OPS Imaging of Human Microcirculation: A Short Technical ReportLindert J.a · Werner J.b · Redlin M.a · Kuppe H.a · Habazettl H.a,b · Pries A.R.a,b
aInstitute of Anesthesiology, Deutsches Herzzentrum Berlin, bDepartment of Physiology, University Hospital Benjamin Franklin, Freie Universität Berlin, Berlin, Germany Corresponding Author
Despite the pivotal role of microcirculation in numerous diseases, techniques for the direct assessment of human microcirculation are limited. A new approach based on orthogonal polarization spectral (OPS) imaging (Cytoscan™ microscope) allows noninvasive observation of human microcirculation in all accessible tissue surfaces. Limitations remain: application of pressure with the instrument affects blood flow, lateral movement of tissue precludes continuous investigation of a given microvascular region, and blood flow velocities above 1 mm/s cannot be measured. We addressed these problems by (a) constructing an attachment to the probe, preventing direct contact of the instrument with the observed tissue area and allowing fixation of the tissue, and (b) implementing a double-flash spatial correlation technique extending the measuring range for blood flow velocities up to ∼40 mm/s. The modified approach was tested in vitro and in vivo. Velocity readings correlated well with velocities of an external standard (r2 = 0.99, range 1.9–33.8 mm/s). Pulsatile flow patterns synchronous with heart rate with maximal velocities of about 10 mm/s could be detected in arterioles of the human sublingual mucosa. The modified instrument may prove useful to investigate the microcirculation in the context of research, diagnosis and therapy control.
Copyright © 2002 S. Karger AG, Basel
Adequate function of the microcirculation is a prerequisite for tissue nutrition and oxygen supply [1, 2]. Numerous diseases, e.g. diabetes mellitus , chronic ulcers, hypertension or sepsis [2, 4], are associated with structural changes of microvessels (e.g. remodeling, rarefaction) and/or functional defects (e.g. endothelial dysfunction). Therapeutic interventions and drugs targeted at the microcirculation may affect arteriolar tone, microvascular blood flow, inflammatory responses, exchange processes or microvessel number/structure.
Previous attempts for assessment of the human microcirculation included direct visualization by microscopy of capillary vessels in the nailfold and the skin [5, 6], as well as investigation of functional parameters like flow, e.g. by laser doppler techniques [7, 8] or oxygen pressure by oxygen-sensitive electrodes/optodes . The first approach is mostly restricted to the capillary bed and provides information on individual microvessels, while the latter measure integrates parameters of microvascular function in a certain tissue volume, but data cannot be attributed to specific microvessels, e.g. nutritive capillaries or arteriovenous shunts  and provides no information on microvascular morphology.
An alternative microscopic approach based on epi-illumination with polarized light was originally described in 1987 by Slaaf et al. . Recently, a modified version of this technique named ‘orthogonal polarization spectral imaging’ (OPS) was introduced  and implemented in a compact transportable intravital microscope (Cytoscan™, Cytometrics, Philadelphia, Penn., USA). The small and lightweight probe of the microscope can be held manually or by simple positioning devices. A sterile disposable lens cap (Cytolens™) allows the investigation of any accessible surface in humans , e.g. skin, mucous membranes or internal organs during surgical procedures.
However, several limitations have already been recognized regarding practicability and analysis options : (1) It is virtually impossible to avoid application of pressure with the tip of the probe during examination, thereby altering the flow velocity in vessels under investigation. Furthermore, relative movements of tissue and probe render recording of selected areas and maintaining optimal focus during observation, as well as offline velocity analysis difficult. (2) Blood flow velocities can only be measured up to a maximum of ∼1 mm/s using conventional illumination, regular video rate recordings, and image analysis techniques. Thus, substantial information on arteriolar flow velocity and pulsatile flow patterns remains hidden.
In the present study these limitations were addressed by: (1) designing a sterilizable device, which can be attached to the sterile plastic cap covering the probe of the Cytoscan. This device maintains a fixed distance between probe and tissue surface, avoiding direct contact with the observed tissue and preventing relative movements of tissue and probe and (2) replacing the standard light source with an asynchronous stroboscopic flashlight triggered by the regular video recorder, and by implementing image analysis according to the principle of spatial correlation, extending the range of measurable blood flow velocities to over 20 mm/s.
materials and methods
Linearly polarized light is emitted from the objective probe into the tissue . A second orthogonally oriented polarizer is used to block light reflected from tissue surfaces with unchanged polarization before visualization. Light which is multiply deflected and scattered in deeper layers of the tissue experiences a change of its polarization direction. This light passes the second polarizer and serves as a virtual light source in the depth of the tissue. Thus, images comparable to those obtained by transillumination are created.
The instrument uses the OPS technique illuminating the tissue with light of a wavelength of 548 nm which is absorbed equally well by oxy- and desoxyhemoglobin to improve visualization of blood vessels. With the 10× probe (334× magnification on the 10.4-inch display), the resolution is about 1.0 μm and thus high enough to allow differentiation of single erythrocytes in capillaries and intravascular optical patterns created by erythrocytes and plasma gaps in larger vessels. These image structures can be used for velocity calculations (see below).
A stainless steel attachment was designed to fit on the sterile disposable plastic cap (CytolensTM) that covers the Cytoscan probe during observation. A defined position of this device is ensured by pushing it towards a protruding edge of the Cytolens. In this position, the attachment exceeds the cap by a predetermined length (e.g. 0.5 mm), thus avoiding direct contact and the application of pressure to the tissue area under observation (fig. 1).
Fig. 1. Schematic drawing of the stainless steel distance holder attached to the disposable plastic cap covering the Cytoscan probe. Suction applied to the connector provides fixation of underlying tissue via small holes.
The front part of the attachment is a hollow circular channel. Suction applied to a connector is transmitted to the underlying tissue via 24 small outlets (diameter 0.5 mm) providing gentle immobilization. The steel attachment can be sterilized by routine techniques.
The shift of intravascular optical patterns in a defined time interval is used for offline velocity calculation by spatial correlation from the videotapes (DSR 20P, Sony, Japan) recorded during patient examination. The maximal velocity that can be measured by this approach depends on the optical magnification and the minimal time delay between the optical recordings. In standard setups, this delay corresponds to an average delay between two consecutive half frames of 20 ms using conventional 50-Hz video systems (or 16.6 ms for 60-Hz systems, e.g. in the US). This time delay and the typical length of intravascular optical patterns (∼10–20 μm) result in an upper velocity limit of about 1 mm/s. In the modified setup used here, the continuous light source is replaced by an asynchronous stroboscopic lamp (Strobex, Model 11360, Chadwick-Helmuth Electronics, El Monte, Calif., USA) that illuminates the first (odd) half-frame of a video field immediately prior to the frame transfer while the second (even) half-frame is illuminated after the frame transfer allowing for a time delay as brief as 0.5 ms, thus extending the upper limit for velocity measurement to approximately 40 mm/s  (fig. 2). In addition to the short delay, velocity determinations are supported by the lack of movement artifacts in the frames due to the very short exposure time (∼20 μs relative to 20 ms for conventional illumination.
Fig. 2. Schematic drawing of the timing of video signals and conventional continuous and asynchronous strobe illumination. The upper panel displays a video field consisting of two half-frames (odd, even) storing image information of 20 ms each divided by the vertical blank during which the frame transfer to the readout storage is performed. Under continuous illumination (middle panel) the entire exposure time of the frames is used to acquire the image information. In contrast, asynchronous strobe illumination (lower panel) results in a short exposure time and the time delay of the exposure between odd and even frame can be reduced to ∼0.5 ms.
The measurement line (straight or curved over a length of ∼50–300 μm) is defined in the focus along the center of the microvessel. Along this line, intensities are recorded for the two corresponding half-frames of any field within a variable number of measuring cycles (e.g. 100 cycles corresponding to 4 s). Calculation of flow velocity using the spatial correlation approach is done automatically and the results are displayed as time course. To enhance measuring robustness and accuracy, up to 5 parallel measurement lines can be defined in a single vessel. In addition, the software is supplemented by a two-dimensional tissue motion analyzer correcting remaining movements over a distance of up to 50 μm in real time.
A disk covered with beads (diameter ∼15 μm) providing optical contrast was rotated by an electric motor at constant speed. The Cytoscan probe was fixed in a micromanipulator, placed at a defined distance from the center of the disk and images were recorded under asynchronous flash illumination. Rotation speed was set to six different levels covering the velocity range expected in vivo. Actual disk velocity at the investigated area was precisely calculated from rotating speed and radial distance and compared with six independent velocity measurements obtained from recordings made with the modified Cytoscan.
After obtaining informed consent according to the declaration of Helsinki healthy volunteers were seated comfortably and the sublingual tissue was examined with the Cytoscan equipped with the distancing device. Once a region of interest was identified, the probe was fixed by applying suction (about –100 mm Hg) and images were recorded for ∼30 s under asynchronous flash illumination. In one experiment, the suction was discontinued in intervals of ∼4 s to evaluate a possible effect of the suction on the blood flow. Velocities with and without suction were compared.
Velocity values obtained by the modified Cytoscan device were compared with the actual disk velocities by linear regression. p < 0.01 was considered to indicate statistical significance.
Validation measurements with the Cytoscan (Vm) against the calculated velocity of the rotating disk (Vd) were performed at six different velocities in the range from 1.90 to 33.80 mm/s. Calculation of linear regression indicated an r2 = 0.99 (p < 0.01) and a bias of –0.34 ± 2.2 % (± SD ) with Vm = 0.0908 + (0.984*Vd).
In the sublingual tissue of 5 healthy volunteers (4 male; 1 female; age 31–48) microvessels with inner diameters from ∼19 to 40 μm were analyzed. Examples of velocity recordings of blood flow are shown in figure 3. Velocities in the range of 0.2–11 mm/s were determined in venules and arterioles, respectively. Recordings in arterioles but also, to a lesser extent, in venules exhibited pulsatile fluctuations in synchrony with heart rate. Investigations were performed with the Cytolens alone or with the attached distancing device. As expected, examination with the Cytoscan alone resulted in irregular tissue movement that made adequate focusing and analysis impossible, if no pressure was applied to the tissue. Moderate pressure reduced tissue movement but impaired the blood flow in the observed superficial vessels. In contrast, moderate application of pressure to the tissue via the Cytoscan probe with the distancing device attached did not lead to relevant changes in blood flow. Immobilization via suction substantially reduced the residual irregular movement of the tissue relative to the probe. Blood flow velocities were not significantly affected by the application of suction compared to levels immediately before the onset of suction (fig. 3c).
Fig. 3.a, b Blood flow velocities of 0.2–11 mm/s were determined in arterioles (a) and venules (b), of sublingual tissue in healthy volunteers. Recordings exhibited pulsatile fluctuations in synchrony with heart rate (ID = inner diameter). c A continuous measurement of the blood flow velocity in an arteriole over a time period of 9 s. The first 4 s were recorded without suction applied to the steel attachment, then a pressure of –100 mm Hg was applied for 4 s. Neither peak nor mean values of flow velocity changed considerably during the suction period.
The Cytoscan intravital microscope, for the first time, provides a relatively easy approach to noninvasive investigation of the human microcirculation of surface tissues both of the intact body and during surgical intervention . However, first experience indicates limitations with regard to practicability (application of pressure with the instrument affecting blood flow, fixation of the tissue) and analysis options (arteriolar blood flow velocities).
The present modifications address these problems. An attachment to the probe maintains a fixed distance between lens and tissue and reduces lateral movement of the tissue by application of suction without affecting the blood flow. Regarding practicability during patient examination this approach is superior to a fixed mounting of the probe in tripods that would also require additional fixation of the tissue itself to prevent movement due e.g. to respiration or peristaltic movement.
In the present approach, the diameter of the suction holes in the device was very small (0.5 mm) to avoid the transmission of pressure effects into deeper layers of the surface tissue and possibly on feeding and draining vessels of the area observed. However, stabler immobilization with reduced suction may be achieved with slightly larger holes.
The suction and distancing device can prevent direct contact of the lens with the top tissue layer investigated and restrict its movement relative to the probe. However, distortion and compression of deeper layers will still occur if higher forces are applied. Therefore it may not be optimally suited for investigation of strongly moving surfaces like the beating heart.
The asynchronous flash illumination combined with a specially developed image analysis software based on the principle of spatial correlation allows the measurement of velocities up to 34 mm/s with an excellent correlation in the in vitro validation experiment.
In the analysis of human sublingual vessels we demonstrated physiological flow patterns in terms of pulsatility of microvascular flow synchronously with the heart rate. Peak velocities in our recordings of arterioles clearly exceeded the range of conventional velocity measurement techniques. Thus, our modifications facilitate patient examination and extend the scope of reliable information about the microcirculation that can be obtained with the Cytoscan to the critical level of arterioles.
The Cytoscan is now available from Rheologics, Exton, Pa., USA. Thus, the OPS imaging technique, which was originally developed for experimental intravital microscopy  may further be used in humans. The presented modifications will be useful for such investigation.
However, for routine use in clinical practice, a customized software solution for analysis, less demanding on user skills, seems indispensable. Such an improved software should allow for the automated selection of interesting vessels based on diameters and morphology, as well as automated velocity determinations. This improved device for the direct visualization of the human microcirculation may provide new diagnostic/therapy control options in the future.
Axel R. Pries
Freie Universität Berlin, Department of Physiology
D–14195 Berlin (Germany)
Tel. +49 30 84 45 16 31, Fax +49 30 84 45 16 34, E-Mail email@example.com
Received: Received: December 3, 2001
Accepted after revision: April 18, 2002
Number of Print Pages : 5
Number of Figures : 3, Number of Tables : 0, Number of References : 14
Journal of Vascular Research
Founded 1964 as Angiologica by M. Comèl and L. Laszt (1964–1973) continued as Blood Vessels by J.A. Bevan (1974–1991)
Vol. 39, No. 4, Year 2002 (Cover Date: July-August 2002)
Journal Editor: M.J. Mulvany, Aarhus
ISSN: 1018–1172 (print), 1423–0135 (Online)
For additional information: http://www.karger.com/journals/jvr