Fig. 1 a, Orthogonal polarization spectral imaging probe. b, Optical schematic of the OPS imaging probe. A typical magnification of ×10 is maintained between the target and its image. This results in a resolution of approximately 1 µm/pixel, which is limited by the dimension of the CCD pixel. The probe can be focused from the target surface to 1.0-mm depth, depending on the type of target and the optics used. In vivo, the typical depth of focus is approximately 0.2 mm. c, Optical density of a graduated gray scale (catalog number 152-7662; Kodak) was measured in the presence of the polarization analyzer (b) or with a 0.5 OD neutral density filter (ć) used in place of the analyzer. Average light intensity for each gray level was converted to OD by the following formula: OD = log 10 ((I m -I d )/(I max -I d )) where I m = measured light intensity, I d = dark light intensity (obtained using a black velvet target), I max = intensity of white target. NEW TECHNOLOGYDifferent disease states, including diabetes, hypertension and coronary heart disease, produce distinctive microvascular pathologies. So far, imaging of the human microcirculation has been limited to vascular beds in which the vessels are visible and close to the surface (for example, nailfold, conjunctiva). We report here on orthogonal polarization spectral (OPS) imaging, a new method for imaging the microcirculation using reflected light that allows imaging of the microcirculation noninvasively through mucus membranes and on the surface of solid organs. In OPS imaging, the tissue is illuminated with linearly polarized light and imaged through a polarizer oriented orthogonal to the plane of the illuminating light. Only depolarized photons scattered in the tissue contribute to the image. The optical response of OPS imaging is linear and can be used for reflection spectrophotometry over the wide range of optical density typically achieved by transmission spectrophotometry. A comparison of fluorescence intravital microscopy with OPS imaging in the hamster demonstrated equivalence in measured physiological parameters under control conditions and after ischemic injury. OPS imaging produced high-contrast microvascular images in people from sublingual sites and the brain surface that appear as in transillumination. The technology can be implemented in a small optical probe, providing a convenient method for intravital microscopy on otherwise inaccessible sites and organs in the awake subject or during surgery for research and for clinical diagnostic applications.At present, the use of microvascular imaging in diagnosis and treatment of human disease is limited. Use has been made of nailfold capillaroscopy in the diagnosis and treatment of peripheral vascular diseases, diabetes and hematological disorders 1-3 . Problems with movement have restricted the use of the bulbar conjunctiva for clinical applications in opthalmology 4-6 . Other locations observed by intravital microscopy include the microcirculation of the skin, lip, gingival tissue and tongue 4 . Laser-sca...

Little is known about the microvascular perfusion of the skin postnatally. Skin microvascular parameters can be assessed noninvasively with orthogonal polarization spectral imaging (OPS), a technique where, through the use of special optics, a virtual light source is created at a depth of 1 mm within the tissue. The light is absorbed by the Hb, yielding an image of the illuminated Hb-carrying structures in negative contrast. In nine term (weight 2100 -4470 g) and 28 preterm infants (weight 550 -2070 g; gestational age 24 -33 wk) red blood cell velocity and vessel diameter and density were determined off-line with the CapImage program in vessels video-recorded by OPS near the axilla on d 1 and 5 of life. Blood pressure, heart rate, hematocrit, and body and incubator temperature were noted. Vessel diameter ranged from 6 to 24 m, vessel density from 219 to 340 cm/cm 2 with no change between d 1 or 5 and no difference between term and preterm infants. Red blood cell velocity increased in preterm infants from d 1 [median 528 m/s, 95% confidence interval (CI) 486 -564 m/s] to d 5 (median 570 m/s; 95% CI 548 -662 m/s; p ϭ 0.001) and correlated with the decrease in median hematocrit from 44% (CI 40%-60%) to 39% (CI 37%-43%) with r 2 ϭ Ϫ0.37 with a 95% CI Ϫ0.59 to Ϫ0.11, p ϭ 0.006. Hematocrit correlates with red blood cell velocity in the microvessels of the skin. The new technology can be used to assess quantitative changes in the microvessels and thus allows noninvasive assessment of tissue perfusion in term and preterm infants. At birth, the skin is richly supplied by a dense subepidermal plexus that shows relatively little regional variation. Even the middle and deep dermis are richly endowed with vasculature. The mature pattern of capillary loops and of the subpapillary venous plexus is not present at birth. With exception of the palms, soles, and nail beds, the skin at birth has almost no papillary loops, but demonstrates a disorderly capillary network. By the end of the first week of life, the capillary network assumes a more orderly pattern. Papillary loops begin to appear as small superficial dilatations or buds in the second week and cooling of the skin appears to encourage maturation. The skin architecture in newborns is notably different from those of an adult. Whereas the latter regularly shows loops of capillaries running orthogonal to the surface of the skin, the neonate has a more horizontal structure, which is readily seen through the very thin upper layers (1-4).Cardiac output normalized to mass is much higher in the newborn than in the adult. Because of the high resting cardiac output of neonates there is limited reserve to further augment blood flow under stress. Perfusion pressure is maintained by redistributing marginal cardiac output and oxygen supply to brain, heart, and adrenal gland. Under no stress conditions the skin has high blood flow in relation to its oxygen requirement. Assessment of skin perfusion is therefore of great interest, but there is only very scant data about the microcirculator...

The use of fluorescent-labeled microspheres (FM) for measurement of regional blood flow is an attractive alternative to the use of radioactive-labeled microspheres. In the FM method the FM have to be completely recovered from the tissue samples in a time- and labor-intensive process. For this reason, a considerable loss of FM is possible. The aim of this study was to develop a filtration device that allows the tissue sample to remain in a single container throughout the procedure to make the process easier and to avoid the loss of FM. The core of the sample-processing unit (SPU) is a single-tube filtration device with a polyamide wire mesh. The protocol for processing tissue from different organs (heart, kidney, liver, spleen, intestine, muscle, bone, lung, brain) was modified and thus shortened significantly. Furthermore, the SPU allows direct filtration of the blood reference sample without previous digestion. Different experiments showed that the SPU in combination with the new protocol excludes the loss of 15-μm FM. The modifications of the whole procedure render it faster and highly standardized.

The Cytoscan^TM Model E-II (Cytometrics Inc., Philadelphia, Pa., USA) is a newly developed instrument which functions as an intravital microscope and is small and easily portable. Through the use of orthogonal polarization spectral (OPS) imaging, the Cytoscan Model E-II delivers images of the microcirculation which are comparable to those achieved with intravital fluorescence videomicroscopy (IFM), but without the use of fluorescent dyes. The purpose of this study was to validate the Cytoscan Model E-II instrument against IFM. The experiments were carried out on striated muscle in the dorsal skinfold chamber of the awake Syrian hamster. The following parameters were measured in identical regions of interest in the same animal under baseline conditions and 0.5 and 2 h after a 4-hour period of pressure-induced ischemia: arteriolar diameter, venular diameter and venular red blood cell velocity. Bland-Altman plots showed good agreement between the two techniques for venular red blood cell velocity. As expected, arteriolar and venular diameters as measured by the Cytoscan were on average 5 µm smaller than the values from IFM, since the Cytoscan measures the red blood cell column width and IFM measures luminal diameter. Thus, OPS imaging can be used to make valid measurements of microvascular diameter and red blood cell velocity in tissues.

Tumour angiogenesis plays a key role in tumour growth, formation of metastasis, detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour angiogenesis is an indispensable prerequisite for developing novel treatment strategies such as anti-angiogenic and antivascular treatment options. Therefore, it was our aim to establish and validate a new and versatile imaging technique, that is orthogonal polarisation spectral TM imaging, allowing for non-invasive quantitative imaging of tumour angiogenesis in vivo. Experiments were performed in amelanotic melanoma A-MEL 3 implanted in a transparent dorsal skinfold chamber of the hamster. Starting at day 0 after tumour cell implantation, animals were treated daily with the anti-angiogenic compound SU5416 (25 mg kg bw 71 ) or vehicle (control) only. Functional vessel density, diameter of microvessels and red blood cell velocity were visualised by both orthogonal polarisation spectral TM imaging and fluorescence microscopy and analysed using a digital image system. The morphological and functional properties of the tumour microvasculature could be clearly identified by orthogonal polarisation spectral TM imaging. Data for functional vessel density correlated excellently with data obtained by fluroescence microscopy (y=0.99x+0.48, r 2 =0.97, R S =0.98, precision: 8.22 cm 71 and bias: 70.32 cm 71 ). Correlation parameters for diameter of microvessels and red blood cell velocity were similar (r 2 =0.97, R S =0.99 and r 2 =0.93, R S =0.94 for diameter of microvessels and red blood cell velocity, respectively). Treatment with SU5416 reduced tumour angiogenesis. At day 3 and 6 after tumour cell implantation, respectively, functional vessel density was 4.8+2.1 and 87.2+10.2 cm 71 compared to values of control animals of 66.6+10.1 and 147.4+13.2 cm 71 , respectively. In addition to the inhibition of tumour angiogenesis, tumour growth and the development of metastasis was strongly reduced in SU5416 treated animals. This new approach enables non-invasive, repeated and quantitative assessment of tumour vascular network and the effects of antiangiogenic treatment on tumour vasculature in vivo. Thus, quantification of tumour angiogenesis can be used to more accurately classify and monitor tumour biologic characteristics, and to explore aggressiveness of tumours.

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Harris, A. G., I. Sinitsina, and K. Messmer. Validation of OPS imaging for microvascular measurements during isovolumic hemodilution and low hematocrits. Am J Physiol Heart Circ Physiol 282: H1502-H1509, 2002 10.1152/ajpheart. 00475.2001 imaging is a new technique that can be used to visualize the microcirculation with reflected light. It uses hemoglobin absorption to visualize the red blood cells (RBCs). Thus the method could fail at low hematocrit (Hct). The aim of this study was to validate OPS imaging for quantitative measurements of diameter and functional capillary density (FCD) under conditions of hemodilution of varying degrees to achieve a wide range of Hcts. The validation was performed in the dorsal skinfold chamber of nine awake Syrian golden hamsters. Measurements of vessel diameter and FCD were performed off-line using Cap-Image on video sequences captured using OPS imaging and standard intravital fluorescence microscopy at baseline, 85, 70, 55, and 40% of the initial Hct. For hemodilution, isovolumic exchange of blood for 6% Dextran 60 was performed. Bland-Altman plots for the vessel diameter and FCD show good agreement between the two methods for both parameters at all studied Hcts. As expected, there was a systematic bias of ϳ4 m in the diameter measurements since the RBC column was measured and not the intravascular diameter. In conclusion, OPS imaging can be used to measure diameter and FCD at a wide range of Hcts.Cytoscan; orthogonal polarization spectral imaging IN RECENT STUDIES it was shown that diameter and functional capillary density (FCD) could be quantitatively measured using the orthogonal polarization spectral (OPS) imaging technology incorporated into the Cytoscan E-II (Cytometrics, Philadelphia, PA) under both physiological and pathophysiological conditions (6, 9). These studies were carried out in the dorsal skinfold chamber of the awake Syrian golden hamster and were performed by comparing the measurements made using intravital fluorescence microscopy images with those made using Cytoscan E-II images.The Cytoscan E-II images red blood cells (RBCs) through the absorbance of hemoglobin (6). The measurement of diameter is dependent on good visualization of the RBC column in the vessel under study. The measurement of FCD requires the observation of RBCs moving through the capillaries. Because the measurement of both of these parameters requires the presence of RBCs, their measurement could be disturbed or influenced during hemodilution. A decrease in the number of RBCs in the venules could lead to an incomplete filling of the vessel, which in turn could result in an underestimation of the vessel diameter. For the measurement of FCD, a decreased number of circulating RBCs could cause too few perfused capillaries to be counted and therefore an underestimation of the FCD.The correct measurement of the vessel diameter is also of critical importance for the noninvasive determination of hemoglobin and hematocrit using the OPS imaging technology incorporated into the Hemoscan (Cytometrics...

OPS imaging can be used to quantitatively measure microcirculatory parameters in the rat liver under both physiological and pathophysiological conditions. Thus, OPS imaging has the potential to be used to make quantitative measurements of the microcirculation in the human liver.