observation of the sublingual microcirculation during sepsis, shock, and resuscitation [Sakr et al., 2004; De Backer et al., 2002, 2004; Boerma et al., 2005]. Results from several medical centers have shown that OPS observation of sublingual microcirculatory alterations provided more sensitive information about patient outcome from sepsis and shock than conventional clinical parameters do. These microcirculatory alterations were shown to be especially present in the capillaries, making their study of particular importance [Goedhart et al., 2007; Dobbe et al., 2007; De Backer et al., 2007].
In addition to the assessment of microvascular morphology and perfusion, OPS imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) can be used for identification and measurement of the capillary glycocalyx, a physiological compartment important for endothelial function and maintaining a barrier function between the circulation and the tissue cells [Nieuwdorp et al., 2005]. The endothelial glycocalyx, a negatively charged gel-like layer, composed of proteoglycans, glycosaminoglycans, glycoproteins and glycolipids, is considered to protect the vascular wall by prevention of direct contact with flowing blood. Hence, the glycocalyx contributes to vascular homeostasis by maintaining the vascular permeability barrier, regulating the shear stress-induced release of nitric oxide (NO) and by inhibition of white blood cell and thrombocyte adhesion to the vascular wall [Henry and Duling, 1999; Weinbaum et al., 2003; Mochizuki et al., 2003; Florian et al., 2003; Thi et al., 2004]. Impaired or damaged glycocalyx is accompanied by a number vascular wall alterations known as the earliest characteristics of atherogenesis, a major cause of cardiovascular diseases [Libby, 2002; Van den Berg et al., 2006; Contantinescu et al., 2003]. Therefore, glycocalyx measurements may hold a promise as a diagnostic tool to estimate cardiovascular risk as well as to evaluate the impact of cardiovascular risk-lowering or even glycocalyx-restoring therapeutic interventions Gouveneur .
Despite the major contribution OPS imaging (Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) has made in the field of intravital microcirculatory imaging, several shortcomings were still present [Lindert et al., 2002; Cerný et al., 2007]. These include suboptimal imaging of the capillaries due to motion-induced image blurring by movement of the OPS device, the tissue, and/or flowing red blood cells. This introduces difficulties in measuring blood flow velocities in these vessels. Driven by the success of OPS imaging and the drawbacks it has, we developed a novel imaging modality for the microcirculation, which we have termed sidestream dark field (SDF) imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) [Goedhart et al., 2007].
In this study, the first aim was to validate SDF imaging by comparison of SDF¬mediated measurements of capillary diameters and red blood cell velocities in the human nailfold microcirculation to OPS-mediated measurements. The second aim of this study was to compare OPS and SDF image quality, in terms of contrast and sharpness. For this purpose, OPS and SDF images of exactly the same sublingual microcirculatory areas were obtained. The third aim was to explore the SDF imaging (Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation)capabilities with respect to imaging individual red and white blood cells and assessment of the endothelial glycocalyx in the sublingual microcirculation.
Ops and sdf imaging technology
For OPS imaging, a Cytocan-II backfocus type device (Cytometrics, Philadelphia, PA) was used [Groner et al., 1999] and for SDF imaging, a MicroScan Video Microscope (MicroVision Medical, Amsterdam, The Netherlands) was employed.
In OPS imaging, the tissue embedding the microcirculation is illuminated with polarized green light [Slaaf et al., 1987, Groner et al., 1999] (Figure 1A). Backscattered (and thus depolarized) light is projected onto a CCD camera after it passes an analyzer, i.e., a polarizer orthogonally-oriented with respect to the incident polarization. The light reflected by the tissue surface, which is undepolarized, is blocked by this analyzer. By elimination of the reflected light and imaging of only the backscattered light, subsurface structures, such as the microcirculation(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), can be observed. The use of green light ensures sufficient optical absorption by the (de)oxyhemoglobin-containing red blood cells (red blood cells) with respect to the lack of absorption by the tissue embedding the microcirculation, creating contrast (i.e., red blood cells are visualized black and tissue is visualized white/grayish).
In SDF imaging, illumination is provided by surrounding a central light guide by concentrically placed light emitting diodes (LEDs) to provide sidestream dark field illumination (Figure 1B). The lens system in the core of the light guide is optically isolated from the illuminating outer ring thus preventing the microcirculatory image from contamination by tissue surface reflections. Light
from the illuminating outer core of the SDF probe(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), which penetrates the tissue illuminates the tissue-embedded microcirculation by scattering. The LEDs emit at a central wavelength of 530 nm, chosen to correspond to an isosbestic point in the absorption spectra of deoxy-and oxyhemoglobin to ensure optimal optical absorption by the hemoglobin in the red blood cells, independent of its oxygenation state. This leads to images similar to OPS images(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), where red blood cells are imaged as dark moving globules against a white/grayish background. To improve the imaging of moving structures such as flowing red blood cells, the LEDs provide pulsed illumination in synchrony with the CCD frame rate to perform intravital stroboscopy. This stroboscopic imaging, (partially) prevents smearing of moving features, such as flowing red blood cells, and motion-induced blurring of capillaries due to the short illumination intervals.
Both the OPS and the SDF devices(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) are fitted with a 5× objective lens system. Illumination intensity and image focus were modulated during imaging to obtain visually optimized images for both techniques. Covered by a sterile disposable cap, the probes can be placed on organ and tissue surfaces to investigate microcirculatory morphology and perfusion under different clinical conditions. To prevent microcirculatory perfusion alterations by applying pressure on the imaged area, the probes were placed onto the tissue and then gently pulled back until contact was lost [Trzeciak et al., 2007; De Backer et al., 2007]. Then the probes were advanced again slowly to the point at which contact was regained and the microcirculation was in focus of the lens systems contained in both probes.

figure 1. A) The OPS imaging device. The green, polarized light is reflected by a half pass mirror to provide dark field illumination. The reflected and backscattered light travel through the hole in the mirror to the second analyzer, termed the analyzer, with orthogonal orientation to the first polarizer. B) The SDF imaging device. Light emitting diodes (LEDs) provide stroboscopic sidestream dark field illumination at 530 nm.
Compared to OPS imaging, SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) has the advantage of low-power LED illumination, which allows battery and/or (portable) computer operation and thereby improved clinical applicability. For OPS imaging, relatively strong light sources and thus mains power supply are required, since a large portion of the illumination light is blocked by the first polarizer and another substantial amount of light is reflected by the tissue surface, which do not contribute to the image formation. These high power light sources limit the portability and clinical applicability of OPS imaging. Since SDF employs low-power LEDs for illumination, no isolation transformers between the device and mains power supply are required to protect current leakage in operating rooms, intensive care units, and emergency rooms. Furthermore, battery operation allows microcirculatory measurements recordings to be made in conditions such as ambulances, and emergency and combat medicine, where mains power is not always available.
validation of sdf imaging
For the validation of SDF imaging by comparison to OPS imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), twenty subjects were screened for a nailfold microcirculation that was clearly visible when applying OPS imaging. Eventually, nine healthy non-smoking male volunteers and one healthy non-smoking female volunteer (mean±SD age was 20±2 year) were selected for this validation study. None of these subjects used any medication and all refrained from drinking coffee at least two hours before the measurements to ensure a stable and uninfluenced nailfold microcirculation.
The OPS device and the SDF device(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) were mounted in a specially engineered universal holder (Department of Instrumentation, Academic Medical Center, University of Amsterdam) for accurate positioning and stabilization of both probes and to enable quick and easy interchanging of the devices. Video output was visualized on a monitor and connected to a computer via a signal converter (Canopus, ADVC110) to directly and digitally record images onto a hard drive as DV-AVI files to enable off-line analysis of the images for capillary diameters and red blood cell velocities using AVA software (Automated Vascular Analysis, Academic Medical Center, University of Amsterdam) [Dobbe et al., 2008].
Validation of SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) was performed in analogy to our previously published protocol, where OPS imaging was compared to intravital capillaroscopy (i.e., the gold standard for microcirculatory imaging prior to the introduction of OPS imaging) [Mathura et al., 2001c]. Briefly, the subjects were seated in a comfortable and stable position with their arms slightly bent at heart level. The fingers of the non-dominating hand were stabilized by pushing them gently into a clay bed. Room temperature was kept between 19 and 22 oC. By random selection, it was decided which device (OPS or SDF) was to be used first. Paraffin oil was applied to make the highly scattering nailfold skin more translucent. The devices were adjusted for optimal focus and contrast. Images were recorded during rest, 2-min venous occlusion, and 2-min arterial occlusion (starting 2 min after release of the venous occlusion) to investigate the response of microcirculatory blood vessel diameter and red blood cell flow to occlusion and release. A cuff, which was inflated in < 5 seconds, was used for venous (cuff pressure = 50 mmHg) and arterial (cuff pressure = 180 mmHg) occlusion. OPS and SDF images(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) were acquired sequentially during each of the physiological stimuli.
From the nailfold microcirculation, four capillaries were arbitrarily selected for further off-line analysis (Figure 2). After stabilization (to eliminate movement artifacts) of isolated video sequences, AVA software was used to analyze microcirculatory blood vessel diameters and red blood cell kinetics. Regression analysis for capillary diameters obtained with SDF imaging and the capillary diameters obtained with OPS imaging showed that the magnification ratio OPS:SDF equals 0.9:1.0 (slope = 0.90, R2 = 0.88). During further analysis, the scaling factor ×0.9 is applied for the SDF-mediated measurements to correct for the magnification difference and to allow comparison of capillary diameters and red blood cell velocities obtained with OPS and SDF imaging.
At rest, mean±SD capillary diameters measured using OPS and SDF imaging were 15.8±4.9 and 16.1±4.2 µm (p=0.71), respectively. During venous occlusion, capillary diameters measured using OPS and SDF imaging (Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation)were 17.4±4.6 and 18.0±4.1 µm (p=0.51), respectively. During arterial occlusion, capillary diameters measured using OPS and SDF imaging were 16.1±4.7 and 16.0±3.8 µm (p=0.93), respectively. Furthermore, Bland-Altman analysis showed an average measurement bias of only 1.3±2.3 µm between OPS and SDF imaging (plot not shown).
OPS imaging allowed the measurement of red blood cell velocities by the use of space-time diagrams [Dobbe et al., 2008] in 36 out of the 40 capillaries and SDF imaging allowed these measurements in 39 out the 40 capillaries. The capillaries in which the red blood cell velocity could not be determined were hyperperfused resulting in velocities beyond the detection of the frame rate of the CCD camera. At rest, mean±SD capillary red blood cell velocities measured using OPS and SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) were 277±94 and 270±96 µm/s (p=0.60), respectively. During venous occlusion, red blood cell velocities measured using OPS and SDF imaging were 83±37 and 89±38 µm/s (p=0.19), respectively. The red blood cell velocities during venous occlusion were significantly lower than during rest for both OPS and SDF imaging (p<0.01). Microcirculatory perfusion completely stopped during arterial occlusion as observed by both OPS as SDF imaging. Furthermore, Bland-Altman analysis showed an average measurement bias of only 14±72 µm/s between OPS and SDF imaging during rest and 3±52 µm/s during venous occlusion (plots not shown). Hence, SDF and OPS imaging provide similar quantitative data on capillary diameters and red blood cell velocities, validating the use of SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) for studying the microcirculation.

figure 2. OPS (A) and SDF (B) image of the same nailfold capillary bed. Four capillaries were selected for further off-line analysis of capillary diameters and red blood cell velocities.
Ops and sdf image quality comparison
To compare image quality for OPS and SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), sublingual images were obtained in two subjects that were trained in OPS and SDF imaging and were able to locate the same sublingual microcirculatory areas on command. To ease location of the sublingual microcirculatory areas, OPS video frames were saved and printed to serve as guides. During the sublingual recordings, images were optimized by illumination and focus modulation. No off-line image enhancement was performed for both image analysis and publication.
After recording the sublingual microcirculation by OPS and SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), three microcirculatory areas were selected for image quality analysis in terms of contrast and sharpness. In these three areas, one video frame was isolated for both OPS and SDF. Since the functional information in microcirculatory images lies in the capillaries and the venules, image quality was determined for each of these vessel types. Therefore, in each of the sublingual microcirculatory video frames, six capillaries and five venules were chosen to perform capillary and venular quality analysis. To determine capillary and venular contrast and sharpness, cross-sectional grayscale profiles (grayscale value 0 corresponds to black and 255 corresponds to white) were obtained using ImageJ (developed at the US National Institutes of Health). The contrast was defined as the absolute difference between the minimum value within the vessel and the maximum value (average of two sides of the vessel). The sharpness was defined as the angle of the grayscale profile at the vessel wall (average of two sides of the vessel).
For OPS and SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), similar capillary (77±7 and 79±6; p=0.37) and venular sharpness (74±8 and 72±7; p=0.23) were found. Capillary (14±8 and 23±12; p<0.01) and venular (47±23 and 56±25; p=0.05) contrast, however, were found to be higher in SDF images compared to in OPS images. In Figures 3A and 3B, the capillary and venular quality for OPS and SDF imaging are illustrated. Figure 3A clearly shows that capillaries have higher contrast when using SDF imaging compared to when using OPS imaging. Venular contrast and sharpness is approximately equal for both techniques as shown in Figure 3B. Additionally, the Figures depict the magnification difference between the OPS and SDF device (Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation)(scale bars Figures 3A and 3B).

figure 3. A) Capillary contrast and sharpness. B) Venular contrast and sharpness. The scale bars indicate the magnification difference between the OPS and SDF device.
imaging individual red and white blood cells Using SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), individual red and white blood cells can be observed. This is exemplified in Figures 4 and 5. Figure 4 shows individual red blood cells, separated by plasma gaps, flowing trough a capillary loop. In Figure 5, most white blood cells travel from the vertical capillary via the T-junction to the right capillary (Figure 5B, 5C, and 5D). As the capillary increases in diameter, the white blood cells start rolling till they reach the capillary-venule junction. Once arrived in the venule some white blood cells remain rolling against the venular wall and others are taken up by the blood flow and slowly flow down-stream.
imaging the endothelial glycocalyx Provided that white blood cells are sufficiently stiff to (temporarily) damage the endothelial glycocalyx during their passage in small capillaries (i.e., < 10 µm), while the glycocalyx in turn is stiff enough to deform red blood cells, the red blood cell column width in before and after white blood cell passage can be used to estimate glycocalyx thickness. Hence, using SDF imaging of the sublingual microcirculation(Renal microcirculation,kidney microcirculation,Tongue microcirculation,lingua microcirculation), estimations of individual capillary glycocalyx dimensions could be obtained. For glycocalyx measurements, it is first important to distinct white blood cells from plasma gaps, which can be done by following the white blood cell/plasma gap to a capillary-venule junction (Figures 6A-6D). At this junction, white blood cells will tend to roll against the venular wall, while plasma gaps will dissolve in the larger blood stream (Figure 6D). Once an image is captured from before

figure 4. Individual red blood cells (numbered from 1 to 7) and plasma gaps flowing through a capillary can be observed in normal view (A) and in the zoomed view (B).

figure 5. SDF imaging enables white blood cell visualization in normal view (A) and in zoomed view (B, C, and D). The red arrows indicate a rolling white blood cell at t=0 ms (A and B), t=400 ms (C), and t=800 ms (D). and after the white blood cell passage, an estimation of the glycocalyx thickness can be made, by subtracting the initial red blood cell column diameter from the diameter after white blood cell passage (Figures 6E and 6F).
discussiOn and cOnclusiOns
This study introduced a novel optical technique for clinical observation and assessment of the microcirculation, termed sidestream dark field (SDF) imaging, and validated it by quantitative comparison to OPS imaging. Results showed that OPS and SDF imaging provided similar values for capillary diameters and red

figure 6. A) Sublingually-acquired microcirculatory image using SDF imaging. The white square indicates the region of interest, which is enlarged in the following panels. B and C) White blood cell flowing through a capillary (indicated with arrows). D) Same white blood cell, rolling against venular wall. E) Enlarged view of the capillary without (left side) and with (right side) the white blood cell. F) Same as panel (E), with enhanced contrast for more clear observation of the widening of the red blood cell column after the white blood cell passage (indicated with arrows).
blood cell velocities in the human nailfold microcirculation. These basic findings validate the use of SDF imaging for clinical measurement of microcirculatory vessel diameters and red blood cell velocity measurements. SDF imaging, moreover, provided significantly higher image quality with more detail and higher capillary and venular contrast and enabled imaging of individual red and white blood cells and measurement of the endothelial glycocalyx thickness.
Increased microvascular quality and observability of granular structures probably originates from the stroboscopic illumination, which prevents smearing of moving features such as red blood cell columns in capillaries and venules. Stroboscopic imaging also reduces image blurring due to movement of the device and/or the tissue. An additional contributing factor to the superior quality of SDF imaging is the shallower focusing depth of the SDF device with respect to the focus of the OPS device(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation). In OPS imaging, underlying vascular (and thus light absorbing) structures (partially) darken the image, lowering image contrast and quality. In SDF imaging however, these underlying structures do not interfere, due to the shallow imaging depth of the SDF device, and therefore provide clear images of the superficial microcirculatory network.
However, although SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) was shown to be superior to OPS imaging, it still suffers from some shortcomings. In the current SDF device, the detectable red blood cell velocity is physically limited (at approximately 1 mm/s) by the length of the observed vessels in combination with the 25 fps (PAL) or 30 fps (NTSC) acquisition rates. Hence, future improvement of microcirculatory imaging will be made by incorporation of more advanced camera technology in terms of resolution and frame rate, which will enable red blood cell velocity measurements in high flow vessels and more accurate vessel geometry determination. This, in conjunction with completely automated software, with (new) microcirculatory scoring systems implemented, will lead to faster and more exact determination of microcirculatory functioning in clinical and experimental settings.
Another point of concern with SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) is the pressure-induced microcirculatory alterations by application of the SDF probe onto organ and tissue surfaces. These pressure-induced effects occur with other surface flow/perfusion measurements, such as OPS imaging and laser Doppler velocimetry, as well and might lead to false interpretation of the actual microcirculatory perfusion. To prevent the microcirculatory measurements from this pressure artifact during OPS imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation), Lindert et al. engineered an extending click-on ring, which was placed around the OPS probe. By applying suction via holes in the ring using a vacuum pump, the tissue in the center, that was imaged through the OPS probe, was inhibited from moving and the pressure on the imaged vasculature was reduced [Lindert et al., 2002]. However, in order to objectify the assessment of the microcirculatory perfusion, the pressure artifact should be characterized, i.e., the distortion of microcirculatory perfusion should be measured as a function of the applied pressure.
In conclusion, the present study has introduced SDF imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation) as a novel imaging modality, incorporated in a hand-held clinically-applicable device. SDF imaging was validated by quantitative comparison to OPS imaging(Renal microcirculation,kidney microcirculation,sublingual microcirculation,Tongue microcirculation,lingua microcirculation). It is anticipated that SDF imaging will serve as a novel and improved imaging modality to contribute to the clinical assessment of the microcirculation in various clinical scenarios and, additionally, allow more reliable application of computer-aided image processing and analysis software for quantification of microcirculatory alterations associated with disease and therapy.

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