JP2003510112A - Medical application of orthogonal polarization spectral imaging - Google Patents

Abstract

  (57) [Summary] The medical application of orthogonal polarization spectrum (OPS) imaging technology is presented. This technique provides high-contrast images of subsurface phenomena such as vascular structures, intravascular blood flow, glandular structures, and high-resolution images of the surface of solid organs. The many clinical (diagnostic and therapeutic) applications and research applications of this technology in the medical and pharmaceutical fields are described.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION The present invention relates to orthogonal polarization spectrum (OPS) imaging analysis. The present invention is more particularly intended to directly, and often non-invasively, image, characterize, evaluate, monitor and / or analyze microvessels and / or vasculature of a subject. In vivo medical and clinical use of OPS imaging. The invention also relates to in vitro and in vivo applications of reflectance spectral imaging analysis for basic and clinical research, and as an educational tool.

2. Related Art Various disease states, including, for example, diabetes, hypertension, many ophthalmological conditions, and coronary heart disease, each give rise to unique microvascular pathologies. To date, imaging the human microcirculation for diagnosis and / or treatment has been limited to vascular beds that are visible to the blood vessels and even closer to the surface (eg, nail fold or conjunctiva).

Nailfold capillary microscopy has been used, for example, in peripheral vascular disease, Raynaud's phenomenon, diabetes and blood disease (Forst, T. et al., Clinical Science).
(Clinical Science) 94: 255-261 (1998); Fagrell, B .; And Bollinger, A .; , Clinical Capillaroscop
y (clinical capillary microscope): A Guide to It's Use in Cl
internal Research and Practice (Guide for Clinical Research and Its Use in Practice), Hogrefe & Huber, Seattle (1990); Fagrell, B .; And Intaglietta, M
． J. Int. Med. 241: 349-362 (1997)). These studies were conducted to obtain experimental data on capillary density, capillary shape and blood flow velocity, and were limited to gross physical measurements on the capillaries. No spectral or individual cell measurements were taken, and Doppler technology was used to assess velocity.

The use of spherical conjunctival tracts for clinical applications in ophthalmology has been limited by kinematic problems (Davis, E. and Landau, J., Clinica).
l Capillary Microscopy (Clinical Capillary Microscope), Tho
mas (Thomas), Springfield (1966); Fenton, B
． M. Others, Microvasc. Res. 18: 153-166 (1979)
Wolf, S .; Hypertension 23: 464-467 (19).
1994)).

Other locations observed by biomicroscopy include the microcirculation of the skin, lips, gingiva and tongue (Davis, E. and Landau, J., supra).

Laser scanning cofocus imaging is one new technique that enables reflected light imaging of in vivo microcirculation (Bussau, LJ et al., J. Anat 1
92 (Pt 2): 187-194 (1998); Rajadhyaksha.
, M .; J. J. et al. Invest. Dermatol. 104: 946-952 (1
995)). This method allows the skin layers to be distinguished and the microcirculation within the skin to be imaged. However, images obtained using a laser scanning co-collection microscope can be collected only at a fraction of the normal video rate (up to 16 to 30 frames / second), and this technique is used. Fluorescent labels for enhancing the contrast are required for the existing microvascular images (Bussau, LJ. Et al., Supra).

Direct observation of other organs in the human body using conventional techniques requires the use of fluoroscopy or fluorescent dyes to enhance contrast, or especially during surgery. This has not been done due to the size of the equipment needed to capture the image.

Recently, several devices have been developed for in vivo analysis based on reflectance spectroscopy. However, these conventional reflective devices are not optimal for several reasons.

US Pat. No. 4,998,533 (James W. Winkelman (1
991)) discloses one non-invasive device for in vivo analysis.
This device utilizes image analysis and reflectance spectroscopy to measure individual cell parameters such as cell size. Measurements are made only in microtubules, such as capillaries, where individual cells can be seen. Since this device only makes measurements in the capillaries, the measurements taken do not accurately reflect the measurements for larger tubes. As a result, the Winkelman device is unable to measure total hemoglobin concentration, which depends on the ratio of the volume of red blood cells to the volume of total blood in a central or true hematocrit, or in large blood vessels such as veins.

The Winkelman device measures the relative number of white blood cells to red blood cells by counting the individual blood cells that flow through the microcapillaries. The Winkelman device relies on accumulating a sufficiently reliable number of white blood cells to estimate the concentration. The Winkelman device does not provide a means by which it is possible to image and count platelets, or to image capillary plasma or to quantify the components of capillary plasma. Also, this device does not provide a means by which abnormal components of blood such as tumor cells can be detected.

Other devices utilize light illuminating means to direct an illumination source directly onto the blood vessels in the detection area. As a result, these devices are extremely sensitive to movement of the device relative to the patient. Such increased sensitivity to movement of the device or patient can lead to erratic results. To suppress this motion sensitivity, these devices require stabilizing and immobilizing means.

Other conventional devices were developed based on traditional dark field illumination techniques. As understood in traditional microscopes, dark field illumination illuminates the specimen but does not accept direct light on the subject. For example, traditional dark-field imaging techniques illuminate the image plane such that the angular distribution of the illuminating light and the angular distribution of the light focused by the object for imaging are mutually exclusive. However, since the illuminating light is incident on the field of view of the detector, these devices that scatter optically active tissue into the image path can produce orientation-dependent backscatter or image brightening that reduces image contrast. There is a nature. Further, the rotation of these devices can change the contrast.

To date, there are no non-invasive methods to image and characterize the microcirculation inside the eye. Of primary interest is the microcirculation of the retina and optic disc. External circulation of eye structures, as well as benign and malignant tumors, and
Also of interest is the changes that occur in association with disease processes such as circulatory disturbances that occur in red blood cell mudification and agglutination that occur in various forms of hemocyte disease.

Retinal circulation is affected by many physical illnesses such as diabetes mellitus, sickle cell anemia, and macroglobulinemia. Various types of macular degeneration are also affected by circulatory changes. In fact, there are many other ocular and physical symptoms that affect the microcirculation of the retina. Glaucoma is an ocular condition usually associated with high intraocular pressure. However, glaucoma in which no elevated intraocular pressure is observed when tested is often referred to as low tension glaucoma. In glaucoma, visual loss is associated with hypofunction and death of nerve fibers within the optic nerve that transmit shock to the brain, which ultimately interprets vision. There is a decline in the function of some nerve fibers, and changes in the microcirculation of the optic disc that contribute to the death of some nerve fibers and affect vision. The optic disc is easily visualized with normal eyes by using equipment such as ophthalmoscopes or low eye cameras of various types. Little is currently known about the effects of treatments on the optic disc microcirculation, surrounding structures, and microcirculation in this area, and other parts of the intraocular structure.

Current techniques image the microcirculation of various intraocular structures only after injecting various dyes into the patient's circulatory system. Then take a picture. If a procedure such as laser ablation of a leaking retinal canal or capillary tube is required, this can be done by comparing a photograph usually placed on the screen next to the laser with what is visible to the treating physician. carry out. Clearly, new methods of imaging and characterizing the microcirculation of the eye and other tissues and organs of other human bodies are needed.

Orthogonal Polarization Spectra (OPS) Imaging OPS imaging is a novel method for imaging and characterizing microcirculation using reflected light, which is non-invasive to the microcirculation of mucosal and solid organ surfaces. Enables dynamic imaging.

OPS imaging is entirely incorporated herein by reference in its entirety.
US Pat. No. 5,983,120 (issued Nov. 9, 1999), US Pat. No. 6,104,939 (issued Aug. 15, 2000), and PCT application WO 97/15229 by Ner and Nadeau. It is described in. Patent No. 5
, 983,120 and 6,104,939 and WO 97/15229, both Groner and Nadeau use high resolution images of microcirculation to perform a complete blood cell count including white blood cell differential (CBC + Diff) ( The main elements of CBC) were directly measured and calculated. CBC + Diff is one of the most frequently requested diagnostic tests and is performed 2 billion times a year in the United States. A combination of OPS imaging, image processing, reflectance spectroscopy and algorithmic calculations was used to determine hemoglobin concentration, hematocrit, red blood cell and white blood cell counts and platelet counts without taking blood samples from the body.

In addition, OPS imaging techniques were used to non-invasively measure other types of blood constituents such as non-cellular constituents present in the plasma constituents of blood (eg blood gas and bilirubin). Clearly, the rapid and non-invasive quantification of various blood and vascular properties allows for the collection of venous blood samples that can pose particular problems for neonates, elderly patients, burn patients, and special care unit patients. Thus eliminating the need to confirm blood characteristics. This type of device also eliminates the delay of waiting for the test results of the evaluation on the patient. The device also has the advantage of providing comfort to the patient while eliminating the risk of exposure to AIDS, viral hepatitis, and other bloodborne illnesses. Non-invasive blood tests will have substantial utility in current medical practice.

An enhanced OPS imaging system for high-contrast in vivo imaging of the vascular system is described in co-pending US patent application Ser. No. 09 / 401,859 (filed Sep. 12, 1999) and is fully Incorporated herein by reference.

Therefore, OPS imaging imparts high image quality to a completely non-invasive in-vivo analysis device for the vascular system. This device uses blood cell components (red blood cells, white blood cells and platelets),
High-resolution imaging and characterization of blood flow, vessels through which blood travels, and vascular analysis through the vasculature. In addition, this device minimizes radiance and other undesirable artifacts that occur in conventional reflectance spectroscopy systems.

While the above document provides a thorough description of OPS imaging, a brief discussion of this technique is provided.

In OPS imaging, tissue is illuminated with linearly polarized light and the tissue is imaged through a polarizer orthogonal to the plane of the illumination light. Only depolarized photons scattered in the tissue contribute to the image. The optical response of OPS imaging is linear and can be used to perform reflectance spectrophotometry over a wide range of optical densities typically achieved by transmission spectrophotometry.

In OPS imaging, while linearly polarized light in one plane is irradiated onto a target medium, its reflection is performed through a second polarizer (analyzer) oriented in a plane strictly orthogonal to this irradiation surface. Image the light. To form the image, light is collected, spectrally filtered to isolate wavelength regions and linearly polarized. The polarized light is then reflected by the beam splitter in the target direction. The objective lens collects light in a region having a diameter of about 1 mm. The length of the objective lens can be varied, and two typical OPS imaging probes have 3 inch and 8 inch objective lenses. The light reflected from the target is collected by the same objective lens,
It then forms an image of the illuminated area in the target with an imaging detector such as a charge coupled device (CCD) video camera or a complementary metal oxide semiconductor (CMOS) video camera. The polarization analyzer is located immediately in front of the camera. It is possible to choose a polarizing beamsplitter to maximize efficiency.
That is, one orthogonal polarization state is reflected while the other orthogonal polarization state is transmitted.

In polarized light, the state of polarization is maintained for normal reflection as well as single scattering events. Typically, 10 or more scattering events are needed to efficiently depolarize light (
MacKintosh, F.M. C. Phys. Rev. B 40: 9342 (
1989); Schmitt, J .; M. Others, Applied Optics (
Applied optics) 31: 6335 (1992)). Therefore, only the reflected light from the object that can pass through the analyzer is generated from the composite scattering (more than 10 times the length of a single scattering) that occurs at a relatively deep inside the medium. This depolarized scattered light back illuminates any absorbing material in the foreground. If wavelengths within the hemoglobin absorption spectrum are selected, OPS imaging can be used for fluoroscopic endoscopy to image blood vessels in the peripheral microcirculation. A wavelength region centered on the isosbestic point (548 nm) of oxy and deoxyhemoglobin was selected for optimal microcirculation imaging. In this wavelength region, the isosbestic point (about 420 nm) of the Sole region where the absorption of hemoglobin is maximized but the scattering length is short is used, or composite scattering occurs in the deep tissue, but the absorption of hemoglobin is smaller in the tube. The isosbestic point (810) in the near infrared region, which is insufficient to give good contrast,
nm) is a compromise.

It is noted that scattered light can be used to observe superficial cell structures, regardless of the absorption properties that allow the imaging and characterization of blood cells.

Conventional reflectance spectroscopy is performed on an analyte-enhanced diffuse diffuse reflectance surface. Typically, reflectance spectroscopy has a much more limited range of optical density (OD) measurement than transmission spectroscopy, which can easily measure changes from 0 to 3 OD. In reflected light spectroscopy, specular reflection and light scattering reduce the apparent OD of the analyte. Reflection and light scattering are due to physical properties rather than chemical concentration of the analyte (Ko
rtum, G.R. Reflectance Spectroscopy, Springer-Velag, New York (1969)).

OPS imaging was developed to remove some of the confounding errors inherent in reflectance spectroscopy due in part to reflection and light scattering. Studies have shown that OPS imaging techniques can be used to measure a wide range of OD in reflection, typically only achieved with standard transmission spectroscopy.

The theoretical description of OPS imaging begins with recalling that both the scattering for illumination and the absorption for contrast are required to image reflected light. In a continuous medium, these are typically a pair of material constants (coefficients)
(Star, WM et al., Phys Med Biol 3)
3: 437-454 (1988)). Both scattering and absorption are linearly dependent on penetration, and reflected light intensity is given by the ratio of their coefficients (Kortum,
G. Reflectance Spectroscopy, Sp
ringer-Velag, New York (1969)). Therefore, high quality imaging is generally not possible with turbid media, as scattering reduces image resolution. However, in OPS imaging, reflected illumination is provided only by complex scattering. This is clearly a non-linear function of penetration depth and thus decouples from absorption. As a result, it is possible to image and characterize the absorbing material at shallow depths with high contrast and resolution.

(Comparison between OPS Imaging and Intravital Microscope) In a hamster, a fluorescent in vivo microscope (Harris, AG et al., Int)
． J. Microcirc. -Clin. Exp 17: 322-327 (19
1997)) and OPS imaging showed comparable physiological parameters measured after ischemic injury under controlled conditions (Harri).
S.A. G. Others, The study of the microcircul
ation using orthogonal polarization
spectral imaging (study of microcirculation using orthogonal polarization spectrum imaging), Yearbook of Intensive Care
and Energy Medicine 2000 (Intensive Care and Emergency Medical Yearbook), Ed. J. -L. Vincent, Springer-Verl
Ag, Berlin, pp 705-14 (2000)), Harris, A .; G.
Others, Validation of OPS imaging (validation of OPS imaging), 21st European Conference on
Microcirculation (European Conference on 21st Century Microcirculation), Ed. B
． Fagrell, Bologna, Monduzzi Editor, pp
． 43-48 (2000).

The ability of OPS imaging to quantify relevant pathological parameters and pathophysiological changes in the microcirculation is demonstrated by functional capillary density (FCD) in the dorsal cutaneous chamber of the Syrian golden hamster during wakefulness Was done. The FCD is defined as the length of the red blood cell perfusion capillary for each observation area and is given in cm / cm ² . Since capillaries contain flowing red blood cells that are counted in that measurement, FCD is a direct measure of vegetative tissue perfusion and an indirect measure of oxygen transfer to tissues (Harris, AG; Others, Am. J. Physiol. 271.
: H2388-2398 (1996)). The hamster dorsal skin fold model has been standardized and has been widely used to investigate ischemic / reperfusion injury (Id .; No).
lte, D.I. Int. J. Microcirc. -Clin. Exp. 15
: 244-249 (1995)).

Standard in-vivo fluorescence video microscopy (IVM) (Harris, AG et al., Int. J. Microci) 0.5 and 2 hours after 4 hours of compression ischemia.
rc. -Clin. Exp 17: 322-327 (1997)) and baseline OPS imaging was used to measure FCD for the same capillary network (N.
olte, D.I. Int J. et al. Microcirc-Clin Exp 15
: 9-16 (1995)). A total of 6 capillaries per animal were imaged and videotaped by both methods (n = 10). Computer-aided microcirculation analysis system (CapImage ™) (Klyscz, T. et al., Bi
Images from both systems were analyzed through iterative reproduction using an OmedTech (Berl) 42: 168-175 (1997)) and FCD was measured using the images from both methods. FCD was measured equally using the two imaging methods. In addition, a Brand-Altman analysis (Bl-Altman)
and J. M. And Altman, D .; G. , Lancet 1: 307-.
310 (1986)) showed that the methods are in very good agreement and that there is no systematic bias throughout the FCD measurement.

Direct comparison of contrast between OPS imaging and fluorescence method for hamster image pairs of dorsal cutaneous microcirculation was performed. There was no significant difference in absolute contrast between the two methods (p = 0.43 vs. t-test). Therefore,
The contrast obtained by OPS imaging without dye is
It was equivalent to that obtained by contrast enhancement by.

In the following tests, standard IVM was used to evaluate CYTOSCAN ™ A / R (a high-contrast OPS imaging device owned by Cytometrics) in physiological and pathophysiological conditions. Was carried out.

Method: In the dorsal skin fold model of the Syrian golden hamster during awakening,
Tube diameter, RBC velocity and functional capillary density (FCD) were measured before and after 4 hours of ischemia. For indoor-growing tumors, measurements were taken 3, 6 and 9 days after implantation of melanin-deficient melanoma cells. In rat liver, blood vessel diameter, RBC velocity and number of perfused sinusoids were measured before and after 60 minutes of ischemia. Analyzing the data using the Brand-Altman graph, two methods (IVM vs. CY
The conformity of TOSCAN ™ A / R) was investigated.

Results: In all three methods, the two methods were in very good agreement for measuring vascular RBC velocity. There is no bias, the distribution form is constant over the entire range, and the 95% confidence interval that is the allowable range is secured.
5% was within this range. Measurement of perfusion (indoor FCD
Good functional agreement between the two methods was also observed for the functional tube density in the tumor, and the number of perfused sinusoids in the liver). In the hamster chambers (muscle and tumor) there was good agreement for tube diameter measurements, but a 4 micron bias for IFM. The two methods measure two different diameters (endothelial cell to endothelial cell (IFM) vs. RBC column (OPS imaging)
), And this is actually to be expected, as the tubes appear larger due to fluorescence dispersion. In the liver, there was good agreement between the two methods for measuring tube diameter. Similar results were obtained in the pancreas, intestine, brain and skin flap, and wound healing.

Conclusion: CYTOSCAN ™ A / R can be used to make precise microvascular measurements of vessel diameter, RBC velocity and perfusion in various organs of animal models. Therefore, the same measurement can be performed on the human body.

(Quantification of Optical Density In Vitro) Quantitative data on local hematocrit can be obtained by a fluoroscopic method using blood hemoglobin, but can also be obtained by using an epi-illumination method without systematic labeling of red blood cells. It is not possible. Thin tissue section (such as rat diaphragm or testis muscle)
Have already been tested for the determination of local hematocrit using an in-vivo microscope (Pittman, RN and Duling, BR, J.
． Appl. Physiol. 38: 321-327 (1975)). The optical systems used in these tests were evaluated and calibrated using glass capillaries filled with hemoglobin solution and / or whole blood (Pitt).
man, R.M. N. And Duling, B .; R. J. Appl. Physio
l. 38: 315-320 (1975); Lipowsky, H .; H. Other, M
icrovasc Res 24: 42-55 (1982)).

The OPS imaging system has been shown to produce images of blood-filled capillaries similar to those obtained by fluoroscopy (Groner, W. et al. Nat.
Med. 5: 1209-1213 (1999)). For a series of measurements performed on glass capillaries filled with a solution of diluted whole blood or hemoglobin,
The optical densities obtained by fluoroscopy and OPS imaging were compared. The light receiving element was the same. Basically, the optical densities were the same (r ² = 0.984). Therefore, OPS imaging can be useful in the quantification of local hematocrit using the optical density approach in epi-illumination.

OPS imaging utilizes the absorption of hemoglobin to produce contrast. Therefore, the tube must contain the RBC to be imaged. For measurements, the tube diameter must exceed the minimum resolution of the camera and optics.
For these particular images, the camera magnification was 1 micrometer / pixel. Therefore, the resolution of the system is a single RBC, and a diameter of about 5
Sufficient to break individual micron capillaries. Finally, there must be sufficient contrast between the RBC and surrounding tissue.

OPS imaging technology is described in US Pat. Nos. 5,983,120 and 6,104.
, 939 (Croner and Nadeau), and an enhanced OPS imaging system for high-contrast in-vivo imaging of the vascular system is described in US patent copending application Serial No. 09 / 401,859 (filed Sep. 22, 1999). However, the medical and clinical applications of this breakthrough technology, as well as the research applications, have not yet been fully realized.

SUMMARY OF THE INVENTION The present invention relates to OPS imaging techniques for imaging and characterizing microcirculation. These new medical applications may lead to the development of new clinical trials for human and / or animal microvascular pathology.

Accordingly, the present invention is a method for detecting circulatory disorders, comprising the steps of (a) using an orthogonal polarization spectrum (“OPS”) imaging probe to prevent circulatory disorders in individuals with circulatory disorders, or in hepatic disorders. In obtaining a single captured image or sequence of images of a microcirculation of an individual suspected of having (i) illuminating tissue in the microcirculation of this individual with light polarized in a first plane of polarization. And (ii) capturing at least one image or sequence of images reflected from the tissue, the reflected image passing through an analyzer having a plane of polarization substantially orthogonal to the first plane of polarization. And obtaining a captured image by generating a raw reflection image, and (b) analyzing the captured image to determine the characteristics of the microcirculation. By differentiating, a method is provided that includes identifying this circulatory disorder.

Further, the present invention is a method of monitoring an individual's microcirculation before, during or after medical treatment, comprising: (a) using an OPS imaging probe before, during or after medical treatment. And (i) irradiating the tissue in the microcirculatory system of the individual with light polarized in a first plane of polarization, in order to obtain a single captured image or sequence of images of the microcirculation of the individual; ii) capturing at least one image or sequence of images reflected from the tissue, the reflected image being passed through an analyzer having a plane of polarization substantially orthogonal to the first plane of polarization. Obtaining a captured image by generating a reflected image of the individual, and (b) analyzing the captured image to determine the individual before, during or after medical treatment of the individual. Methods are provided that include monitoring microcirculation.

Optical probes of small size impact the microcirculation, such as (bleeding, septic) shock, hypertension, altitude sickness, diabetes, sickle cell anemia, and many other red or white blood cell abnormalities. It is easy to use as a non-invasive diagnostic tool in the experimental and clinical setting to assess and monitor the status of microvascular sequelae known to give. In addition to non-invasive applications, surgery (transplantation, heart, blood vessels, orthopedics, nerves, plastic surgery, etc.), wound healing, tumor diagnosis and treatment, and imaging and quantitative analysis of microcirculation during intensive care are also applied by OPS imaging technology. Is. Capillary density, tubing (and microtubules) in many previously inaccessible areas, due to the ability to obtain high-contrast images of human microcirculation using reflected light
Morphology, tube density, vasospasm, red blood cell (RBC) velocity, cell morphology, tube diameter, leukocyte-endothelial cell interaction, vasodynamics such as vasomotor, functional tube density, functional capillary density,
Parameters such as blood flow, area marginal ratio, hemoglobin concentration, hematocrit can be quantified. Preferably, OPS imaging is used to measure more than one parameter. These measurements provide a tool in developing high precision tools to assess perfusion during clinical treatment of those diseases that impact tissue viability and microvascular function. This approach allows physicians to follow the progress and development of microvascular disorders as well as directly monitor the effects of treatments on the microcirculation.

The microcirculation OPS image can be a single acquired OPS image, or a sequence of images, depending on the parameter being measured.

Parameters that can be measured by a single acquisition image include tube diameter (tubes can include arteries, capillaries and veins), tube morphology, cell morphology, capillary density, tube density, area marginal ratio. And vasospasm.

Since it is dynamic, the parameters that can be measured only by the sequence of images include red blood cell velocity, functional capillary density, functional tube density, blood flow, leukocyte-endothelial cell interaction (rotating leukocyte and adhesion (adhesion (adhesion)). Sex) including white blood cells), and vasodynamics such as vasomotor.

Accordingly, the present invention is directed to US Pat. Nos. 5,983,120 and 6,104.
OPS imaging probe apparatus (also referred to herein as a "standard" probe), as described in US Patent Application No. 09 / 401,8.
No. 59 (filed Sep. 22, 1999) is directed to new medical applications of the OPS imaging probe device (also referred to herein as a "high contrast" probe). The medical applications described herein are believed to be applicable to both standard OPS and high contrast imaging systems. However, in many cases high contrast images are preferred.

The OPS imaging techniques described herein are useful for imaging subjects and animals and for in vitro applications in research and education.

The equipment used includes a light source, an illumination system and an imaging system.
The light source provides an illumination beam that propagates along an illumination path between the light source and the surface on which the object is located (the object surface). The illumination system converts the illumination beam into a high-contrast illumination pattern and projects the illumination pattern onto a subsurface object. The illumination pattern has a high intensity part and a low intensity part. The imaging system uses blood or tissue under the patient's skin, as well as internal organs (heart, brain, etc.) exposed during surgery.
An image capture device for detecting an image of a subsurface object, such as blood or tissue of the colon).

For inaccessible parts or solid organs in humans and animals,
In vivo imaging with a transmission microscope is not possible. The OPS imaging method employs a small size optical probe and produces a clear image of the microcirculation by reflection from the surface of solid organs and from sites such as the human lower tongue during awakening. Therefore, OPS imaging is capable of clarifying the difference between normal and pathological microdefect structures and functioning non-invasively. The diagnosis and progression of the disease, as well as the effectiveness of the treatment, are monitored for diseases in which changes in microvascular function are recorded.

In one embodiment of the present invention, a standard or high contrast OPS imaging probe is used for in vivo diagnosis and diagnosis of cancer, or as an adjunct to cancer surgery.

In one aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to diagnose epithelial or epithelial cell tumors, or precancerous conditions. Examples of epithelial or epithelial cell tumors that can be diagnosed by OPS imaging include the cervix,
Includes skin, esophagus, bronchial, intestinal or conjunctival tumors.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to assess tumor boundaries or tumor margins before, during, or after cancer treatment.

In another aspect of this embodiment, standard or high contrast OPS imaging probes are used to diagnose various types of tumors based on their vasculature.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to effect the effects of cancer radiotherapy, such as peripheral vasodilation, or other microvascular or vascular effects on irradiated tissue. Monitor.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used for in vivo wound healing and wound healing management. In this embodiment, the OPS imaging probe will be used to image, characterize, evaluate and manage various types of wounds, such as venous ulcers, pressure ulcers, traumas, non-healing surgical wounds and burns. . Since venous ulcers result from circulatory disorders of the lower part, knowledge of the microcirculation is essential for effective treatment.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to image and characterize microvessels inside or around a wound.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to image, characterize and quantify perfused microvessel density in venous stasis or diabetic ulcers.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to observe the necrotic tissue and determine the debridement method required for the wound.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to assess the margins of the wound to determine the probability of healing.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to assess the viability of injured tissue to effectively support the skin graft.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to more accurately and objectively determine the cut line. In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to measure and compare wound revascularization and healing with different wound treatments.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to monitor capillary bifurcation through wound healing.

In another embodiment of the invention, a standard or high contrast OPS imaging probe is used in vivo during orthopedic surgery.

In one aspect of this embodiment, standard or high contrast OPS imaging probes are used to monitor blood flow (perfusion) during and after formation, reconstruction, reattachment or microsurgery.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to determine healthy and necrotic or dead tissue surrounding the flap.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used post-operatively to continuously monitor the microcirculation and identify any potential problems with reperfusion. Early indication of reperfusion problems helps avoid the need for reoperation.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used in vivo in the field of heart and cardiac surgery.

In one aspect of this embodiment, a standard or high contrast OPS imaging probe is used to enhance imaging and characterization of the cardiac microcirculation and
Confirm reperfusion during a minimally invasive surgical procedure that avoids cardiopulmonary bypass devices such as “keyhole” surgery (ie, heart port) or thoracotomy.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to monitor changes in a patient's blood flow (microcirculation) during open heart surgery while the patient remains connected to a cardiopulmonary device. And detect. Therefore, it is possible to use the OPS imaging probe to monitor changes in the condition of a patient connected to a cardiopulmonary device.

In another aspect of this embodiment, during coronary artery bypass graft (CABG) surgery, O
The PS imaging probe can be used to determine if the implant has good blood flow and is providing good blood flow to downstream blood vessels. That is, in cardiac surgery, it is possible to monitor blood flow to the capillaries after bypass surgery using an OPS imaging probe that is in direct contact with the heart.

In another aspect of this embodiment, an OPS imaging probe is used to monitor heart risk. Imaging and characterizing the microcirculation using OPS imaging probes helps non-invasively determine high-risk cardiac profiles. It will be possible to investigate the microvascular sequelae of hypertension and use it for the determination of heart risk.

In another aspect of this embodiment, an OPS imaging probe is used to image lung tissue.
Used for characterization and evaluation.

In another embodiment of the invention, a standard or high contrast OPS imaging probe is used in vivo prior to or during neurosurgery.

In one aspect of this embodiment, a standard or high contrast OPS imaging probe is applied directly to the brain to image and characterize the microcirculation during diagnostic or therapeutic neurosurgery. Together, parameters such as diameter, flow rate and functional capillary density are measured.

In another embodiment of the invention, a standard or high contrast OPS imaging probe is used to detect vasospasm following neural deficiency or subarachnoid hemorrhage.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used to detect tumor boundaries in the brain.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used to determine and classify brain tumors based on the vasculature of different brain tumors and differences in microcirculation.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used to image and characterize vascular changes due to neural tumors.
OPS imaging probes can also be used to determine the extent of neural tumors.

In another embodiment of the invention, a standard or high contrast OPS imaging probe is used in vivo during organ transplantation.

In one aspect of this embodiment, a standard or high contrast OPS is used during organ transplant surgery.
An imaging probe is used to measure perfusion after connecting the transplanted tissue / organ.

In another embodiment of the invention, a standard or high-contrast OPS imaging probe is used in vivo during vascular graft surgery, such as peripheral arterial occlusion (PAOD).

In one aspect of this embodiment, a standard or high contrast OPS is used during vessel transplant surgery.
An imaging probe is used to measure perfusion rate after connecting the implant.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used in vivo during orthopedic surgery and in the field of orthopedic treatment.

In one aspect of this embodiment, a standard or high contrast OPS is used during orthopedic surgery.
Imaging probes are used to identify and observe necrotic tissue for surgical resection. The probe will be located at the site of the tumor.

[0087]   The probe can also be used to image bones, tendons and ligaments.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to image and characterize the microcirculation around the periosteum (bone). Differences in microcirculation were observed when images were taken before and after the fracture.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used in vivo in the field of gastroenterology as well as in gastrointestinal (GI) or gastroesophageal surgery.

In one aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to image and characterize the large intestine and also to treat inflammatory bowel disease, ulcerative colitis, localized ileitis, or Diagnose and treat other gastrointestinal disorders that affect the microcirculation. It is possible to insert the probe into the rectum and make direct contact with the wall of the large intestine.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe can be used during gastrointestinal (GI) surgery to perform colon imaging and characterization during intestinal resection. Other GI organs can also be visualized.

In another aspect of this embodiment, standard or high-contrast OPS imaging probes can be used to determine the boundaries of cancerous GI tumors as well as to visualize and characterize necrotic tissue. Thus, during surgery, the affected tissue can be more easily resected from the stomach and / or esophagus.

In another aspect of this embodiment, the OPS imaging probe can also be used to image and characterize the rectal mucosal microcirculation, eg, in patients with inflammatory bowel disease.

In another embodiment of the invention, standard or high contrast OPS imaging probes are used in vivo in the field of ophthalmology.

In one aspect of this embodiment, standard or high contrast OPS imaging probes are used to image and characterize the microcirculation of the eye. The visualization tool can be used to provide information regarding diagnostic purposes and treatments, as well as the effects of various types of treatments on the circulatory system of the study area.

In another aspect of this embodiment, OPS imaging techniques can be used to diagnose macular degeneration, retinopathy (retinopathy) and glaucoma.

In another aspect of this embodiment, the OPS disk technique is used to
Imaging and characterization of changes in the retina, sclera, conjunctiva, and vitreous humor can be performed.

In another aspect of this embodiment, OPS imaging techniques are used to early diagnose and treat diabetes by observing changes or differences in the microcirculation of the eye, particularly the sclera and / or aqueous humor of the eye. It can be performed.

In another embodiment of the invention, OPS imaging probes are used to monitor female microvascular function in normal or complex pregnancies.

In one aspect of this embodiment, standard or high-contrast OPS imaging probes are used to detect or monitor complex pregnancies in women with preeclampsia (PE).

In another embodiment of the invention, OPS imaging probes can be used for neonatal monitoring.

In one aspect of this embodiment, standard or high-contrast OPS imaging probes are used to quantitatively measure changes in the neonatal microcirculation in various disease states such as sepsis and meningopathy, thus Diagnose those symptoms. Neonatal hemoglobin levels can be monitored non-invasively.

In other embodiments of the present invention, OPS imaging can be used to perform high altitude studies or space physics studies.

In one aspect of this embodiment, standard or high contrast OPS imaging probes are used to observe and evaluate microcirculatory changes at high altitudes to investigate, diagnose and / or treat altitude sickness.

In another embodiment of the invention, the OPS imaging probe can be used in vivo for critical care or intensive care.

In one aspect of this embodiment, a standard or high contrast OPS imaging probe is used as a sublingual measurement or monitoring device for critically ill patients to diagnose, treat or prevent sepsis and shock (hemorrhagic or septic). .

In another embodiment of the invention, the OPS imaging probe is used in the field of drug development.

In one aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to determine if circulation to the tumor is blocked for anti-angiogenic agents or for angiogenic agents. Various drugs, such as determining whether blood vessels extend to organs and improving circulation, or the mechanism of action of new treatments against antihypertensive drugs or the cause of microvascular hypertension. To investigate the effect of the on the microcirculation.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to study hemoglobin-based oxygen gallium (ie, DCL Hb, synthetic hemoglobin), for example using product to result in RBC flux. Determine their effects on microcirculation, such as whether or not

In another aspect of this embodiment, the OPS imaging probe can be used to study the effect of ultrasound circular anxiety (ie, injectable dye) on microcirculation.

In another aspect of this embodiment, an OPS imaging probe can be used to image and detect the leakage of injectable dyes or other injectable contrast-producing agents from blood vessels into tissues.

In another embodiment of the invention, a standard or high contrast OPS imaging probe is used, as was previously done with a standard capillary microscope.
Visualize and characterize the capillary bed in the nail fold.

In one aspect of this embodiment, standard or high contrast OPS imaging probes are used to investigate, diagnose and assess patients with circulatory disorders such as Raynaud's phenomenon, osteoarthritis or systemic sclerosis.

In another embodiment of the invention, OPS imaging probes are used in the field of anesthesiology.

In one aspect of this embodiment, a standard or high contrast OPS imaging probe is used to monitor hypovolemia during surgery. For example, OPS imaging can be used to non-invasively and continuously monitor hemodynamic parameters of anesthetized patients, such as hemoglobin concentration and hematocrit.

In another embodiment of the invention, a standard or high-contrast OPS imaging probe is used in anesthetized patients to monitor red blood cell aggregation and early embolization during surgery.

In another embodiment of the invention, a standard or high contrast OPS imaging probe is used in anesthetized patients to treat patients who may develop as secondary complications of infection, childbirth, malignancy and other serious illnesses. Visualize, characterize, identify, and / or monitor disseminated intravascular coagulation (DIC).

In one aspect of this embodiment, standard or high-contrast OPS imaging probes are used to image, characterize, identify, and / or monitor infection, and more specifically DIC due to meningitis. .

In another embodiment of the invention, standard or high-contrast OPS imaging probes are used to image, characterize, and monitor changes in leukocyte-endothelial cell interactions such as occur during inflammation or infection. To do.

In another embodiment of the invention, any or all of the above fields (heart disease, heart surgery, wound healing, diabetes, hypertension, ophthalmology, neurosurgery, plastic surgery, transplantation, anesthesia and pharmacology). OPS for in vitro or in vivo basic or clinical studies in
Use an imaging probe.

In another embodiment of the invention, an educational tool for medical students and / or science students learning, for example, physiology, anatomy, pharmacology, microcirculation, and disease states affecting the microcirculation. As the above, an OPS imaging probe is used.

It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory, and are intended to more particularly describe the invention as claimed. Should.

Detailed Description of Embodiments The present invention relates to OPS imaging techniques for imaging and characterizing microcirculation. This medical application may lead to the development of new clinical trials for human and / or animal microvascular pathology.

Accordingly, the present invention provides a method for detecting circulatory disorders, comprising the steps of (a) using an orthogonal polarization spectrum (“OPS”) imaging probe to prevent circulatory disorders in individuals with circulatory disorders, or in hepatic disorders. In obtaining a single captured image or sequence of images of a microcirculation of an individual suspected of having (i) illuminating tissue in the microcirculation of this individual with light polarized in a first plane of polarization. And (ii) capturing at least one image or sequence of images reflected from the tissue, the reflected image passing through an analyzer having a plane of polarization substantially orthogonal to the first plane of polarization. And obtaining a captured image by generating a raw reflection image, and (b) analyzing the captured image to determine the characteristics of the microcirculation. By differentiating, a method is provided that includes identifying this circulatory disorder.

Further, the present invention provides a method of monitoring an individual's microcirculation before, during or after medical treatment, comprising: (a) using an OPS imaging probe before, during or after medical treatment. And (i) irradiating the tissue in the microcirculatory system of the individual with light polarized in a first plane of polarization, in order to obtain a single captured image or sequence of images of the microcirculation of the individual; ii) capturing at least one image or sequence of images reflected from the tissue, the reflected image being passed through an analyzer having a plane of polarization substantially orthogonal to the first plane of polarization. Obtaining a captured image by generating a reflected image of the individual, and (b) analyzing the captured image to determine the individual before, during or after medical treatment of the individual. Methods are provided that include monitoring microcirculation. The term “tissue” as used herein encompasses blood.

Small size optical probes impact the microcirculation, such as (bleeding, septic) shock, hypertension, altitude sickness, diabetes, sickle cell anemia, and many other red or white blood cell abnormalities. It is easy to use as a non-invasive diagnostic tool in the experimental and clinical setting to assess and monitor the status of microvascular sequelae known to give. In addition to non-invasive applications, surgery (transplantation, heart, blood vessels, orthopedics, nerves, plastic surgery, etc.), wound healing, tumor diagnosis and treatment, and imaging and quantitative analysis of microcirculation during intensive care are also applied by OPS imaging technology. Is.

The ability to obtain high-contrast images of human microcirculation using reflected light allows for capillary density, tube (and microtubule) morphology, tube density, vasospasm, in many previously inaccessible sites. Erythrocyte (RBC) velocity, cell morphology, tube diameter, leukocyte-endothelial cell interaction, vasodynamics (such as vasomotor), functional tube density, functional capillary density, blood flow, peripheral area ratio, hemoglobin concentration, hematocrit Quantitation of one or more such parameters is possible. Preferably, OPS imaging is used to measure more than one parameter. These measurements provide a means to develop high precision tools to assess perfusion during clinical treatment of those diseases that impact tissue viability and microvascular function. Using this technique, the doctor
It will allow the progress and development of microangiopathy to be followed and the effect of treatment on the microcirculation directly monitored.

The term “capillary density” as used herein means the ratio of the length of the capillary to the total observed area and is expressed in cm / cm ² .

As used herein, the term “tubular (and / or microtubule) morphology” means the physical or structural characteristics of a tube (or microtubule). This term refers to an individual tube or network of tubes.

As used herein, the term “tube density” means the area occupied by vasculature. Tube density is expressed as the ratio of the area occupied by the tube structure to the total observed area. Numbers are expressed as percentages. Tube density can also be referred to as the "vascular analysis index."

The term “vasospasm” as used herein refers to trauma and / or long-term abnormal stenosis of the duct due to the presence of extravascular blood in the area adjacent to the duct. For example, vasospasm is well known due to cerebral subarachnoid hemorrhage.

As used herein, “red blood cell (RBC) velocity” means the observed velocity of red blood cells within a blood vessel.

The term “cell morphology” as used herein refers to the cell shape characteristic of white blood cells, red blood cells and / or epithelial cells.

The term “tube diameter” as used herein means the distance between observable vessel walls.

As used herein, the term “leukocyte-endothelial cell interaction” means any interaction between leukocytes and the inner surface of blood vessels (endothelial cell layer). These interactions may include rolling along the wall of the blood vessel at less than the RBC velocity, staying in place for a period of time, or moving through the vessel wall to the site of inflammation. .

The term “vasodynamics” as used herein refers to temporary (time-dependent) changes in vessel diameter. This can be a spontaneous change (ie vasomotor) or a change in response to a neural, hormonal or pharmacological stimulus. Furthermore, the normal response of the duct to a given stimulus may be influenced by other factors. These other factors will be described as affecting vasodynamics.

As used herein, the term “functional tube density” means the ratio of perfusion tube length to total observed area. A perfusion tube is a tube through which RBC can be observed to flow.

As used herein, the term “functional capillary density” means the ratio of perfused capillary length (number) to total observed area. A perfusion capillary is a capillary through which RBC can be observed to flow. It is expressed in cm / cm ² (or number / cm ² ).

The term “blood flow” as used herein means the observed movement of red blood cells that flow through a vessel.

The term "regional marginal ratio" as used herein means a measure of red blood cell aggregation or heterogeneity in the vessel. The ratio is defined as the ratio of the area of erythrocyte aggregates detected in the tube to the perimeter or contour length of the erythrocyte aggregates. Fully perfused tubes with no plasma space have a higher area margin ratio. The plasma space or partially filled tube has a smaller area marginal ratio. For example, this parameter can be used to make an assessment of congestive blood flow or aggregated cells.

As used herein, the term “hemoglobin (or Hb) concentration” means the concentration of iron-containing protein pigment (hemoglobin) present in red blood cells. Hemoglobin, the main component of red blood cells, is a complex protein that acts as a vehicle for transmitting oxygen and carbon dioxide throughout the body. Retain 1.34 ml of oxygen per gram of hemoglobin. The red blood cell mass of an adult contains about 600 g of hemoglobin and can carry 800 ml of oxygen. The primary function of hemoglobin is to transfer oxygen from the lungs, which have high oxygen tension, to tissues with lower oxygen tension.

The term “hematocrit” as used herein means the ratio of the volume of red blood cells of a blood sample to the volume of red blood cells of whole blood. It is expressed as a percentage, or preferably a percentage.

Accordingly, the present invention provides US Pat. Nos. 5,983,120 and 6,104.
OPS imaging probe apparatus (also referred to herein as a "standard" probe), as described in US Patent Application No. 09 / 401,8.
No. 59 (filed Sep. 22, 1999) is directed to new medical applications of the OPS imaging probe device (also referred to herein as a "high contrast" probe). The medical applications described herein are believed to be applicable to both standard OPS and high contrast imaging systems. However, for certain medical applications, high contrast images are preferred.

The OPS imaging techniques described herein are useful for imaging subjects and animals, and for in vitro applications in research and education.

The equipment used includes a light source, an illumination system and an imaging system.
The light source provides an illumination beam that propagates along an illumination path between the light source and the surface on which the object is located (the object surface). The illumination system converts the illumination beam into a high-contrast illumination pattern and projects the illumination pattern onto a subsurface object. The illumination pattern has a high intensity part and a low intensity part. The imaging system includes blood and / or tissue under the patient's skin, blood and / or tissue from the sublingual region, blood and / or tissue from the surface of solid organs (heart, brain, colon, lungs) exposed during surgery.
Or an image capture device that detects images of subsurface objects such as tissue, or epithelial or epithelial intercellular tissue (ie, the neck). In-vivo imaging by transmission microscopy is not possible for inaccessible sites or solid organs in humans and animals. The OPS imaging method employs a small size optical probe and produces a clear image of the microcirculation by reflection from the surface of solid organs and from sites such as the human lower tongue during awakening. Therefore, OPS imaging is capable of clarifying the difference between normal and pathological microdefect structures and functioning non-invasively. The diagnosis and progression of the disease, as well as the effectiveness of the treatment, are monitored for diseases in which changes in microvascular function are recorded.

The image of the microcirculation can be a single acquired OPS image, or a sequence of images, depending on the parameter being measured.

Parameters that can be measured with a single acquisition image include tube diameter (tubes can include arteries, capillaries and veins), tube morphology, cell morphology, capillary density, tube density, area marginal ratio. And vasospasm.

Parameters that can be measured only by the sequence of images because they are dynamic include erythrocyte velocity, functional capillary density, functional tube density, blood flow, leukocyte-endothelial cell interaction (rotating leukocytes and adhesion (adhesion). Sex) including white blood cells), and vasodynamics such as vasomotor.

The invention is illustrated by the numerous examples and experimental data presented below. This information is provided to aid the understanding and practice of the present invention and is not a limitation thereof.

(Application to Cancer) In this embodiment of the present invention, the high-contrast OPS imaging probe is used for diagnosing or predicting cancer in vivo, or as an adjunct to cancer surgery.

In one aspect of this embodiment, a standard or high contrast OPS imaging probe is used to diagnose epithelial or epithelial cell tumors, or precancerous conditions. Examples of epithelial or epithelial cell tumors that can be diagnosed by OPS imaging include the cervix,
Includes skin, esophagus, bronchial, intestinal or conjunctival tumors.

Regarding cervical intraepithelial neoplasia (CIN), the incidence and mortality of invasive cervical cancer have been significantly reduced over the past 50 years, but both the reported and actual incidence of CIN are increasing. . As a result, the mortality rate of cervical cancer may increase by 20% from 2000 to 2004 if the screening technique for CIN is not improved.

Current screening for CIN and cervical cancer is relatively inexpensive but labor intensive, initially with reliance on Papanicolaou smears, and has a false negative error rate of 20.
~ 30% is associated with inadequate sampling and / or poor reading of the Pap smear. On the other hand, in the Papanicolaou smear classification “ASCUS” (representing atypical cells of undetermined significance based on the newer “Bethesda” classification system), false positive results for Papanicolaou smear occured in 70% to 80% of women. Requires a needless, time-consuming and costly follow-up test (colposcope and / or biopsy), which adds extra concern to the patient.

Therefore, there is a need for a more reliable method of classifying cervical tissue into normal or abnormal tissue, in the latter case distinguishing inflammatory or benign HPV infection from CIN.

An image of the excellent microcirculation obtained by the OPS imaging technique was overlaid with a grid-like "mosaic" of epithelial cell shape. They were rhombohedral, pentahedral, hexahedral, and heptahedral, extremely flat and regular. The diameter was approximately 10-40 microns based on RBC and comparison with arterioles and venules in the same field. They clearly overlapped the vasculature. Illuminated cell edge (refraction)
. The cells were extremely "flat", implying that they are superficial epithelium rather than deep epithelium and not the more cuboidal basal layer cells.
Furthermore, it was possible to distinguish the size and shape of the cell nucleus. These features are of particular importance in the assessment of all malignant and malignant conditions.

With the information that the focal “plane” of the constructed system is 150-200 microns from the surface of the optical probe, which corresponds to the depth at which the vasculature is present, the mechanism of light scattering and back lighting or other effects. Theorized that the upper layer epithelium would be described by. The optics of the OPS imaging probe were then optimized so that the epithelium was the main feature of the image. Thus, providing epithelial images of varying depths using the OPS imaging probe allows direct diagnosis of epithelial cell-to-epithelial lesions, including carcinoma in situ or early erosive cancer.

In the field of gynecologic oncology, the recognition and treatment of carcinoma in situ is a challenge. Current practice usually involves a Papanicolaou smear test, which may be associated with papillomavirus detection, followed by biopsy colposcopy, followed by excision surgery. This is time consuming and requires frequent patient visits for pelvic examinations, cytological and wet test (for papillomavirus), and pathological examination of biopsies. It is also extremely expensive.

In this embodiment of the invention, OPS imaging technology or its derivation technology can be used to directly image, characterize, and quantify pre-malignant and malignant epithelial lesions. For example, in the case of carcinoma in situ of the neck, it was possible to expose the epithelial cell layer using an OPS imaging probe immediately after visual gynecological examination. The lesions described above are defined as a regular pattern of disruption by cells with prominent features, including abnormal size, abnormal shape, and cellular characteristics such as abnormal nucleocytoplasmic ratios. They lead to abnormal "architecture" (histopathology term), which previously required the examination of biopsy material by a pathologist. By reconstructing a three-dimensional image of the tissue from the OPS image collected while focusing on the tissue, it was possible to distinguish the abnormal destructive architecture of pre-malignant and malignant epithelial lesions from normal cells.

Using the OPS imaging probe, it was possible to make a direct diagnosis by comparing the normal and “suspect” areas of the unaffected cervix.
It was possible to characterize the difference in the regularity of the mosaic by image analysis of the mosaic. Cell size, shape and other morphological features can be quantified to observe and quantify blood flow and velocity differences between normal and suspicious regions, as well as between different tumor types. This has never been done before, and pathological diagnosis is not statistical, but still impression and subjectivity. Needless to say, direct diagnosis using OPS imaging technology does not preclude conventional biopsy.

The approach to epithelial cell-to-cell diagnostics further calls for optimizing OPS imaging probes. For example, one skilled in the art would like to emphasize mosaic pattern image analysis, different magnification, different and possibly continuously variable focus, different light source dimensions, masking of apertures to the dark field, and possibly epithelial cell boundaries and internal structures. It is necessary to optimize the epithelial illumination (perhaps at different angles), image quantification of normal vs. abnormal areas under examination, etc.

FIG. 1 is a block diagram of an OPS imaging probe 200 with an elongated objective lens 214 (approximately 8 inches) that can be used for screening and / or diagnosis of cervical tumors or precervical cancer lesions. The probe 200 includes a light source 202, a condenser lens 204, a relay lens 208, a detector 260 and an objective lens 21.
Including 7. One or more bandpass filters 206 can be placed in the optical path to enhance the quality of the acquired image. As one of ordinary skill in the art will be familiar with, the type of filter used depends on the object being imaged.

The light source 202 illuminates a tissue region of interest (generally indicated at 224). Although one light source is shown in FIG. 1, it should be understood that the invention is not limited to one light source and more than one light source can be used. In one embodiment using multiple light sources, each light source can be monochromatic or polychromatic. Light source 202 can be pulsed light, a non-pulsed light source that provides continuous light, or a light source capable of either operation. The light source 202 can include, for example, a pulsed xenon arc light or lamp, a mercury arc light or lamp, a halogen light or lamp, a tungsten light or lamp, a laser, a laser diode, or a light emitting diode (LED). The light source 202 can be a coherent light source or an incoherent light source.

A folding mirror or beam splitter 218 is used to source the light source 202 and the target 224.
Form an optical path between. According to one embodiment of the present invention, the coated plate has a reflectance of 50% for the illumination beam 209. Another embodiment of beam splitter 218 is
It is well known to those engaged in related technology.

In a preferred embodiment, a first polarizer 210 is placed between the light source 202 and the object 224. The first polarizer 210 polarizes the light from the light source 202. A second polarizer or analyzer 220 is placed between the object 224 and the image capture means 260 along the image path 207. The polarizers 210 and 220 preferably have their planes of polarization oriented 90 degrees with respect to each other. Polarizers such as polarizers 210 and 220 having their planes of polarization oriented at 90 degrees to each other are referred to herein as "crossed polarizers."

Preferably, the image from the object 224 originates from a depth less than the composite scatter length and travels along the image path 207 to the image capturing means 260. However, the imaging system of the present invention can also capture images formed from depths greater than the composite scatter length. The objective lens 217 is used to magnify the image of the object 224 on the image capturing means 260. The objective lens 217 is arranged coaxially with the illumination path 209 and the image path 207. The image capturing means 260 is arranged on the enlarged image plane of the objective lens 217. One of ordinary skill in the art will appreciate based on the description herein that the objective lens 217 can include one or more optical elements or lenses depending on the space and imaging requirements of the device 200.

Suitable image capture means 260 comprises a device capable of capturing the high resolution images described above. The image capturing means captures all or part of the image according to the purpose of analysis. Suitable image capturing means are cameras, film media, photosensitive detectors, photocells, photodiodes, photodetectors, or CCDs or Cs.
Including but not limited to MOS cameras.

The image capturing means 260 can be attached to the image correcting and analyzing means (not shown) for correcting and analyzing the image. The resolution required for the image capture means can depend on the type of measurement and analysis performed by the in-vivo device.

Preferably, the objective lens 217 can be one or more lenses selected by the lowest magnification required to image the illuminated object. The required magnification is a function of the size of the object in the illuminated tissue being imaged, as well as the size of the pixels used in the image.

According to a preferred embodiment, the illumination path 209 and the image path 207 share a common axis. This coaxial nature allows the objective lens 217 to be used for more than one purpose.
Is taken into consideration. First, the objective lens 217 acts as an objective lens for the image capturing means 260. In other words, it collects the image beam emanating from the object 224 on the image capturing means 260. Secondly, the objective lens 217 is
It serves to collect high-contrast illumination patterns on the target surface. Illumination beam 209
The high-intensity part of is guided outside the field of view (FOV) of the image capturing means 260.

The combination of the optical characteristics of the objective lens 217 and the image capturing means 260 determines the FOV of the device 200. The FOV of the image capturing means will be limited by a number of parameters including the numerical aperture of its objective lens (here objective lens 217), the entrance pupil, the exit pupil, and the area of the detector comprising the image capturing means 260. .

The cervix is a particularly important goal because of the frequency of disease and the regularity of cancer tests, and because of its thick, flat and regular cervical epithelium, other interepithelial lesions and the basal layer And the subepithelial extension of epithelial malignancies through the basement membrane is also a good target for this diagnostic approach. For example, the difference between benign active nevus and malignant melanoma results in a serious diagnostic dilemma that usually requires a biopsy.
The OPS imaging techniques of the present invention allow direct diagnosis and differentiation of the skin lesions, or their pre-cancerous precursors. It is possible to visually record colored lesions on the body of an individual and use archival images and quantitative measurements as a "baseline".

In another aspect of this embodiment, a high-contrast OPS imaging probe is used to assess tumor boundaries or tumor margins before, during, or after cancer treatment. For example, OPS imaging can be used to image and characterize the margins of true skin cancer that is clinically invisible to the naked eye dermatologist. This makes it possible to avoid the need for further surgery.

In another aspect of this embodiment, high contrast OPS imaging probes are used to diagnose various types of tumors based on their vasculature.

In another aspect of this embodiment, a high contrast OPS imaging probe is used to monitor the effects of cancer radiotherapy, such as peripheral vasodilation (swelling of smooth blood vessels). High contrast OPS imaging probes can also be used to investigate other microvessels or vascular effects on irradiated tissue, such as changes in perfused microtubule density with changes in radiation dose.

(Application to Wound Treatment and Wound Healing Management) In this embodiment, the OPS imaging probe uses venous ulcers (due to chronic venous insufficiency and diabetes), pressure ulcers (sometimes referred to as bed sores, It is formed when blood flow is blocked by chronic pressure, blocks oxygen and nutrients and causes tissue ulceration and necrosis), traumatic wounds (wounds received during accidents and assault cases), and Imaging of various types of wounds, including healing surgical wounds (incisions made during a surgical procedure that do not heal within the expected time frame), burn wounds, etc.
Used in characterization, evaluation and management. Since venous ulcers form as a result of underlying circulatory problems, microcirculatory knowledge is essential for effective treatment.

In each of the following aspects of this embodiment, capillary density, blood vessel (and microvessel) morphology, blood vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, blood vessel diameter, leukocyte endothelial cells. Quantitatively determine one or more parameters such as interaction between cells, vasodynamics (eg vasomotor), functional vessel density, functional capillary density, blood flow, intra-area to marginal ratio, hemoglobin concentration, hematocrit value, etc. I have something to do.
The number of parameters to be determined is preferably two or more.

In one aspect of this embodiment, standard or high contrast OPS imaging probes are used to image and characterize microvessels in and around the wound.

In another aspect of this embodiment, the standard or high-contrast OPS imaging probe is for imaging, characterizing, and quantifying perfused microvessel density within venous stasis ulcers and diabetic ulcers. used. Using OPS imaging on chronic wounds in diabetic patients, a relative microvascular depletion within the wound bed and adjacent tissues is observed compared to unrelated skin.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to observe necrotic tissue as well as to determine the extent of debridement required by the wound.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to assess the margins of the wound and determine its potential for future healing.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to assess the viability of wound tissue and to support successful skin grafts.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to more accurately and more objectively determine the cut line.

In another aspect of this embodiment, standard or high contrast OPS imaging probes are used to measure and compare revascularization and healing for wounds with various wound healing therapies.

In another aspect of this embodiment, a standard or high-contrast OPS imaging probe monitors capillary “budding” (ie, creation of new capillaries) during wound healing. Used for.

In the following studies, OPS imaging was used to image and characterize the microcirculation of the skin. The purpose of this study is to provide an OP for making microvascular measurements in the skin under normal and diseased conditions (in this case in the process of wound healing).
Confirm the effectiveness of using S-imaging in contrast to standard fluorescence video microscopy (Groner, W. et al., Nat. Med. 5: 1209-1213 (
1999)). It is clear that assessing the microcirculation of the skin has important clinical implications, as some of the dermatological pathological conditions are associated with changes in the microvasculature. Quantitative analysis of skin microcirculation makes it possible to identify vascular abnormalities, angiogenesis during wound healing, as well as decreased blood flow after skin flap creation.

Materials and Methods: Experiments were performed on the ears of hairless (SKH-1hr) mice (n = 9). Circular wounds are described by Bondar and coworkers (Bondar, I.
． Res. Ex. Med. 191: 379-388 (1991)). OPS imaging device (CYTOSCAN ™ A / R
) Was attached to an in-vivo microscope so that a computer controlled XY plate was available. Observations were performed using both in vivo fluorescence microscopy (IVM) and OPS imaging in the exact same region of interest at 4, 7, 7, 10 and 15 days after baseline and wound generation. In the measurement by IVM, FITC-De
xtran i. v. 0.02 ml of a 3% solution of was used as a plasma marker. The images acquired from both devices are computer-aided analysis system (CapImage (
Trademark)) (Klyscz, T. et al., Biomed. Tech. (Berl) 42.
168-175 (1997)) during video tape playback.
Measurements of arteriole diameter, venule diameter, red blood cell velocity and functional capillary density (FCD) were performed during wound healing as well as at baseline.

Conclusion: OPS imaging yielded high quality images of skin microcirculation with optical contrast comparable to that achieved by IVM. In addition, the use of OPS imaging allowed accurate quantitative measurements on vessel diameter, venule RBC velocity and functional capillary density during the physiological state of wound healing and during the course of wound healing. Due to the miniaturization and portability of the equipment and the quality of the images acquired without the need for fluorescent dyes, OP
It is suggested that S-imaging can offer great potential for use in diagnostic measurements on human skin.

In another study, wound-induced angiogenesis was observed using OPS imaging.

Overview: Wound-related trauma first creates a signal to the tissue to initiate its own repair. This initial trauma initiates inflammation, which gives rise to a series of molecular signals that promote angiogenesis. Conheim is one of the first to describe the early changes in vasculature that occur following wounds in recent history (Conhei
m, J.M. , The pathology of the circulatio
n, Section 1 in Lectures in General P
Athology: A Handbook for Practitioner
s and Students, trans. by A. B. McKee, ed
． ． , New Sydenham Society, London. V. 126
, 129, 133, pp. xvii-528, 1889-1890 (1889)). More recently, Clark and Clark have suggested that angiogenesis is a key step in initiating wound healing (Clar.
k, E. R. And Clark, E .; L. , Am J Anat 64: 251.
-299 (1939)).

The window chamber is one of the widely used experimental models in studying wound healing. Hashimoto and Prewitt placed a chamber in the ear of a rabbit and recorded the changes that occur in this model with respect to the healing wound (Hashimoto, H. et al., Int. J. Microcirc: Cl.
in. Exp. 5: 303-310 (1987)). Dewhirst and others (D
ewirst et al., Rad. Res. 112: 581-591 (1987)).
A number of investigators have placed window chambers in the dorsal skin folds of rats, indicating that this arrangement is an excellent model for testing early angiogenesis. However, such chambers cannot be used clinically on humans. This study utilized a similar principle with wounds for the simulation of angiogenesis.

A reproducible model for assessing angiogenesis would be useful in a variety of clinical situations, such as testing the effectiveness of a particular treatment to prevent angiogenesis in cancer patients. Let's do it. Auerbach and others (Pharmacol Therapy
utics 51: 1-11 (1991)) point out in a recent review that an understanding of angiogenesis is important for improving knowledge of wound healing. Similarly, Folkman proposes that an understanding of angiogenesis allows the development of treatments to suppress normal angiogenesis (
Folkman, Adv. Cancer Res. 19: 351-358 (19
1974)). The purpose of this study was to characterize the development of microvessels around the entire thickness of the wound in humans.

Materials and Methods: Subjects underwent local anesthesia on the volar side of the forearm, followed by a full-thickness, 4 mm diameter skin punch biopsy that extended to the underlying fat or fascia. The wound was dressed with an antibiotic ointment. Wounds were examined every other day using various optical devices such as a surgical microscope with a video camera and CYTOSCAN ™ A / R. On each day of observation, the bandage was removed and the tissue adjacent to the skin wound was recorded for several seconds at each location, moving clockwise around the biopsy margin. Approximately 5 minutes was required to completely go around the wound edge. This video image was stored in either analog or digital form.

In the analysis, a semi-quantitative scale with four grades was set up. Grade 0 is characterized by a complete lack of vessel segmentation and color. Grade 1 is characterized by a very small number of microvessels clearly discernible in the tissue adjacent to the wound, scattered in pink or red. Grade 2 is characterized by the fact that the short vertical microvascular loops found at the ends are unordered, with no preferential orientation or micro-branching. Grade 3 is characterized by longer curved microvessels that are generally curved but begin to extend radially outward from the wound edge.
Some tiny branches were also seen. Grade 4 is composed of long microvessels that are radially aligned to have interconnected loops or anastomotic structures like sun rays. Some limited branching was also observed. After defining the semi-quantitative scale, the images were viewed by a blind observer and the images were scored using these detailed criteria.

Results: The grading of the observed vascular patterns was reproducible based on well-defined criteria. The coefficient of variation was approximately 10% or less. It took only a few days for the vascular grade to rise to an average of 1.5. A score of 2.0 was achieved less than 5 days following wounding. The average grade change from 2.0 to 2.5 was more gradual, requiring approximately 10 days post wounding. Then the newly formed blood vessels appear,
It rapidly expanded and began to align radially, which achieved a grade of 3.0 in less than a full day. A score of 1.0 was achieved shortly after the first wound.

Microvasculature was also observed in patients with diabetes and venous ulcers. Video microscopy was performed on each patient using the CYTOSCAN ™ A / R for a few weeks of conservative non-surgical treatment prior to treatment to observe vascular grade patterns. In the most difficult wounds, a general lack of vascularity was seen adjacent to the wound (grade 1). Especially in diabetic patients, the number of microvessels found on the skin surface immediately adjacent to the wound was extremely low. The healing wound had a slightly increased number of microvessels (grade 2), although it did not show a clear orientation. In parallel with the clinical improvement of the wound, microvascular prolongation and the appearance of preferential microvascular orientation (grades 3 and 4) were seen.

Discussion: A semi-quantitative assessment of wound-induced angiogenesis appears to be possible using instruments such as CYTOSCAN ™ A / R, which allows for in-vivo microscopy. To be Over the centuries, many researchers have observed microvessels associated with various types of wounds. However, clinical trials in patients have been extremely difficult due to the large and complex equipment required. Even in the case of experimental animals, in-vivo microscopy is difficult. OPS imaging greatly facilitates acquisition of microscopic images of living microvasculature. Minimal training is required for the practitioners to be able to accurately perform the video microscopy protocols for these clinical trials.

In the following case report, OPS imaging was used to assess microcirculation of burn wounds.

Overview: Selective treatment of deep second and third degree burns is aimed at early resection and transplantation. Indispensable for such aggressive surgical treatment is accurate diagnosis of the burn site. This emphasizes the importance of accurately assessing the severity of burn wounds as the basis for early policy making. Not only are clinical examinations based on the appearance of wounds not a robust method for these procedures, but clinical evaluation of burns performed by a clinically experienced burn surgeon is not always satisfactory. (Robson, MC et al., Clin. Pla
st. Surg. 19: 663-671 (1992); Heimbach, D.
． M. J. J. et al. Trauma. 24: 373-378 (1984)). Therefore, ancillary diagnostic techniques may help the surgeon to accurately analyze the severity of the burn, and thus may help the surgeon to make a quick bedside decision. However, even with all the technological innovations, the ideal device for such applications has not yet been routinely established.

Recently, a novel microscopy technique called Orthogonal Polarization Spectral (OPS) Imaging has been introduced (Groner, W. et al., Nat. Med. 5: 120).
9-1212 (1999); Messmer, K .; , Ed. , Orthogo
nal Polarization Spectral imaging: a
New Tool for the Observation and Mea
maintenance of the Human Microcirculati
on, Prog. Appl. Microcirc. , Karger, Basel
(2000), pp1-117). OPS imaging provides one method that can be used to directly image the microcirculation of animal and human organs.
OPS imaging uses polarized reflected light with a wavelength of 548 nm to image hemoglobin carried in microvessels without the use of fluorescent dyes, thereby avoiding the effects of phototoxicity (Saetzler, RK. J. Histoche et al.
m. Cytochem. 45: 505-513 (1997)). The OPS imaging technique has been shown to be effective in the measurement of functional capillary densities in the context of fluorescence in-vivo microscopy, the standard method for the measurement (Harr).
is, A. J. J. et al. Vasc. Res. (In Press) (2000))
. Functional capillary density is a parameter that reflects the perfusion of capillary tissue,
Obtained as capillary length with red blood cell perfusion per observed area (Harri
S.A. G. , Am. J. Physiol. 271: H2388-H2398 (
1996)).

Tissue blood flow in human burns has been the subject of several studies, and the extent of diminished cutaneous blood flow in heat-damaged skin correlates with its level of destruction. (Micheels, J. et al., Scand)
． J. Plast. Reconstr. Surg. 18: 65-73 (1984)
Years); Alsbjorn, B .; Others, Scand. J. Plast. Recons
tr. Surg 18: 75-79 (1984); O'Reilly, T .; J
． J. J. et al. Burn. Care Rehabil. 10: 1-6 (1989)
). However, direct visualization of microcirculation in human burns has not yet been possible due to methodological difficulties. This case study introduced OPS imaging as a novel technique for assessing microcirculation in burn wounds.

Patient: The patient was a 26 year old male patient with a left hand burn. This burn was caused by boiling oil during cooking and had been washed with cold water immediately before coming to the emergency room. After excision of the inactivated tissue at the beginning, a second clinical observation
He was diagnosed with severe burns. The blisters were open but remained intact. The burns were treated conservatively with topical 1% silver sulfadiazine in a semi-fluid oil in an aqueous emulsion, and each finger was individually wrapped with a soft gauze bandage. The hands were raised for the first 48 hours after the burn, and an analgesic was orally administered to control pain.

Test Protocol: The OPS imaging technique is CYTOSCAN ™ A /
It is built into a small, easy-to-use device called R (Cytomet
rics Inc. , Philadelphia, PA). After obtaining informed consent, observation of microcirculation was started from the third day after injury using OPS imaging. The measurements were taken at a certain room temperature (27
C.). After cleaning the wound by rinsing with sterile saline to remove topical agents, the OPS imaging probe was placed at six different sites of interest at the dorsal surface of the hand, including the fingers, for approximately 10 minutes. Addressed to Microcirculation images are Super VHS videotapes (Sony, Cologne, Germany)
) Recorded on. O covered with disposable sterile plastic cap
The working distance of the PS imaging probe was approximately 2 mm. A sterile ultrasonic gel was applied between the probe and the tissue to improve the refractive index matching. The patient did not experience pain or discomfort during the administration of the OPS imaging probe, which required a duration of approximately 5 minutes. The probe was immobilized during observation with the help of a custom-made holder, and the circulation of the capillaries was not stressed. The measurement of microcirculation was continued on days 6, 12, 20, 23, 26 and 30 days after burn.
Done daily and always repeated for the same site within the injured area. Quantitative analysis of microcirculation was performed off-line during playback of videotapes using the CapImage ™ computer program (Klyscz, T. et al., Biomed. Te.
ch. (Berl.) 42: 168-175 (1997)). Functional capillary density was evaluated from a single image digitized from a continuous video sequence. The assessment included capillaries that were in focus and clearly identifiable as perfused red blood cells. Data are obtained as the number of capillaries [n / cm ² ] perfused per area of observation (Bollinger, A. and Fagrell, B., Clinica), as described above in the clinical capillary observation device test.
l Capillaroscopy-A Guide to it's Use
in Clinical Research and Practice, To
ronto, Hofgrefe & Huber Publishers, pp
． 1-166 (1990).

Results: OPS imaging produced high quality images for microcirculation of burn wounds with a total magnification of 254x on-screen. Capillary morphology was clearly discernible, and these images allowed quantitative analysis of the number of capillaries with red blood cell perfusion. The functional capillary density (FCD) measured on the third day after injury was 11.2 ± 4
． It was 6 n / mm ² (mean ± SEM). During the early phase of healing, microcirculatory changes are characterized by a slow but stable increase in FCD, showing a marked increase from day 12 post-burn (16.6 ± 6.9). The maximal FCD (48.2 ± 19.7) measured at day 23 started to decrease from this time point, and finally reached 25.2 ± 10.3 n / mm ² at the end of the observation. did.
The patient was able to tolerate this measurement well and was enthusiastic about seeing their capillaries in the healing wound. Wounds that had a surface area of 122 cm ^{2 on} the day of injury as calculated by digital areometry showed uncomplicated healing within 3 weeks without scarring.

Discussion: The ability to precisely and as early as possible determine the severity of thermal injury is essential for planning surgical treatment, ie for optimizing patient function and cosmetic outcomes ( Kao, CC and Garner W. L., Plas.
t. Reconstr. Surg. 105: 2482-2492 (2000)
Heimbach, D .; Et al., World J. Surg. 16: 10-15 (
1992); Germann, G .; Pediatr. Surg. Int.
12: 321-326 (1997)). Early burn wound ablation and closure with grafted skin provide several benefits (Heimbach, D. et al. Wor.
ld J. Surg. 16: 10-15 (1992); Engrav, L .; H
． J. J. et al. Trauma. 23: 1001-1004 (1983); Gray
, D. T. Am. J. Surg. 144: 76-80 (1982); Sc.
hiller, W.W. R. J. J. et al. Trauma. 43: 35-39 (1997)). However, clinical assessments are often inaccurate and are estimated to not exceed 64% even for experienced burn surgeons (Heimbach,
D. Others (supra). Due to the limitations of clinical observations, there has been interest in supporting clinical observations over the last decades, especially in devices and methods capable of distinguishing between burns that heal without complications and those that require surgery. It's no surprise that they have been sent. Many modalities are available for grading burns, including vital dyes, fluorescein fluorometry, and nuclear magnetic resonance imaging (Heimba).
ch, D.I. Others (supra). However, to date, none of these techniques have been applied to a wide range of clinical applications. One of the reasons for this is the additional damage that occurs with some of these invasive approaches. However, its main reason is that these techniques have not been proven to give reliable results, except in the case of laser Doppler (LD) techniques. Heimbach, D. et al. Et al., World J. Surg. 16: 10-15
(1992), and Shakespeare, P .; G. , Burns 18
: 287-295 (1992).

The LD technique was used to evaluate the extent of skin destruction in human burns by Mich.
First used by eels and coworkers (Michels, J. et al.
Others, Scand. J. Plast. Reconstr. Surg. 18: 65-
73 (1984)). The principle of using LD for such tests lies in the hypothesis that the amount of skin blood flow correlates with the level of destruction (O'Reilly, TJ.
J. J. et al. Burn. Care Rehabil. 10: 1-6 (1989))
. According to Braverman's description, the nutrient component of the skin is 1-2 m below the skin surface.
formed by the dermal papilla loop located at m. (Braverman, IM.
, Microcirculation 4: 329-340 (1997)).
Therefore, when the teat loop is broken (as in the case of deep skin burns), microcirculatory blood flow cessation, or at least diminished blood flow, is unavoidable.

In this context, LD measurements can be useful in obtaining blood flow data such that a predictive description of the severity of the burn site can be obtained. LD measurements have already been used in laboratory tests and have been used in adult patients (Alsbjorn, B. et al., Scand.
J. Plast. Reconstr. Surg. 18: 75-79 (1984); O'Reilly, T .; J. J. J. et al. Burn. Care Rehabile
． 10: 1-6 (1989); Schiller, W .; R. J. J. et al. Trau
ma. 43: 35-39 (1997); Green, M .; J. J. et al. Burn.
Care Rehabil. 9: 57-62 (1988); Niazi, Z .;
B. Burns. 19: 485-489 (1993); Yeong, E .;
K. J Trauma. 40: 956-961 (1996)), and children (Atiles, L. et al., J. Burn. Care Rehab. 16:
596-601 (1995)), a significant relationship between burn blood flow and clinical final outcome was demonstrated. However, despite these promising outcomes, movement and agitation can make measurements difficult to analyze or useless, so the use of LD is patient-exciting and collaborative. (Micheels, J., Scand. J. Plast. Reco
nstr. Surg. 18: 65-73 (1984); Park, D .; H. Plast. Reconstr. Surg. 101: 1516-1523 (1
998)). In addition, measurements are reported to be relatively time consuming for patients and laboratory staff (Michels, J., Scand. J. Plas).
t. Reconstr. Surg. 18: 65-73 (1984)), not all patients are eligible for this type of test. In addition, the need for additional staff and the unquestionably high cost of treating patients are significant. Furthermore, a limitation of the LD technique is the force exerted by the probe on the skin surface, which means that the measurement itself affects the parameter under test (Obei).
d, A.D. N. J. J. et al. Med. Eng. Technol. 14: 178-181
(1990)).

A clinical trial using LD on the finally healed wound showed a steady increase in mean perfusion levels during the first few days after burn. On the other hand, when the burn wound was deeper, such a clear pattern could not be detected in the restoration of blood flow (O'Re
illy, T .; J. J. J. et al. Burn. Care Rehabil. 10: 1-
6 (1989); Green, M .; J. J. et al. Burn. Care Rehab
il. 9: 57-62 (1988); Atiles, L .; J. J. et al. Burn.
Care Rehabil. 16: 596-601 (1995)). From these studies, the extent of damage to skin microvessels is one of the limiting factors in burn healing.
Became clear.

The hydrodynamic changes in the microvascular network following burns were also tested in animal models using in vivo fluorescence microscopy and fluorescent agents for contrast enhancement (B).
oykin, J .; V. Plast. Reconstr. Surg. 66: 1
91-198 (1980)). Although these studies have allowed detailed observations of dynamic changes in the microcirculation after burn injury, this type of study is so far limited to the laboratory.

In this case report, the microcirculation of human burn wounds was first evaluated by OPS imaging. The technique allows repeated direct and non-invasive visualization of the microcirculation of the skin and dermis during the healing process. The study was conducted within minutes and patients were forgiven for acclimatization periods to specified environmental conditions as required by studies using LD (Michelels, J. et al., Supra). Compression artifacts are eliminated because there is no need to contact the surface of the OPS imaging probe. Unlike LD measurements, OPS imaging allowed the visualization of individual capillaries in burn wounds, probing their morphology, and video recordings to quantitatively assess kinetics during the healing process.

Microcirculation data obtained from OPS imaging showed that during the healing process, FCD showed similar recovery to that reported in previous studies on perfusion data using LD.
(O'Reilly, TJ, et al., J. Burn. C.
are Rehabil. 10: 1-6 (1989); Heimbach, D.
． Et al., World J. Surg 16: 10-15 (1992); Green.
n, M. J. J. et al. Burn. Care Rehabil. 9: 57-62 (19
1988)). FLD may also be a useful parameter for predicting whether or not the burn site will be healed, if the correlation between the perfusion and the healing potential of the burn wound is obtained from the LD measurement. Is high.

The results obtained in the patients reported here demonstrate that OPS imaging can provide a reliable tool for quantitatively assessing functional capillary density within burn wounds. The device is easy to operate, portable and does not require any special features. In addition, the probe can be wrapped in a sterile plastic foil, allowing for intraoperative measurements. These results regarding the evaluation of microcirculation in burns using OPS imaging are promising, and it is expected that this technique will develop knowledge about the dynamics of microcirculation in the pathophysiology of heat injury. To be done. Moreover, OPS imaging, in combination with clinical observation, appears to be a promising diagnostic tool for assessing the severity of burn sites.

Applications in the Plastic Surgery Field In this embodiment of the invention, a high contrast OPS imaging probe is used in vivo during plastic surgery.

In each of the following aspects of this embodiment, capillary density, blood vessel (and microvessel) morphology, blood vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, blood vessel diameter, leukocyte / endothelium. Quantitatively quantify one or more parameters such as cell-cell interactions, vasodynamics (eg vasomotor), functional vessel density, functional capillary density, intra-area to marginal ratio, blood flow, hemoglobin concentration, hematocrit, etc. I have to decide. The number of parameters to be determined is preferably two or more.

In one aspect of this embodiment, the high contrast OPS imaging probe is used to monitor blood flow (perfusion) during plastic surgery, reconstructive surgery, reattachment surgery or microsurgery as well as after these surgeries. To be done.

In another aspect of this embodiment, the high contrast OPS imaging probe comprises:
Used to determine healthy tissue around the flap for necrotic (ie, dead) tissue. Skin flap reconstruction uses the subject's own tissue to surgically remove the tumor or reconstruct or replace tissue lost during trauma. It is important that the plastic surgeon monitor blood flow (perfusion) to the valve both intraoperatively and postoperatively to ensure that there is adequate perfusion in the tissue. This procedure can be accomplished by using functional capillary densitometry, RBC velocity and diameter.

In the following study, flap flap perfusion was monitored using OPS imaging. Despite recent advances in technology, there is currently no monitoring system for reliably detecting human skin flap perfusion failure. However, there is a great need for an objective method to detect a lack of perfusion in transplanted tissue. OPS imaging allows polarization to be used to directly image and characterize microcirculation. The purpose of this study was to confirm the effectiveness of OPS imaging for measuring microvessels in the flap. This confirmation was performed in contrast to in vivo fluorescence microscopy (IFM), which is a standard technique for quantitatively measuring microcirculation.

Materials and Methods: A ready-made skin flap model of male hairless mice (hr / hr) was used (Galla, TJ et al., Br. J. Plastic Surg. 45).
: 578-585 (1992)). After valvular wound creation (n = 9), skin microcirculation examination using both OPS imaging and IFM was performed at 1, 6, and 24 hours following the valvular wound creation. FITC-Dextr for IFM measurement
The application of an (5 mg / kg BW) was essential, but not essential for OPS imaging. To compare the two techniques, the same region of interest was monitored continuously. The valve was scanned from the distal portion to the base. A total of 60 regions of interest were captured on each time videotape. Functional Capillary Density (FCD) is Cap
It was measured off-line using the Image ™ computer program. All procedures were performed under general anesthesia with isoflurane.

Results: By using OPS imaging, the tracer (FITC-De
It was possible to image and characterize the flap microcirculation without xtran). From these images, quantitative analysis of FCD could be realized. FCD values were significantly lower in the distal portion of the valve compared to the valve base (171.8 ± 34.7 vs 62.0 ± 2).
5.6, mean ± SD; 1 h data). Comparison of OPS imaging and IFM showed significant correlation with FCD values (p <0.001, Spearman's rank sum test) at all time points. A Brand-Altman plot showed good agreement between the two methods.

Discussion: OPS imaging allows quantitative analysis of skin flap perfusion. In addition to the results of the efficacy confirmation test for the mouse skin flap, it is necessary to further prove that OPS imaging can be successfully used in humans by the test. Performing this novel technique in reconstructive surgery will provide further insight into the function of the flap microcirculation and provide a new tool for intraoperative and postoperative monitoring of flap perfusion.

In another aspect of this embodiment, standard or high-contrast OPS imaging probes are used to image and characterize microcirculation in skin and muscle flaps and free flaps exhibiting complications. It For example, in the free flaps of interest, direct observation of red blood cell flow within the capillaries using OPS imaging has shown that the valves are viable.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to monitor the microcirculation after plastic surgery or microsurgery and identify any potential problems with reperfusion. . The early indication of reperfusion problems can help avoid repeated surgery.

Cardiac Application In this embodiment of the invention, a high contrast OPS imaging probe is
Used in vivo in cardiology and / or heart surgery.

In each of the following aspects of this embodiment, capillary density, blood vessel (and microvessel) morphology, blood vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, blood vessel diameter, leukocyte endothelial cells. Quantitatively determine one or more parameters such as interactions between cells, vasodynamics (eg vasomotion), functional vessel density, functional capillary density, blood flow, intra-area to marginal ratio, hemoglobin concentration, hematocrit value, etc. Sometimes. The number of parameters to be determined is preferably two or more.

In the following study, epicardial microcirculation was detected during focally induced ischemia,
To confirm whether OPS imaging could be used for visualization and characterization, OPS imaging was applied to beating porcine heart using an epicardial stabilization device.

Method: Eight pigs (70 kg body weight) underwent a midline sternotomy under general anesthesia.
After exposing the heart, an epicardial suction device (Octopus, Medtroni
c Inc. ) Was attached. Two fork-like extensions of this suction device were placed on top of the left anterior descending coronary artery (LAD) and a suction of 400 mmHg was applied to stabilize the myocardial area. An OPS imaging probe was placed in this stabilized region to visualize epicardial microvessels. Focal ischemia for this area was achieved using a single-fiber tourniquet suture around the proximal LAD.

Results: It was possible to collect epicardial microvascular images in all 8 animals. Semi-quantitative analysis showed a reduction in microvascular blood flow due to LAD occlusion.

Conclusion: By using OPS imaging with a local myocardial wall anchor,
The epicardial microcirculation can be imaged and characterized. From the images obtained, it is possible to quantify changes in epicardial blood flow and to uniquely assess microcirculatory changes during focal ischemia. The ability to detect and image changes in epicardial microcirculation relative to the beating heart is of particular interest and could be a useful tool during coronary revascularization.

In one aspect of this embodiment, the high contrast OPS imaging probe is
Imaging of the microcirculation of the heart so that reperfusion can be confirmed during minimally invasive cardiac surgery avoiding cardiopulmonary bypass devices such as "keyhole" surgeries (ie Heartport) or thoracotomy. Used to enhance characterization and characterization.

In another aspect of this embodiment, the high contrast OPS imaging probe comprises:
It is used to monitor and detect changes in the patient's blood flow (microcirculation) while subjecting the patient to a cardiopulmonary device during open heart surgery. During monitoring, the OPS imaging probe is placed in the patient's oral cavity. The observation of the difference in blood flow was performed after placing the patient on the cardiopulmonary device and after blocking the device compared to before the surgery. An increase in the appearance of blood vessels was also observed when white blood cells were visible. In particular, the longer the patient spends on the cardiopulmonary system, the worse the microcirculatory flow. Thus, it is possible to use the OPS imaging probe to monitor the progress of the patient not only while on the cardiopulmonary device, but also when the device is removed.

In the following study, OPS imaging was used to directly image and characterize microvascular changes in patients undergoing cardiac surgery.

Methods: OPS imaging was used on 12 male patients undergoing cardiopulmonary bypass (CPB) surgery (mean age 61.1 years) to examine changes in microvascular perfusion during CPB. The interaction between leukocyte endothelial cells was also examined. Immediately after induction of anesthesia (T1), early phase of CPB (T2), late phase of CPB (T3)
), And 1 hour after reperfusion (T4), microvascular diameter (DIA [μm]), red blood cell velocity (VEL [mm / s]), and functional capillary density (T4) in images collected from the sublingual mucosa. FCD [cm / cm ² ]) was measured.

Results: DIA increased significantly at T3 and T4. VEL decreased at T2 and increased at T3. There was no significant change in FCD (Table 1).

[0233]

[Table 1]

Median (25% -75%); ^* p <0.05 vs T1.

Conclusion: This data provides evidence for microcirculatory changes in CPB. However, since only a decrease in FCD was not significant, it is likely that trophic blood flow is well maintained in uncomplicated CPB.

In another study, described below, OPS imaging was used to image and characterize changes in epicardial microcirculation in human heart-lung machines.

Methods: In a preliminary study, OPS imaging was used on 3 humans who underwent median sternotomy under general anesthesia and underwent cardiopulmonary placement for various cardiac procedures.
For all patients, L during each cycle of cardioplegic perfusion with hematological cardioplegic solution
The region under perfusion was visualized by AD. Microvessel diameter (DIA [mm]) and red blood cell velocity (VEL [mm / s]) in images acquired directly from the heart surface
) Was measured.

Results: Epicardial microvessel images were acquired in all patients by using OPS imaging. The diameters of arterioles and venules ranged from 10 to 70 m. Erythrocyte velocities can be up to 0 during the use of cardioplegic solution in these vessels.
． It reached 6 mm / sec. Evidence of vasomotion was also detected, as arteries and arterioles were contracting and dilating. Many areas with unclear flow of red blood cells were detected, indicating that the myocardial protective solution did not reach this part of the myocardium.

Conclusions: This data provides a first in-vivo microscopy image of the surface of the human heart when the cardioplegic solution was used in humans. Semi-quantitative analyzes performed to date suggest that there are significant differences in microvascular blood flow. OPS imaging can be very important during cardiac surgery because it allows direct assessment of microvascular changes in the human heart.

In another aspect of this embodiment, the OPS imaging probe is also used during coronary artery bypass graft (CABG) surgery to determine if the site provided by the graft has sufficient blood flow. can do. That is, the cardiac surgeon can use the OPS imaging probe in direct contact with the heart to monitor blood flow to the capillaries after bypass surgery.

In another aspect of this embodiment, the OPS imaging probe can be used to monitor a patient's microcirculation during cardiac surgery to repair a birth defect of the heart. For example, OPS imaging probes have been used to image the heart surface of two infants during repair of a ventricular septal defect (VSD) during pediatric cardiac surgery. The OPS imaging probe had the size of a large pen and the probe was applied to the surface of the heart. The probe was hand-held and manually focused in response to images viewed on a high resolution monitor. The image shows a cardioplegia solution (Bret) used to protect the heart during ischemia associated with open heart surgery.
It was recorded while using the tschneider solution). At first, my heart is still
He was beating at 20 beats / minute, followed by a myocardial protection period. The well-established CapImage ™ analysis program was used for quantification of in-vivo microscopy images.

Using OPS imaging made it possible to acquire images from the surface of the heart and to calculate the diameter of the observed microvessels. Brett
Although the schneider solution did not contain red blood cells, blood vessel imaging was possible due to the red blood cells remaining in the blood vessels. In general, the cardioplegic solution reached the majority of microvessels, but some of the microvessels did not reach these microvessels because they contained a column of erythrocytes and the flow of erythrocytes was not clear. Therefore, areas of the heart that were not covered by the cardioplegic solution remained unprotected from surgery-induced ischemia. Moreover, significant vasomotion was observed in arterioles with diameter changes ranging from 70 μm to 32 μm while using the cardioplegic solution.

Therefore, in view of these findings, as well as previous reports, in yet another aspect of this embodiment, OPS imaging is performed using cardioplegic solution perfusion during any cardiac surgical procedure. It can be used to qualitatively and quantitatively assess efficacy. In addition, microvascular phenomena such as vasomotion can be examined using OPS imaging techniques.

In another aspect of this embodiment, the OPS imaging probe can be used to monitor a patient's microcirculation during any type of cardiac surgery. Examples of surgery that can be used include surgery to replace or repair a heart valve.

In another aspect of this embodiment, the OPS imaging probe can be used to monitor heart risk. OP to visualize and characterize microcirculation
The use of S imaging probes can help non-invasively determine high risk heart profiles. Microvascular sequelae due to hypertension can be examined and used in determining heart risk.

(Respiratory Medicine Application) In this embodiment, an OPS imaging probe is used for imaging, characterization and evaluation of lung tissue.

In each of the following aspects of this embodiment, capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter, leukocyte endothelial cells. Quantitatively determine one or more parameters such as interaction between cells, vasodynamics (eg vasomotor), functional vessel density, functional capillary density, blood flow, intra-area to marginal ratio, hemoglobin concentration, hematocrit value, etc. I have something to do.
The number of parameters to be determined is preferably two or more.

Pig lungs were imaged using a CYTOSCAN ™ A / R with two objectives, an objective with 5 × optical power and an objective with 10 × optical power. By using a 10-fold A / R, considerable details such as the blood vessels in the corners (blood vessels around the alveoli) and the capillaries performing gas exchange in the alveoli were observed. Multiple blood vessels were also imaged through a thin membrane covering the lungs themselves. 5 times A /
In the case of R, the alveoli could be visualized more clearly, but the "corner" vessels were too small in this case (approximately 10-15 micrometers) to be hardly quantified. The higher definition when observed at 5 times as much as 10 times is due to the different depth of field between the two objective lenses.

Application to Brain Surgery In this embodiment of the invention, a high contrast OPS imaging probe is
In vivo before or during brain surgery.

In each of the following aspects of this embodiment, capillary density, blood vessel (and microvessel) morphology, blood vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, blood vessel diameter, leukocyte endothelial cells. Quantitatively determine one or more parameters such as interaction between cells, vasodynamics (eg vasomotor), functional vessel density, functional capillary density, blood flow, intra-area to marginal ratio, hemoglobin concentration, hematocrit value, etc. I have something to do.
The number of parameters to be determined is preferably two or more.

In one aspect of this embodiment, the high contrast OPS imaging probe is applied directly to the brain during diagnostic or therapeutic brain surgery to observe cortical or pial microcirculation and determine vessel diameter. , Parameters such as flow rate, functional capillary density are measured.

[0252] A specially prepared OPS imaging device was used to image pial microcirculation in anaesthetized humans prior to brain surgery. To allow the surgeon to position the light guide on the brain surface and hold the image guide in place for stable video recording, a stainless steel surgical arm with three axes of rotation was developed. This arm can be made rigid by twisting a number of fixing knobs. Prior to surgery, this arm was secured to the operating table rail with the OPS imager. The arm was covered by a sterile foil used to cover the endoscope, which was further attached to a sterile Teflon sleeve covering the light guide. Following the craniotomy, the imager was slowly positioned on the surface of the buffy coat. The surgeon was able to observe the microcirculation of the brain on the TV monitor.

Although it is difficult to see a static image, it was possible to observe red blood cells flowing in a row through the capillaries during video playback. The blood vessels could be easily identified as arterioles, capillaries or venules. The video images were of sufficient quality to quantify the flow of red blood cells using commercially available image processing software originally developed to analyze the video images obtained by in-vivo microscopy ( Kly
scz, T.S. Biomed. Tech. (Berl.) 42: 168-17.
5 (1997)). In addition, clear images of the human esophagus and the microcirculation layer in the stomach were also obtained.

In another aspect of this embodiment, the high contrast OPS imaging probe comprises:
Used to detect aneurysms and vasospasm following subarachnoid hemorrhage.

In another aspect of this embodiment, the high contrast OPS imaging probe comprises:
Used to detect tumor boundaries in the brain. Brain tumors are typically not located on the surface of the brain and are therefore not visible. In another neurosurgical application of OPS imaging, OPS imaging probes can be used during surgery to detect changes in functional capillary density within the "normal" tissue overlying the tumor, and thus the brain tumor margin. Can be used to evaluate

Using OPS imaging technology, microcirculatory abnormalities in human brain tumors were observed. Thus, in another aspect of this embodiment, the high contrast OPS imaging probe can be used to identify and type brain tumors based on differences in vasculature and microcirculation of various brain tumors.

[0257] Human tumor microcirculation is one of the areas of intense research as tumor hypoxia and angiogenesis are important. In the following study, OPS imaging was used to image and characterize the microcirculation of human brain tumors in 11 patients (age 55.9 ± 5.17 years; mean ± SEM) during surgery. These patients were screened because the tumors in the brain are superficial. From this, microcirculation of the tumor was observable with minimal treatment to the tumor and surrounding tissues.
The microcirculation was compared between the patient's healthy cortex and the tumor by randomly imaging various sites.

Benign meningioma (n = 5), glioblastoma multiforme (n = 4) and metastasis (n =)
We examined three types of brain tumors called 2). In meningioma, the background was extremely dark compared to normal, there was almost no blood flow, and the blood vessel pattern was disorganized and dilated. In glioblastoma, the background was similar to normal, blood flow was reduced, and there were fewer blood vessels than normal. Metastases had a much darker background, almost no blood flow, and disordered vascular patterns compared to normal. This study found that OPS imaging could be used to identify in situ for pathological microcirculation within brain tumors during surgery.

The microcirculation parameters are computer-aided microcirculation analysis system (CapImag).
e (trademark), Dr. Zeintl Ingenieurburo, Heidel
Berg, Germany). Significant differences were found between various brain tumors and healthy cortex in microcirculation parameters (red blood cell velocity (RBCV), functional vessel density (total length of vessel per site), etc.). RBCV of glioblastoma is 0.40 ± 0.10 mm / s, whereas it is 1.59 ± 0.3 in arterioles.
2 and 0.64 ± 0.09 for venules. There was a difference (p <0.05) between healthy cortex and various tumor types in blood vessel length P (vascular density) at each site, and meningioma (control; tumor): 68.10 ± 5. .65 cm / cm ² ; 41.75 ± 6
． 39; glioblastoma multiforme (control; tumor): 88.47 ± 5.70; 50.
02 ± 6.91; metastasis (control; tumor): 72.96 ± 17.04; 25.04 ±
It was 6.02). This study represented the first recording of human brain tumor microcirculation. OPS imaging is expected to provide deeper insights into possible treatments for pathogens and brain tumors.

The following study reports intraoperative observations using OPS imaging on human brain microcirculation.

One of the findings for the human microcirculation was primarily histological examination (Hunzi.
ker, O.I. J. Geront. 34: 345-50 (1979); Cra.
igie, E .; H. , Bid. Rev. 20: 133-146 (1945))
Conclusions derived from observations of biological objects in animals (Uhl,
E. Stroke 30: 873-879 (1999)).
Implementation of OPS imaging with a small handheld device (CYTOSCAN ™ A / R) now allows observation of human cerebral microcirculation during neurosurgery (Groner, W. et al. Nature Medicine 5). : 120
9-1213 (1999)). The technique is based on illuminating the tissue with linearly polarized light, while its reflected light is imaged using a cross polarization analyzer. It is not necessary to use fluorescent dyes. The purpose of this study was to verify the feasibility of OPS imaging during brain surgery and to collect basic quantitative data on human cortical microcirculation under normal and pathological conditions.

Patients and Methods: The study was approved by the local ethics committee and informed consent was obtained from each patient prior to surgery. To date, 12 patients (6 male patients and 6 female patients) are undergoing the study. Four patients had an accidental intracerebral aneurysm surgery and were treated as a control group. Three patients had subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm and underwent surgery to clip the aneurysm. Five patients underwent surgery for brain tumors. The average age was 47.3 ± 9.7 years. None of the patients had previously undergone surgery. These operations consisted of sufentanil (1 μg / kg per induction of anesthesia b.
w. ), Propofol (400-700 mg / h), remifentan
It was performed under general anesthesia using standard procedures with il (0.25-0.5 ng / kg / min). In addition, all patients received 8 mg dexamethasone and 250 ml 20% mannitol prior to performing craniotomy. Patients with SAH were also given continuous infusion of nimodipine (0.5-2 mg / h). Systemic parameters were routinely measured during surgery.

Intraoperative measurement: In-vivo microscopy was performed using a CYTOSCAN ™ A / R equipped with a 5 × magnification lens and a CCD camera. The technical details of this system are described in various places (Groner, W. et al. Nature Medicine).
ine 5: 1209-1213 (1999)). This device has a sterile plastic cap (CYTOLENS ™, Cytometrics I).
nc. , Philadelphia, PA) and sterile plastic foil, the device was adapted to a Leyla retractor that allowed stable positioning of the probe on the brain surface during the measurement. On-line observation of cortical microvessels was performed shortly after opening the dura and prior to each clipping of the aneurysm or excision of the tumor. Second before closing the dura at the end of surgery
Was measured. The time difference between the first and second measurements will vary depending on the type and difficulty of the surgical approach. In each patient, at least 5 locations of interest (SOI) on the surface of the cortex were randomly selected. Each SOI was scanned for 30 seconds and the images were videotaped for offline evaluation. Due to intraoperative shifts and excision of brain tissue, it was not possible to evaluate the same vessel segment. Microcirculation parameters are evaluated by computer-aided microcirculation analysis system (
CapImage ™, Dr. Zeintl Ingenieurburo
, Heidelberg, Germany) at a final magnification of 260 (Klyscz, T. et al., Biomed. Tech (Berl) 42:16.
8-175 (1997)). The diameter of blood vessels [μm], red blood cell velocity in arterioles and venules [mm / s], and functional capillary density [cm ⁻¹ ] were measured. The latter is defined as the length (per cm ² ) of all capillaries that are perfused with red blood cells at the time of observation.

The results were as follows: Morphology of cortical vessels: In the OPS system arterioles with typical structures, capillaries and draining venules were clearly distinguishable (Fig. 2 and 3). Capillaries appeared irregularly, but almost circular. The number of capillaries within a particular area range is variable, with a reduced number near the junction of venules and arterioles. During extravasation of SAH, red blood cells were observed in the subarachnoid space (
(Fig. 4). In these patients, segmental vasospasm in arterioles was detected, leading to a 50% reduction in vessel diameter. In some of the arterioles, this vasospasm was confined to only one segment, whereas in other arterioles, multiple spasms caused the blood vessels to look like a pearl string (Fig. 4). Furthermore, SA
Initially, changes in vascular endothelium were observed at the border of tumor resection in patients with H as well as patients with brain tumors (Fig. 5). In these blood vessels, an opaque layer was observed along the vascular surface of the lumen, resulting in a reduction of the intracavitary space that resulted in reduced red blood cell flow.

In the case of superficial tumors, it was possible to distinguish neoplastic tumor blood vessels from normal cerebral microcirculation. Tumor vessels were irregular and tortuous, often sinusoidal, and formed a normal microcirculation and clumps with intervening vessels at specific sites (FIG. 6). These tumor vessels were in close contact with venules, but no clear association with arterioles at this site was found.

Functional Capillary Density: The initial functional capillary density is 94.7 ± 9.
1 cm ⁻¹ (mean ± SEM), and 79.1 ± 5.7 in tumor patients. S
In AH patients, the initial value was smaller than that in the other patients, and the average value was 6
It reached 1.7 ± 12.5. In all patients, functional capillary density increased at the end of surgery compared to the first measurement (FIG. 7), of which the most significant increase was observed after aneurysm surgery in SAH patients.

Vessel diameter: The observed vessel diameter is 10-150 μm for arterioles / small arteries.
And the range of 10 to 210 μm for venules / venules. Because blood vessels are randomly selected, it is not possible to measure the same vessel segment at the beginning and end of surgery. Therefore, the actual changes in specific blood vessels could not be deduced from our observations. Therefore, the data distribution is presented rather than the average value (FIG. 8). No significant changes in vascular diameter distribution were observed during the procedure, except for segmental microvasospasm in SAH patients.

Erythrocyte Velocity: By combining the line shift diagram with the CapImage ™ system, RBC velocity measurements up to 2 mm / s are possible. In many cortical vessels, especially arterioles, RBC velocities exceed this value and accurate values cannot be collected. For this reason, RBC velocities are classified into 6 grades, providing the percentage of vessel classifications with a particular RBC velocity at each time point (FIG. 9). Blood vessels with too high an RBC velocity to be measured were included in the velocity classification above 2 mm / s. From our data, RB> 2 mm / s in all three patient groups
The number of vessels with C velocity increased at the end of surgery, of which SAH
It can be seen that the most significant changes were shown in the subsequent aneurysm surgery.

Discussion: From this experience with the OPS imaging system, CYTOSCAN (
The Trademark A / R proves to be a suitable device for intraoperative observation of human cortical microcirculation. Although visualized for brain capillaries, arterioles and venules, a quantitative analysis of microcirculation parameters was also collected. However, in some patients at high magnification, there is brain movement primarily caused by breathing rather than heartbeat transmission, and good image quality adversely affects postoperative offline quantification of microcirculation parameters. Was affecting. In a system that works by the reflectivity of light, a semitransparent thin buffy coat brain is the ideal organ to be examined, but the quality of the image depends largely on the underlying lesion. Patients with occasional aneurysms or tumors had good or excellent image quality, while patients with subarachnoid hemorrhage had a negative impact on imaging due to extravasation of red blood cells and swelling of the brain. In addition to extravasation of red blood cells, arterioles (10 to 95 μm
m) and small arteries (100 to 150 μm), clear microvasospasm could be observed. These microvasospasms were observed in patients with normal transcranial Doppler values and no clinical signs of vasospasm at the time of surgery. Whether this intraoperative finding could be prognostic for developing clinically significant vasospasm later in the course of the disease will need to be evaluated.

In addition, an opaque layer associated with microvascular endothelium was also observed at the resection border of SAH patients, as well as brain tumor patients. The pathology underlying this observation is unknown. This finding implies either swelling of the endothelium or a thick layer of plasma proteins along the inner surface of the endothelium, which can be speculated to cause a reduction in intracavity space. This results in reduced red blood cell flow in certain blood vessels.

Intraoperative visualization of brain capillaries, arterioles and venules, as well as quantitative analysis of microcirculation parameters are obtained. FCD was found to be higher at the end of surgery in all patients. The reason for this is probably a reduction in intracranial pressure, either due to drainage of cerebrospinal fluid in patients with aneurysms or due to reduced tumor mass in patients with brain tumors. The resulting relaxation of brain tissue may also be the reason for the increased number of microvessels (especially venules) with RBC velocities above 2 mm / s. Unlike the case of mean vessel diameter, which essentially depends on the observer's choice of SOI, functional capillary density and red blood cell velocity appear to be constant largely unaffected by quantitative selection parameters.

OPS imaging is expected to be one of the useful tools in brain tumor surgery.

In another aspect of this embodiment, the high contrast OPS imaging probe comprises:
It is used to directly image and characterize the vascular effects of neural trauma and to determine the extent of neural trauma.

In the following study, it was found that cortical hypoperfusion was followed by hyperperfusion after controlled impact injury of the cortex in rats.

[0275] Impaired cerebral perfusion results in traumatic brain injury followed by tissue damage. This long-term study demonstrates that controlled impact injury (CCII) of the cortex is followed by in-vivo microscopy (CYTO) using a laser Doppler flowmeter and OPS imaging.
SCAN ™ A / R) was utilized to examine the duration of cortical hypoperfusion.

Method: Pre-CCII, 30 min, 4 h, 24 h, and 48 h after CCII, crushed by moving the laser Doppler probe in 50 × 0.2 mm steps over the trauma hemisphere in 6 rats. Peripheral and traumatic uninjured cortical perfusion was determined. Arteriole and venule diameters and flow velocities were evaluated in the same rat using orthogonal polarization spectral imaging.

Results: At 4 hours post CCII, cortical perfusion was significantly reduced by 33% (p <0.05) compared to pre-traumatic levels. The normal value of paCO ₂ is (average value:
42.1 ± 1.0 mmHg), but cortical perfusion was significantly increased by 43% and 107% (p <0.005) at 24 and 48 hours after CCII, respectively. In vivo microscopy revealed corresponding changes. In the early posttraumatic phase, vessel diameter was reduced by 24% in arterioles, while venule diameter remained unchanged. Most of the change in the flow rate occurred in the venules, and the flow rate decreased by 39%. In late phase, vessel diameter is +39 in arterioles
% And + 75% in venules, significantly increased (p <0.005). In venules, the flow rate was above measurable and was the same as that determined at all time points in arterioles.

Conclusions: The cortical hypoperfusion seen in the early phase following CCII appears to reverse as the long-lasting hyperperfusion phase follows. These findings may be due to changes in tissue mediators (acidosis, NO, serotonin).

(Application to Organ Transplantation) In this embodiment of the present invention, a high contrast OPS imaging probe is
Used in vivo between organ transplants.

In one aspect of this embodiment, a standard or high contrast OPS imaging probe is used during transplant surgery to determine the amount of perfusion after connecting the transplanted tissues / organs. For example, any transplanted organ can be imaged including liver, lung, pancreas, intestine, kidney, heart. During liver transplant surgery (as opposed to, for example, kidney transplant), it may be difficult to assess perfusion other than liver transplant,
This imaging becomes particularly useful. In the liver, it is possible to measure the number of perfused sinusoids. The OPS probe can be placed directly on the transplanted part of the organ.

Capillary density, vascular (and microvascular) morphology, vascular density, vasospasm, red blood cell (RBC) velocity, cell morphology, vascular diameter, interactions between leukocyte endothelial cells, vasodynamics (eg vasomotor) ), Functional blood vessel density, functional capillary density, blood flow, intra-area marginal ratio, hemoglobin concentration, hematocrit value, etc. may be quantitatively determined. The number of parameters to be determined is preferably two or more.

Vascular Graft In this embodiment of the invention, standard or high contrast OPS imaging probes are used in vivo during vascular grafts such as peripheral arterial occlusive disease (PAOD).

In each of the following aspects of this embodiment, capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter, leukocyte endothelial cells. Quantitatively determine one or more parameters such as interaction between cells, vasodynamics (eg vasomotor), functional vessel density, functional capillary density, blood flow, intra-area to marginal ratio, hemoglobin concentration, hematocrit value, etc. I have something to do.
The number of parameters to be determined is preferably two or more.

In one aspect of this embodiment, a standard or high-contrast OPS imaging probe is used to determine the amount of perfusion after connecting the graft during the graft surgery.

Orthopedic Application In this embodiment of the invention, a standard or high contrast OPS imaging probe is used in vivo during orthopedic surgery and in the field of orthopedic practice.

In one aspect of this embodiment, a standard or high contrast OPS imaging probe is used during orthopedic surgery to detect and observe necrotic tissue that is surgically removed. The position of the probe will be on the traumatized site.

The probe can also be used to image bones, tendons and ligaments.

In another aspect of this embodiment, a standard or high contrast OPS imaging probe is used to image and characterize the microcirculation around the periosteum (bone). Differences in microcirculation are observed when images are taken before and after fracture.

Capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter, interactions between leukocyte endothelial cells, vasodynamics (eg vasomotor) ), Functional blood vessel density, functional capillary density, blood flow, intra-area marginal ratio, hemoglobin concentration, hematocrit value, etc. may be quantitatively determined. The number of parameters to be determined is preferably two or more.

Gastrointestinal Application In this embodiment of the invention, a standard or high contrast O orthogonal polarization spectral imaging imaging probe is used in vivo in the field of gastrointestinal and gastrointestinal (GI) or gastroesophageal surgery. .

In each of the following aspects of this embodiment, capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter, leukocyte endothelial cells. Interaction, vasodynamics (vasomotor etc.), functional vessel density,
One or more parameters such as functional capillary density, blood flow, area-to-surround ratio, hemoglobin concentration, and hematocrit value may be quantitatively measured. Preferably two or more parameters are measured.

According to one aspect of this embodiment, the large intestine is imaged and characterized using standard or high contrast orthogonal polarization spectral imaging probes, and inflammatory bowel disease (IBD), ulcerative colitis, clones. Diseases or other gastrointestinal disorders are diagnosed and treated. Orthogonal polarization spectral imaging can be used to distinguish Crohn's disease and ulcerative colitis, and thus aids in accurate diagnosis. This probe can be inserted into the rectum and brought into direct contact with the wall of the large intestine.

In the following study, orthogonal polarization spectral imaging (CYTOSCAN ™ A) was used to image and characterize the colonic microcirculation using a mouse model.
/ R) and an in-vivo fluorescence microscope were compared. In this test, under the control of conditions and D
After inducing SS-induced colitis, the colonic microcirculation of Balb / c mice was examined (
Each group tested was n = 7). Postcapillary vein diameter, red blood cell velocity, and functional capillary density were analyzed for the outer wall of the colon (serosa / muscle) as well as the luminal wall (mucosa) as parameters of microcirculation. To verify the agreement between both methods, linear regression, Spearman's correlation coefficient and the Bland-Altman plot were verified.

In the control and colitis groups, all measured parameters were significantly correlated by the two methods. Orthogonal Polarization Spectral Imaging can be used to image and characterize colonic microcirculation without the use of fluorescence dose and quantify the associated microcirculation of mouse colon under physiological and pathophysiological conditions. It has been demonstrated that it is possible to measure in a positive way. The results obtained were found to be significantly correlated with the results obtained for IVM. Orthogonal polarization spectral imaging was superior in quality and sharpness to IVM.

In the next study, orthogonal polarization spectral imaging detected a porcine microcirculation shunt with mesenteric aortic block.

Changes in microcirculation due to ischemia and reperfusion injury are believed to cause the "no perfusion" phenomenon and organ failure after aortic block (AoX). Until now, in vivo observation of microcirculation was possible only in rodents using an in-vivo microscope. In this study, porcine ileal serosa microcirculation after AoX was observed using orthogonal polarization spectral imaging.

Six pigs were anesthetized and blood flow was thoroughly monitored. Ultrasound probe to the superior mesenteric artery (
SMA) placed around. After stabilizing for 60 minutes, AoX was performed on the SAM for 45 minutes. During baseline, 5 minutes after AoX, and after 2 hours of reperfusion, ileal serosa microcirculation was observed using OPS imaging. Video images were computer analyzed using CapImage ™ software. The number of perfused vessels per imaged microcirculation area (3 / pig) was unchanged from 9 ± 1.79 at baseline to 10.5 ± 1.378 5 minutes after block opening. However, the flow of SMA has almost doubled. Two hours after reperfusion, the number of perfused vessels was significant at 5 ± 2.1 (p <0.05). RBC velocities 5 minutes after AoX and 120 minutes after reperfusion were significantly lower than baseline (p <0.05). These results show that, despite increased superior mesenteric artery flow, capillary density and RB of ileal microcirculation after AoX
It shows that the C speed became low. These results suggest that the shunt pathway exists during the "no reflux" phenomenon.

In the next study, it was revealed by using orthogonal polarization spectrum imaging that endotoxin-derived ileal mucosal acidosis is involved in impaired villous microcirculation in pigs.

The following abbreviations will be used. PA-cath. Is a pulmonary artery catheter for measuring mean pulmonary artery pressure and cardiac output. ECG is an electrocardiogram for monitoring heart rate. Flow V.F. "portae" means V.V. measured by an ultrasonic flow probe. The local blood flow over the V. porta. A. fem. -Cath. Is the arterial route of the femoral artery for measuring blood pressure and collecting blood samples. pCO 2 _- sensor and is an optical fiber intestinal mucosa sensor for measuring the local pCO ₂ of the intestinal mucosa wall in ileum continuously. CYTOSCAN ™ A / R is an orthogonal polarization spectral imaging device for imaging and characterizing the gastrointestinal mucosal microcirculation in the ileum.

Introduction: Gastrointestinal mucosa-arterial pCO ₂ -gap (Δr-aPCO ₂ ) blood pressure measurements can be used to monitor gastrointestinal perfusion and indicate mucosal acidosis (Fr.
iddian-Green, R.M. G. , Br. J. Anaesth. 74:59
1-606 (1995); Brinkman, A .; Other, Intensive
Care Med. 24: 542-556 (1988)). However, several different pathophysiological conditions impair local blood flow, oxygen delivery and consumption, and CO ₂ productivity and distorbencies of cellular energy metabolism.
s) and other effects on local pCO ₂ homeostasis (S).
chlictig, R.A. J. J. et al. Crit. Care 11: 51-56 (1
996); Vandermeer, T .; J. Crit. Care Med
23: 1217-1226 (1995). With respect to the role of villus microcirculation in promoting the increase in Δr-aPCO ₂ and ileal mucosal acidosis, because no suitable device was commercially available to image the intestinal mucosal microcirculation in situ,
Long-term studies using large species animal models have not been conducted so far. CYTOSCAN ™ A / R, a new non-invasive method based on orthogonal polarization spectrum (O orthogonal polarization spectral imaging) imaging, allows previously inaccessible visualization, characterization, and recording of this site Became.

Therefore, the purpose of this study was to analyze the effect of villus microcirculation on the development of mucosal intestinal acidosis in hyperenhanced endotoxic shock in pigs and to compare this analysis with local blood flow. It is in. CY for gastrointestinal microcirculation analysis
Consider the applicability and viability of TOSCAN ™ A / R.

Materials and Methods (1) Animal Model: The porcine intracellular toxin model has been described in detail so far (Santak, B. et al., Br. J. Pharmacol 124: 1).
689-1697 (1998)). In this experiment, 16 pigs (mean weight 4
8 kg) was tested. All pigs were anesthetized and mechanically ventilated. A pulmonary artery catheter was inserted through the right jugular vein to measure average pulmonary artery pressure and cardiac output using the thermodilution method. One of the femoral arteries was catheterized for continuous blood pressure recording and blood collection. Lactated Ringer's solution (10 ml /
(kg / hr) was intravenously injected to maintain the body fluid equilibrium.

A midline incision was made and a pre-calibrated Doppler ultrasonic flow probe (Trans
onic Systems, Ithaca, NY) was placed around the portal vein. T2
Blood flow was continuously recorded using a 06 flow meter (Transonic Systems). Fiber optic CO ₂ sensor (Multiparameter Intravascula) connected to a monitor (Paratrend 7; Pfizer)
r Sensor; Pfizer, Karlsruhe) and insert CYT
Ileal fistuloplasty was performed to record in-vivo images of the ileal microcirculation using OSCAN ™ A / R.

[0304]   After the instrument was installed, it was left for 8 hours to stabilize before baseline measurements.

(2) Protocol: 2 of endotoxin (ENX) group (n = 10) and sham group (n = 6)
Animals were randomly assigned to groups. Endotoxin (Escherichia coli lipopolysaccharide B0111: B4 strain [
Difco Laboratories] 20 mg / l in 5% dextrose
Solution) or intravenous injection of saline was started. Mean pulmonary artery pressure (MPAP) is 50 mm
After reaching Hg, the infusion rate of endotoxin was increased until MPAP was adjusted to 35-50 mmHg of moderate pulmonary hypertension. Hydroxyethyl starch (HES)
To stabilize hemodynamics and keep mean arterial pressure (MAP) above 60 mmHg. In addition, 12 hours and 24 hours after the start of the infusion of endotoxin or saline, the hemodynamics of the microcirculation of the ileal mucosa or the measurement of Δr-aPCO ₂ and
A YTOSCAN ™ A / R recording was made. At the end of the measurement, the animals were sacrificed using a potassium chloride injection.

[0306] was calculated Δr-aPCO ₂ by using the difference between the pCO ₂ and pCO ₂ of the artery of the ileal mucosa. At each measurement, using CYTOSCAN ™ A / R, 6 1-minute continuous images (6 vi) of each minute of villus microcirculation from randomly selected ileal mucosal locations.
Deo sequence) was recorded. Count all villi, perfused, anatomically perfused (ie, with both perfused and non-perfused capillaries in the same villus), and unperfused It was classified semi-quantitatively. Due to technical difficulties, the microcirculation of the two animals in the sham group was not recorded.

(3) Statistical analysis: All values shown are mean range and interquartile range, unless otherwise specified. F to change according to rank to make multiple comparisons
riedman repeated measurements analysis and Study
Differences between groups were tested using the nt-Newman-Keuls test. p <0.0
Five was considered significant. Mann-W for non-obstructed samples
Differences between groups were analyzed using hitney-Rank-Sum-Test.

Results: Endotoxin significantly increased mean arterial blood pressure from baseline until 12 and 24 hours after onset of endotoxemia, while maintaining a significant increase in cardiac output. Although there was a decrease, no effect was observed in animals in the sham group (Table 2).

[0309]

[Table 2]

[0310]   #P <0.05 vs baseline; §p <0.05 ETX alone vs Sham

Table 2 Cardiac output of sham and endotoxin treated pigs
Output: CO) time-dependent changes and mean arterial blood pressure (MAP). EXT is the endotoxin group (n = 10): animals that received endotoxin injection after recording baseline data, Sham
Is a sham-treated group (n = 6): animals that have been treated in the same manner as described above, but have received saline injection instead of endotoxin.

The blood flow in the portal vein did not change in both groups, and there was no difference between the groups (FIG. 10). In the endotoxin group, (Δr-aPCO ₂ ) was significantly increased 12 hours and 24 hours after the baseline, and cardiac output was significantly increased, but no effect was observed in the sham group (Fig. 11).

12 and 13 show changes in ileal mucosal microcirculation during the study. In the endotoxin group, at baseline all villi were perfused, but after 12 hours of infusion of endotoxin, the microcirculation was quite heterogeneous. That is, half of the villi were not perfused or were anatopically perfused, whereas in the sham group only 5% of the classified villi were not perfused (p <0.05). Substantially the same pattern was observed in the endotoxin group after 24 hours.

DISCUSSION: The purpose of this study was to identify macrocirculatory during long-term porcine endotoxemia that mimics the clinical manifestations of septic shock in humans.
on) and microcirculation on the development of ileal mucosal acidosis.

Previous studies have shown acidosis of the ileal mucosa during endotoxemia in pigs, but local blood flow was maintained and therefore changes in microcirculation were expected. Unfortunately, to date, there are no commercially available instruments that can be incorporated into experimental settings to image and characterize changes in the small intestinal mucosal microcirculation in situ. Therefore, when compared to microscopic examination of capillaries, CYTOSCAN ™
A / R is the product of choice, is inexpensive and easy to use, and does not require the isolation of the ileum from the animal. Because this technique affects the perfusion of the capillaries, it should be used to avoid over-pressing the probe against the tissue.

An important finding is the overt structuring of the villus microcirculation 12 and 24 hours after developing endotoxemia, respectively. Because of this finding it was advantageous to use low magnification and identify as many villi as possible in a field. Furthermore, a decrease in the villous capillary network was observed during endotoxemia, which was also seen by other experts in a smaller animal model of septic shock and described as a decrease in capillary density. (Farquhar I. et al., J. Surg. Res. 61: 190.
-196 (1996)). Unfortunately, there is no simple, time-sparing device to quantify such changes. It was not possible to measure the flow velocity in the villus due to some intestinal movements that cannot be corrected by the movement control mechanism of the analysis software.

In addition to this, changes in the microcirculation were evident, so a comparison of perfused villi and non-perfused villi was central to the evaluation.

These changes were accompanied by a significant increase in the pCO ₂ gap in the endotoxin group with intramucosal acidosis (Leverve, XM, Intensiv).
e Care Med. 25: 890-892 (1999)). Given the unaltered portal blood flow, ie the availability of well-maintained microcirculatory oxygen, the gradual increase in Δr-aPCO ₂ is mainly due to villus capillary perfusion. This finding argues that a reduction in arterial blood supply of at least 60-70% is required to reduce pCO ₂ -gap in mucosal acidosis Kn
Crit., recently published by ichwitz et al. Care Med. 26: 1
This is in sharp contrast to the data of 550-1557 (1998).

In conclusion, by using orthogonal polarization spectral imaging incorporated into CYTOSCAN ™ A / R, long-term porcine swallowing was achieved even when the portal blood flow in both intestinal walls was well preserved. In an endotoxin-induced hyper shock model, it was clarified that the progression of ileal mucosal acidosis is involved in the major changes in villus microcirculation.

In another aspect of this embodiment, an orthogonal polarization spectral imaging probe can be used during gastrointestinal (GI) surgery to perform colon imaging and characterization during intestinal resection.

In yet another aspect of this embodiment, orthogonal polarization spectral imaging probes can be used to measure the boundaries of cancerous GI tumors and to image and characterize necrotic tissue. Therefore, ablation of affected tissue from the stomach and / or esophageal region is easily accomplished during surgery.

In yet another aspect of this embodiment, orthogonal polarization spectral imaging probes can be used to image and characterize the microcirculation of the rectal mucosa of, for example, inflammatory bowel disease (IBD) patients.

There is strong evidence that the microcirculation is involved in IBD. Orthogonal polarization spectral imaging was used to image and characterize the rectal mucosal microcirculation in 11 healthy volunteers and 25 IBD patients. A few of these (3 healthy, 6 IBD patients) were selected and a well-known image analysis algorithm was applied to quantify the images. In addition, microscopic images were taken using a conventional endoscope during biopsy collection. Detailed imaging of the rectal mucosa, which was imaged by orthogonal polarization spectral imaging, was possible, and the patient was well tolerated. Observed in red blood cell motility and capillary venules. The crypt mucosa where the mucosa is renewed and formed is clearly identified. A characteristic of the normal rectal mucosa is a well defined vascular pattern of elevated crypts surrounded by hexagonal rings of capillaries. A large difference was seen between the rectal microcirculation of healthy volunteers and that of IBD patients. The clear mucosal and capillary structures were completely deformed to the extent that the crypts were no longer discernible, the capillaries were swollen and more tortuous than normal. Polyhedron-recognition algorithm (expressed as Euler-number)
In the image analysis using, a large difference was found in the results between the IBD of the normal group (15.7), the severe group (-434) and the moderate group (-36.3).

In another aspect of this embodiment, orthogonal polarization spectral imaging probes can be used to assess and characterize microcirculation in hard tissues such as liver and pancreas. Other organs within the intestinal tract, such as the kidney, small intestine, gallbladder, mesentery, bladder, diaphragm, stomach and esophagus can be similarly imaged using orthogonal polarization spectral imaging. In addition, using an orthogonal polarization spectrum imaging probe in ileostomy,
The villi within the ileum and villus microcirculation can be visualized.

The purpose of the next study is to validate orthogonal polarization spectral imaging against standard in vivo fluorescence microscopy (IFM) under normal and pathophysiological conditions in rat liver. .

Background: Quantitative analysis of hepatic microcirculation with IMF in animals has enhanced our knowledge of ischemia / reperfusion injury. However, human liver microcirculation cannot be observed due to the size of the instrument and the need for a fluorescent metal for contrast enhancement. Orthogonal polarization spectral imaging is a recently introduced technique that can be used to image microcirculation without the need for fluorescent dyes. This technique is a small and portable device and could potentially be used to test human liver microcirculation in a clinical setting. However, the ability to quantitatively measure microcirculatory parameters needs to be validated before implementation in clinical use.

Method: The livers of Sprague-Dawley rats (n = 9) were removed and images were taken using orthogonal polarization spectral imaging and IMF in the same microcirculation region before and after the induction of lung ischemia for 20 minutes. . Images were videotaped for later off-line analysis using a computer.

Results: Sinusoidal perfusion rate (sinus) using orthogonal polarization spectral imaging.
The ocular perfusion rate, vessel diameter and venule red blood cell velocity can be accurately quantified. The correlation coefficient is significant, and
The tman analysis revealed good agreement from the data obtained using the two methods at baseline as well as during reperfusion.

Results: In rat liver under both physiological and pathophysiological conditions, orthogonal polarization spectral imaging can be used to image liver microcirculation and quantitatively measure microcirculation parameters. Thus, orthogonal polarization spectral imaging may be used to quantitatively measure human liver microcirculation. As can be seen from the correlation parameters and the Brand-Altman analysis, orthogonal polarization spectral imaging and standard methods for such measurements, ie, data obtained from in vivo fluorescence microscopy, showed statistically significant agreement. To be
In contrast to the fluorescence method, orthogonal polarization spectral imaging can be used to image human microcirculation because orthogonal polarization spectral imaging does not require fluorochromes to enhance contrast. Also, because of the small size of the orthogonal polarization spectrum imaging device, it can be readily used during clinical settings, such as during transplant surgery.

One of the most frequent complications after liver transplantation is transplantation failure resulting from severe alteration of the liver microcirculation. Therefore, the ability to assess human hepatic blood flow is of great clinical importance.

Also, to image rat pancreas, the following validation study was performed using orthogonal polarization spectral imaging. Quantification of Human Pancreatic Capillary Perfusion During Transplantation and Surgery Correlates With Laboratory Testing Results and Provides New Findings for Acute Pancreatitis and Pancreatic Ischemia / Reperfusion Injury Pathology You can

Materials and Methods: Eight rats (n = 8) were anesthetized and monitored as described above. After a lateral incision, the short gastric artery was tied and dissected. A larger rope was then removed from the larger curvature of the stomach to access the pancreas. The microsurgical technique carried out has already been described in detail (Hoffman et al., Res. Exp. M.
ed. (Berl.) 195: 125-144 (1995)). After indirect mobilization, the pancreas and attached spleen were gently excised on a movable stage, covered with a thin plastic foil and kept in the air from drying out. To image the capillary network, plasmamaker's FITC (fluorescein isothiocyanate, Sigma-Aldrich Chemie, Deisenhofe).
n, Germany) and 0.3 ml of 5% bovine serum albumin labeled with IV was injected intravenously. To compare capillary perfusion using the two techniques, the same settings as for liver were used. In each animal, the same 6 microcirculation areas were evaluated using IVM and orthogonal polarization spectral imaging. Functional capillary density, defined as the length of red blood cell perfused per observation area (cm / cm ² ), was analyzed by off-line computer evaluation of images recorded using the CapImage ™ computer program. .

Results and Discussion: A typical honeycomb-like network of pancreatic capillaries is CYTOSCAN.
It can be visualized using T ™ A / R. Quantitative analysis of orthogonal polarization spectral imaging shows an average functional capillary density of 385.4 ± 45 cm / c
It was found to be m ² , which is in agreement with the previous test using the fluorescence microscope. As can be seen from the Brand-Altman analysis, these values are
On average, it was less than only 2.2% (8.7 cm / cm ² ) obtained from IVM images.

Also, the 95% confidence interval was only two data points, and the regression line coincided with the median. Thus, it is clear that there is a very good agreement between the two methods (IVM and orthogonal polarization spectral imaging) for measuring FCD of rat pancreas under baseline conditions. IVM and CYTOSCAN (
The contrast and quality of the images obtained with the ™ A / R were qualitatively the same, so the time required for image analysis was comparable for both techniques.

Therefore, orthogonal polarization spectral imaging is a suitable instrument for the quantitative analysis of perfusion of pancreatic capillaries during baseline. The CYTOSCAN ™ A / R is a useful device for the scientific and clinical evaluation of pancreatic microcirculation during human surgery and transplantation. Pancreatic perfusion injury indicates the degree of damage to organs after ischemia (H
offman et al., Res. Exp. Med. (Berl.) 195: 125-4.
4 (1995); Hoffman et al., Microsc. Res. Tech. Three
7: 557-571 (1997)), measurement of FCD is a useful diagnostic parameter for monitoring pancreatic status.

Ophthalmic Application In this embodiment of the invention, a standard or high contrast orthogonal polarization spectral imaging probe was used in vivo in the field of ophthalmology.

In each of the following aspects of this embodiment, capillary density, blood vessels (and microvessels)
Morphology, blood vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, blood vessel diameter, leukocyte-endothelial cell interaction, vasodynamics (vasomotor etc.), functional blood vessel density, functional capillary density, blood flow , The area-to-ambient ratio, hemoglobin concentration, and hematocrit value may be quantitatively measured. Preferably two or more parameters are measured.

According to one aspect of the invention, a high contrast orthogonal polarization spectral imaging probe is used to image and characterize the microcirculation inside the eye, particularly the retinal microcirculation. In one embodiment, orthogonal polarization spectral imaging can be utilized in a non-contact method. In this embodiment, the light of the device is set so that the relaxed eye (focused in the infinite region) is in focus and images the orthogonally polarized spectral imaging rays to the retina. Alternatively, the imaging probe can be used in direct contact, as in the case of using a special corneal contact lens for diagnosis. Such an imaging device can be used for diagnosis and therapy.

In a preferred embodiment, a fundus imaging device, a laser that delivers a light beam of the appropriate wavelength to treat a particular intraocular region, and orthogonal polarization spectral imaging are functionally integrated. In addition to diagnostics and therapeutics, devices of this type can be used to provide information regarding the effectiveness of various agents on the circulatory system of the area being tested.

In order to make an accurate measurement, it is necessary to once again easily find the exact area (point) in the eye to be measured and treated or examined and reexamined. Therefore, centering, tracking and grid devices may be incorporated into the system. This tracking device is important in order to allow the area to be evaluated and treated to always be in the same position relative to the device, despite good eye movements.

Further, in optimizing such a device, it is important to incorporate a recording device that displays the sizes of blood vessels and lesions regardless of the magnification. Currently, when used externally as a direct contact device, the area imaged by this device is about 0.5 mm. Dioptic p of the eye to be examined
Depending on the power and power of the corneal contact lens when used, the size of the intraocular region may vary somewhat.

Image multiplication (im) so that the image can be defined with a minimum of light of the desired wavelength.
Age intensification) can be incorporated. Ideally, this image is color and real-time. This device allows the rapid diagnosis of pathological problems of the microcirculation. It is possible to take real-time videos and photos for permanent recording. Laser or other types of rapid treatments such as photochemotherapy of diseases can be performed in real time.

The optimization of this device includes some or all of the following features. That is, this includes a centering device, a method of visualizing the same point or region, a device that tracks eye movements and is always at the same position, a storage device that displays the size of a blood vessel at each magnification, that is, a calibration device, An image enhancement device and a magnification means for enlarging the size of an image. Color images are arbitrary.

In another aspect of this embodiment, orthogonal polarization spectral imaging techniques can be used to diagnose macular degeneration, or retinal disease (retinopathy) and glaucoma.

In another aspect of this embodiment, orthogonal polarization spectral imaging techniques can be used to image changes in the optic disc, retina, sclera, and vitreous humor.

In another aspect of this embodiment, orthogonal polarization spectral imaging techniques are used for early diagnosis and treatment of diabetes by looking at changes and differences in the ocular microcirculation, particularly the ocular sclera and / or aqueous humor. be able to.

In another aspect of this embodiment, orthogonal polarization spectral imaging techniques can be used to analyze the eye during internal carotid artery (ICA) surgery. The purpose of the next study is intraoperative imaging and quantitative analysis of the microcirculation of the internal carotid artery (ICA) and external carotid artery (ECA) blood flow regions using orthogonal polarization spectral imaging.

Materials and Methods: CYTOSCAN ™ A / R was used to image and quantify the anterior (scleral) microvascular perfusion of 15 eyes during surgery. FCD
, Capillary diameter, and blood flow velocity were measured. (I) under controlled conditions, (II) ECA
During ischemia, (III) ICA ischemia, (IV) occlusion perfusion, (V) after removal of occlusion (ischemia), or after ECA reperfusion, and (VI) during ICA, (VII)
5 minutes after reperfusion and (8) 20 minutes after reperfusion, I on the ipsilateral side of the microvascular network
DCD, capillary diameter and blood flow velocity were measured in the CA occlusion area and the opposite side.

Results: Transient ischemia during shunt implantation resulted in a significant reduction of about 50% in perfusion of nutritive ocular capillaries compared to preischemic baseline values. . Furthermore, this short period of ischemia was associated with a significant decrease in capillary density. This change resulted in ICA and ECA when compared to simple short ECA ischemia.
Was even more pronounced after clamping. On the other hand, the characteristics of reperfusion of the shunt of ICA after ischemia were characterized by FCD, capillary diameter and R compared with those of I and III.
The BC rate was significantly increased (p <0.05, paired sample t-test). The emergence of transient ischemia during shunt removal resulted in the same lack of perfusion as during shunt implantation. On the other hand, rapid reperfusion (VI) after ischemia was associated with a significant increase in FCD, capillary diameter and RBC velocity (p <0.05). In addition, increased perfusion may be achieved by reperfusion during shunt perfusion during the reperfusion phase and in the contralateral eye.
Observed although not obvious both after the minutes.

Conclusion: Orthogonal polarization spectral imaging allows us to first directly image and quantify the ocular microcirculation. This allowed rapid and reliable intraoperative ischemia monitoring (lack of perfusion), where reperfusion altered cerebral microcirculation during carotid remodeling. Disorders of the eye microcirculation, such as capillary congestion and occlusion,
We show that ischemia-induced microcirculatory disturbances develop immediately, as well as oscillating blood flow that occurs shortly after short ICA and ECA ischemia. Proper maintenance of perfusion in the ICA perfusion area during remodeling significantly improves and protects ipsilateral and contralateral ocular microcirculation.

Pregnancy Application In this embodiment of the invention, a female is used to monitor microcirculatory function using a orthogonal polarization spectral imaging probe during normal pregnancy and during pregnancy with complications.

In each of the following aspects of the invention, capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter, leukocyte-endothelial cell interaction Quantitatively measured one or more parameters such as action, vasodynamics (vasomotor etc.), functional vessel density, functional capillary density, blood flow, area-to-surround ratio, hemoglobin concentration, and hematocrit value Good. Preferably two or more parameters are measured.

In one aspect of the invention, standard or high contrast orthogonal polarization spectral imaging probes are used to monitor women with preeclampsia (PE). As used herein, "preeclampsia" is a complication of current or recent pregnancy, characterized by hypertension with proteinuria and / or edema. Preeclampsia is a systemic disease involving endothelial cell dysfunction. To investigate how PE affects skin microcirculation, microvascular function was examined using orthogonal polarization spectral imaging.

The purpose of the next study is to examine in vivo microcirculatory function during pregnancy and during pregnancy with preeclampsia.

Summary: For 10 pregnant women with preeclampsia and 10 pregnant women with normotension,
The skin microcirculation was examined. Resting red blood cell velocity (rCBV) and local sympathetic venous-arteriole reflex (VAR) were studied using orthogonal polarization spectral imaging while testing nail fold vein occlusion using orthogonal polarization spectral imaging. Tested. A laser Doppler flux meter was used to evaluate the function of skin microcirculation during venous occlusion, arterial occlusion and at rest.

As a result of using the laser Doppler flux meter, no difference was observed between the normotensive group and the preeclampsia group. In general, there was no difference in absolute velocity between the two groups at rest and during venous occlusion, but using orthogonal polarization spectral imaging, eclampsia was compared to 84% (74-89) in the control group. Decrease in erythrocyte velocity was observed after vein occlusion in both groups with presymptomatic symptoms (58% (40-94) (mean and range)
) (P = 0.003).

In this study, the use of orthogonal polarization spectral imaging revealed a venous-arteriole reflex that was undetectable by a laser Doppler fluxmeter. Orthogonal polarization spectral imaging also makes it possible to test the effects of preeclampsia on other organ beds.

Introduction: Maternal cardiovascular system decreases peripheral vascular resistance and systemic blood pressure in response to pregnancy,
Blood volume and cardiac output rise to normal blood pressure (NT). Such adaptations are needed to meet the requirements of normal pregnancy. However, this response does not work properly in pregnancies with pre-eclampsia (PE). The pathophysiological findings of PE include hypertension,
There is proteinuria, decreased plasma volume, increased peripheral vascular resistance, vasoconstriction, and decreased organ perfusion (Roberts, JM and Redman, CWG, La.
ncet 341: 1447-1451 (1993); Bosio, P .; M.
Obset. Gynacol. 94: 978-984 (1999)). The coagulation cascade begins, an imbalance in the prostacyclin / thromboxane ratio results in platelet aggregation, and increased vasoconstriction (Walsh, S.W.
, Am. J. Obstet. Gynocol. 152: 355-340 (198
5 years)). Collected evidence suggests that endothelial cell dysfunction is common in PE (Roberts, JM and Redman, C).
． W. G. , Lancet 341: 1447-1451 (1993)). The cause of endothelial dysfunction is difficult to detect, but it has been proposed that it is caused by blood products from the placenta, which has insufficient blood flow (Roberts, JM and R.
edman, C.I. W. G. , Lancet 341: 1447-1451 (19).
1993)).

In PE, vascular dysfunction was observed in arteries isolated from various organs (Aa
lkjaer, C.I. Clin. Sci. 69: 477-482 (1985); MacCarthy, A .; L. Am. J. Obstet. Gynaco
l. 168: 1323-1330 (1993); Vedernikov, Y .;
Others, Semin. Perinatol. 23: 34-44 (1999). NT
PE showed endothelial cell-dependent swelling when compared to (MacCarth
y, A. L. Am. J. Obstet. Gynacol. 168: 1323
-1330 (1993); Knock, G .; A. And Poston, L, A
m. J. Obstet. Gynacol. 175: 1668-1674 (199
6 years)), vascular smooth muscle cells of PE women have increased sensitivity to pressor drugs (G).
ant, N.N. F. J. J. et al. Clin. Invest. 52: 2682-2689
(1973)). Further, in PE, an increase in sympathetic nerve activity was observed in blood vessels of skeletal muscle in vivo (Schobel, HP et al., N. Engl. J. Med.
335: 1480-1485 (1996)). Such findings indicate that endothelial dysfunction is not the only cause of altered PE vascular responses.

However, most of these tests have been performed in vitro, and it is difficult to test vascular function in vivo, so in vivo evaluation of vascular function has not been performed so often. It was In vivo studies with a laser Doppler fluxmeter (LDF) using iontophoresis of vasoactive agents showed no changes in the vascular function of the skin microcirculation in PE women (Eneroth-Grimfors, H. et al., Brit. J).
． Obstet. Gynacol. 100: 469-471 (1993)).
The response after arterial occlusion (congestive response, endothelium-dependent vasodilation) tested with LDF was increased in PE hand skin compared to NT, but not in arm skin. Not observed (Beinder, E. and Lang, N., Geburt
shu Frauenheil 54: 268-272 (1994)).
The vascular response to local cooling was vasoconstrictive and was significantly higher in both areas of PE (Beinder, E. and Lang, N., Gebu).
rtshu Frauenheil 54: 268-272 (1994).
). However, using a capillary microscope on the skin of the nailfold showed no difference in hyperemic response among PE women (Rosen, L. et al., Int. J. Micro).
circle. Clin. Exp. 8: 237-244 (1989)), using high-resolution and Doppler ultrasonography, there was a change in hyperemic response in the brachial artery of PE women compared to NT. Seen (Yoshida)
, A. Et al., Hypertension 31: 1200 (1998); Vei.
lle, J .; C. J. J. et al. Soc. Gynacol. Invest. 5: 38-
43 (1998)).

Microcirculatory dysfunction is described by Rosen, L. et al. Proven by others (Int. J. Mi
crocirc. Clin. Exp. 9: 257-266 (1990)), a response to venous occlusion produces a venous-arteriole reflex (a local sympathetic reflex leading to an endothelium-dependent vasoconstrictor response), using a capillary microscope. When tested with PE, the skin of PE showed a decrease. However, as this study was not repeated, the cause of venous-arterioleopathy with increased sympathetic reflexes remains unknown. This is the reason why this test was not conducted. This study also tested a hyperemic response, as PE produced opposite results with respect to vascular response to arterial occlusion using different methods and different vascular beds.

Orthogonal polarization spectral imaging was used to observe the vascular function of PE skin microcirculation in vivo in a simple and non-invasive manner. To implant a small, portable device, this technique allows in vivo observation of human organs as well as skin microcirculation at bedside and during surgery (Groner, W. et al., Natu.
re in Medicine 5: 1209-1212 (1999)). This technique was verified by comparing it with a conventional, concave capillary microscope. A laser Doppler flux meter (LDF) was used to compare these results with other methods for measuring microcirculatory function in vivo. Venous-arteriole reflex (VAR) using both methods
Was tested to control vascular function by the sympathetic nervous system (Hen
riksen, O .; , Acta Physiol. Scan 143: 33-
39 (1991)). The control of endothelium-dependent vasoconstriction was tested for hyperemic response using LDF (Ostergren, J. and Fagrell, B.
Int. J. Clin. Microcirc. Clin. Exp. 5: 37-5
1 (1986)).

Materials and Methods: (1) Subjects: After written informed consent, 10 PE women and 10 pregnant women with normotension (control group) were enrolled in the roster. PE is defined as 20 weeks pregnant with diastolic blood pressure> 90 mmHg and proteinuria> 300 mg / 2
4 hours, or urinalysis value is ++ / ++++ (National High Blood
d Pressure Education Program Working
Group Report on High Blood Pressure
in Pregnancy, Am. J. Obstet. Gynacol. 16
3: 1689-1712 (1990)). Diastolic blood pressure was measured using a Karottoff V. Six of the PE women were taking the drug, three using infedipine (Adalat, 3-4 × 10 mg) and three using methyldopa (Aldomet, 3 × 250 or 500 mg).
Women from the control group were recruited by advertisement in the obstetric outpatient department of the hospital. None of the women in the control group used the drug. Women who were diagnosed with diabetes, hypertension and Raynaud's disease before pregnancy, or who had a fever were excluded from this study. The characteristics of the test subject are shown in Table 3.

(2) Method: Laser Doppler flux meter (LDF) (Perifleu
x40001, PF408 standard probe time constant 0.2 seconds, Perimed,
The total blood flow in the skin was examined using Sweden) (Stern, MD, et al.,
Am. J. Physiol. 232: H441-H448 (1977)). Two probes were attached to the medulla of the finger with double-sided adhesive tape using a probe holder (Perimed, Sweden). A laser beam having a wavelength of 780 nm is introduced into the skin through an optical fiber, and this beam penetrates the skin to a depth of 1 to 1.5 mm,
Some were reflected. When moving an object (red blood cells) causes backscattering, the light beam undergoes a frequency shift proportional to the velocity and number of moving objects, and is given in arbitrary units (
Bolts) and is called flux. Data were recorded and analyzed off-line (AcqKnowledge III and MP 100 WSW,
Biopac System Inc. , Santa Barbara, California).

Orthogonal Polarization Spectral Imaging (Version 2, Cytometrics,
Nailfold microcirculation was tested using Philadelphia PA). This device utilizes green polarized light that has passed through an optical waveguide and is placed on the organ to be measured. By cross-polarizing the reflected light, all surface reflections are removed and a very sharp image can be recorded (Figure 14). A 75 W precision halogen lamp with a wavelength of 540 nm was used as a light source. A movable objective lens with a magnification of 5 times was used for the analysis, which gave a final on-screen magnification of 325 times. All images were recorded using a digital video recorder (Sony DSR-20P) and off-line analysis of the images was performed with a software program (CapImage ™, Dr. Zein).
tl Software engineering, Heidelberg, Germany) (Klyscz, T. et al., Biomed Tech (Berl) 42).
: 168-175 (1997)).

(3) Measurement method: Using LDF to measure VAR, the average flow rate for 5 minutes (
RF) was measured for 2 minutes and mean flow of venous occlusion after 1 minute of occlusion (VOF) for 2 minutes. In addition, the maximum flux (PF) 1 minute after arterial occlusion and the time until PF were measured. The increase and decrease of flux normalized to the resting value ((RF-V
It was calculated as OF) / RF) and ((PF-RF) / RF), and expressed as%. The remaining LDF (biologically zero) during arterial occlusion (Caspary, L. et al., In
t. J. Microcirc. Clin. Exp. 7: 367-371 (199
8 years)) was measured, which was subtracted from all other measured LDF values. At this time, since the laser Doppler device was not available, LDF measurement was performed on all but 2 females in the control group.

Capillary blood cell velocity (rC at rest) using orthogonal polarization spectral imaging
BV) was measured as the average velocity for 30 seconds, and VAR was measured as the average velocity for 30 seconds after venous occlusion (roCBV). Both speeds are line-shift
It was measured using the diagram method (Klyscz, T. et al., Biomed T.
ech (Berl) 42: 168-175 (1997)). The decrease in red blood cell velocity during venous occlusion, normalized to the resting value, was calculated and expressed as% ((rCBV-v
oCBV) / rCBV). In all subjects, 3 capillaries were tested and the median was used for comparison.

(4) Protocol: All subjects were asked not to drink caffeine-containing beverages for 2 hours prior to the study. 15 tests in an air-conditioned room at 23-25 ° C
It was performed after acclimatization for a minute. The subject is on the dominant side (on the
r domining side) Lying in the lateral decubitus position, the arms were slightly bent on the pillow at the position of the heart. A sphygmomanometer band was placed around the upper arm. First, orthogonal polarization spectral imaging was used to test the capillaries of the fourth finger, different from the dominant hand, but the device was placed just above the nail fold surface. The fingers were embedded in a lump of clay to stabilize the hand and a drop of paraffin oil was dropped on the nail fold to allow more light to pass through the hand. Digital thermometer taped to the skin near the nail fold (K
Skin temperature was continuously measured using an ethley 871 A). Three well imaged capillaries were selected and measured from the capillaries of the nail fold. Each capillary was recorded for 2 minutes to measure rCBV, after which the sphygmomanometer band was expanded to 50 mmHg for 2 minutes to measure voCBV.

After recording these, measurement was performed using LDF. Two probes were attached to the middle phalanx of the same finger, one on the palm side and the other on the dorsal side. After resting for a minimum of 5 minutes to measure RF, the sphygmomanometer band was inflated to 50 mm Hg and VOF was measured for 3 minutes. The sphygmomanometer band was then inflated to 200 mmHg to occlude the artery to measure biological zero and 1 minute later, air was removed. Flux was recorded for an additional 2 minutes to measure PF and time to peak. Blood pressure was measured at the end of the test.

(5) Statistics: Measured values are median with range unless otherwise specified.
with range), and the p value was calculated using the Mann-Whitney U test and the Wilcoxon sign rank test. A p-value <0.05 was considered statistically significant.

Results: There were no differences in patient characteristics between the groups except for blood pressure (Table 3). The results of the laser Doppler flux meter are shown in Table 4 and FIGS. 15A and 15B. There were no significant differences in palm or dorsal digit changes in flux or rate during venous occlusion or after arterial occlusion among PE patients at rest or controls. In the control group, no significant changes in flux were observed during palmar vein occlusion, whereas in the PE patient group, dorsal vein occlusion did not cause a significant reaction.

Analysis of capillary erythroid cell kinetics obtained from orthogonal polarization spectral imaging images showed no difference between rCBV and voCBV between the two groups. However, in PE women, the reduction in the percentage of blood cells during vein occlusion was significantly smaller than in the control group, 58% (40-94) versus 84% (74-89) (p = 0.
． 003, see FIG. 16).

Comments: This study confirmed PE using orthogonal polarization spectral imaging.
It supports the VAR disorder in. In addition, Rosen et al. Described above discovered this reflex disorder using a capillary microscope. VAR is one of the reflexes that causes arteriole constriction to maintain arterial pressure in the standing position. This is a local sympathetic axon reflex (Henriksen, O., Acta Physiol. Sc.
and. 143: 33-39 (1991); Vissing, S .; F. Other, A
cta Physiol. Scand. 159: 131-138 (1997)
), The role of endothelial cells in VAR remains unclear (Henrikse
n, O. , Acta Physiol. Scand. 143: 33-39 (19
1991)). This VAR is derived from the dilation of the vein that causes arteriolar occlusion, resulting in diminished blood flow. In addition, this reflex can be induced by inflating the transmural pressure to 25 mmHg or more by inflating the sphygmomanometer band around the arm to cause vein occlusion.

Reflex disturbances cannot be detected using LDF. This difference is similar to the case of using orthogonal polarization spectrum imaging. In LDF, not only blood flow in skin capillaries for nutrition but also blood flow at deeper positions in the subcapillaries (for thermoregulation) are measured. Explained by the fact. It has been suggested that the VAR of such vessels is not very clear (Tooke, JE et al., Int.
． J. Microcirc. Clin. Exp. 2: 277-284 (1983)
Year)). This may explain why there is no difference in VAR with LDF as opposed to the results with orthogonal polarization spectral imaging.

Further, no blood vessel was observed between PE and NT in the hyperemic reaction after arterial occlusion.
This hyperemic reaction is highly endothelium-dependent and is a vasodilator response to arterial occlusion. When the sphygmomanometer band is released, blood flow starts again, rises in a few seconds until the maximum velocity is reached, and returns to the resting value.

Previous studies have shown that the brachial artery in PE women has impaired hyperemic response compared to NT (Yoshida, A. et al., Hypert).
ENSION 31: 1200 (1998); Veille, J .; C. J. J. et al.
Soc. Gynacol. Invest. 5: 38-43 (1998)). Such results cannot be found in the skin microcirculation when using LDF. LDFs are often used extensively in assessing vascular disease and diabetes (Schaubauer, A.M.A. and Roke, TW, Mayo.
Clin. Proc. 69: 564-574 (1994)). However, there are significant physiological changes in flux, which are reproductive (reproductive).
Ivity) (Schabauer, AM. A. and Look.
e, T. W. Mayo Clin. Proc. 69: 564-574 (199
4 years)). This variability is suppressed with some indicators and hyperemia that the subject controls in order to cause VAR as well as vein occlusion.

In PE, sympathetic hyperactivity was found in skeletal muscle vessels. It plays a role in enhancing vasoconstrictor activity of sympathetic nerves and increasing peripheral vascular resistance (Schob
el, H .; P. N.N. Engl. J. Med. 335: 1480-1485 (
1996)). Therefore, the excessive activity of the sympathetic nerve can explain the reaction disorder to the venous occlusion. In PE, the greater the arteriolar contraction, the greater the vasoconstriction disorder due to the stimulation of the sympathetic nerve. Methyldopa is a centrally acting drug that reduces sympathetic outflow and is the first-line drug for long-term control of PE blood pressure, suggesting that it is actually involved in the sympathetic nervous system. Another hypothesis is the excessive activity of the sympathetic nerves that suppresses smooth muscle cell receptors' response to stimulation, resulting in weak vasoconstriction when stimulated.
In heart disease, the down-regulating effect of myocardial α ₁ and α ₂ adenoreceptors (down-reg)
However, this condition is associated with an increase in the activity of sympathetic nerves (Brodde, OC, Pharmacol. Rev. 43:20).
3-242 (1991)). It is unclear what happens to the receptors in this circulation during excessive activity of the sympathetic nerve. However, it cannot be ruled out that VAR impairment occurs as a result of functional or structural changes in PE microcirculation and contributes to vascular dysfunction independent of the sympathetic nervous system.

In this study, skin microcirculation was examined using orthogonal polarization spectral imaging and LDF. As a result, LDF to assess vascular function during pregnancy
Orthogonal polarization spectral imaging provided a more sensitive technique compared to.
Vein-arteriole reflex defects in PE women were detected using orthogonal polarization spectral imaging, but not with LDF. The results suggest that sympathetic vasoconstriction is present in the skin microcirculation. The ability to use orthogonal polarization spectral imaging to observe other organ beds can define vascular dysfunction in PE.

[0379]

[Table 3]

Results are shown as median and range. P <0.001 in comparison with the control group. Mann-W
hitney U test.

[0380]

[Table 4]

[0382]   Results are shown as median and range.

^* Significantly different values compared to RF.

In another aspect of this embodiment, a standard or high contrast orthogonal polarization spectral imaging probe is used to monitor intact placental microcirculation in preeclampsia / fetal growth retardation.

It is well known that fetal nutrition and oxidation are dependent on well-developed and capillary-formed, terminal villi of the placenta. Fetal growth retardation is a common symptom in preeclampsia. Capillary hypoplasia (hypohyperplasia) using a scanning electron microscope.
It has been clarified that pocapillarisation is present in terminal villi (Habashi S. et al., Placenta 4: 41-56 (1).
983)).

Ex Vivo Studies Using Orthogonal Polarization Spectral Imaging of the Surface of Maternal Placental Sites from 10 Cases of Normal Blood Pressure with Pregnant Suitable Neonates and 10 Cases of Preeclampsia with Small Neonates During Pregnancy did. The purpose of this study is to determine whether images of microvessels are available and whether the terminal villi of the latter group are subcapillary compared to the first group.

Within 10 minutes after placental removal, umbilical arteries and veins were cannulated and perfused with a mixed solution of 50 ml Ringer glucose, 5 ml heparin, and 50 ml Custodiol. Next, 5 ml of ink and synthetic barita 10-1
Perfusion with 3 ml. An orthogonal polarization spectral imaging image of the terminal villi was found to be comparable to the images obtained by SEM showing hypocapillary formation, normal capillary formation or hypercapillized. Therefore, orthogonal polarization spectral imaging adds a new and versatile feature for examining the nature of fetal growth retardation (ie, due to heredity or due to placental defects such as preeclampsia).

Neonatal Monitoring In this embodiment of the invention, orthogonal polarization spectral imaging is used for neonatal monitoring.

In each of the following aspects of this embodiment, capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter,
One or more parameters such as leukocyte-endothelial cell interaction, vasodynamics (such as vasomotor movements), functional blood vessel density, functional capillary density, blood flow, area-to-ambient ratio, hemoglobin concentration, and hematocrit value It may be measured quantitatively. Preferably two or more parameters are measured.

In one aspect of this embodiment, changes in neonatal microcirculation during different disease states such as sepsis and meningitis are quantitatively measured using standard or high contrast orthogonal polarization spectral imaging probes. . Neonatal hemoglobin levels can be monitored non-invasively.

In another aspect of this embodiment, a standard or high contrast orthogonal polarization spectral imaging probe is used to measure changes in the neonatal microcirculation before, during, and after a shock (bleeding or sepsis) condition. To be done.

[0390]   The application example described above is also useful for monitoring preterm infants.

High Altitude Testing / Space Physiology In this embodiment of the invention, the orthogonal polarization spectral imaging probe can be used for testing at high altitude or can be used to study space physiology.

Capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter, leukocyte-endothelial cell interaction, vasodynamic (
One or more parameters such as vascular motion), functional blood vessel density, functional capillary density, blood flow, area-to-surround ratio, hemoglobin concentration, and hematocrit value may be quantitatively measured. Preferably two or more parameters are measured.

In one aspect of this embodiment, changes in microcirculation at high altitudes are observed to test, diagnose and / or treat altitude sickness using standard or high contrast orthogonal polarization spectral imaging probes. And will be evaluated. Changes in the microcirculation include
It includes an increase in the number of blood vessels, an increase in the number of WBCs found in such blood vessels, as well as leukocyte-endothelial cell interaction. Further, it can be judged that the change in Hb is a result of high altitude.

Application in Clinical Blood Rheology In this embodiment of the invention, the rheology of blood is tested using an orthogonal polarization spectral imaging probe.

Capillary density, blood vessel (and microvessel) morphology, blood vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, blood vessel diameter, leukocyte-endothelial cell interaction, vascular dynamics (
One or more parameters such as vascular motion), functional blood vessel density, functional capillary density, blood flow, area-to-surround ratio, hemoglobin concentration, and hematocrit value may be quantitatively measured. Preferably two or more parameters are measured.

In one aspect of this embodiment, a standard or high-contrast orthogonal polarization spectral imaging probe is used to cause excess blood, either due to a number of clinical disorders such as sickle cell anemia, or in malaria-infected patients. Changes in the patient's blood rheology due to coagulation are observed and evaluated. This information can be used to test for diseases that alter blood coagulation, ie red blood cell coagulation, or alter blood rheology.

Application in Critical Care or Intensive Care In this embodiment of the invention, an orthogonally polarized spectral imaging probe is used for critical care or intensive care in vivo.

In another aspect of this embodiment, a standard or high-contrast orthogonal polarization spectral imaging probe provides a tongue of a critically ill patient to diagnose, treat, or prevent sepsis and / or shock (hemorrhagic or septic). Used as a lower monitoring device. It has been found that there is either a dramatic reduction in microcirculatory blood flow in the case of sepsis and / or shock, or no microcirculatory blood flow. Orthogonal polarization spectral imaging techniques can be used to prevent these two conditions in critically ill patients, or to try several therapies that alter outcome. Orthogonal polarization spectral imaging can also be used to monitor the effectiveness of treatment.

In this patient population, capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter, leukocyte-endothelial cell interaction, vasodynamics ( One or more parameters such as vascular motion), functional blood vessel density, functional capillary density, blood flow, area-to-surround ratio, hemoglobin concentration, and hematocrit value may be quantitatively measured. Preferably two or more parameters are measured.

In the next study, orthogonal polarization spectral imaging will be used on patients undergoing intensive care.

Introduction: Cardiovascular shock is one of the key challenges in intensive care, whatever the cause. Despite improved deep cardiovascular changes, multiple organ failure often occurs. Even when global blood flow changes appear to be stabilized by solutions and vasoactive agents, large changes in microcirculation may persist and contribute to the development of multiple organ failure (Garrison). , RN et al., Ann. Su
rg. 227: 851-860 (1998)).

Microvascular changes are a direct cause of shock conditions that contribute to a significant loss of blood supply, such as that seen in severe heart disease or hypovolemia. In septic shock, this change is complex and problematic. Using an in-vivo microscope under experimental conditions, several researchers (Lam, CJ, et al., Clin. Invest.
94: 2077-2083 (1994); Piper. R. D., et al., Am. J.
Respir. Crit. Care Med. 157: 129-134 (199
8 years); Farquhar, I .; J. J. et al. Surg. Res. 61: 190-1
96 (1996); Wang, P .; , Am. J. Physiol. 263: G
38-G43 (1992); Madorin, W .; S. Other, Crit Car
e Med 27: 394-400 (1999); Pannen, B .; H. J
． Am. J. Physiol. 272: H2736-H2745 (1997
)) Observed that the heterogeneity of blood flow increased after endotoxin administration. Sepsis Increases the number of capillaries that are not perfused in a short period of time, which may be due to activation of various cellular elements: leukocytes and platelets aggregate and erythrocytes become more rigid (McCu
Skey, R .; S. Others, Cardiovasc. Res. 32: 752-763
(1996); Piper, R .; D., et al., Am. J. Respir. Crit.
Care. Med. 154: 931-937 (1996)). Some investigators have demonstrated that sepsis is associated with a heterogeneity of blood flow and reduced oxygen extraction capacity (Walley, KR, J. Appl. Physiol. 81:88.
5-894 (1996); Humer, M .; F. J. J. et al. Appl. Phys
iol. 81: 895-904 (1996); Drazenovic, R .; J. J. et al. Appl. Physiol. 72: 259-265 (1992)). Also, because of the short blood flow, focal ischemia / reperfusion injury occurs in the regions angiated by these capillaries. In the case of patients, there is still no evidence of involvement in microcirculatory disorders. Access to the human microcirculation has long been restricted to the nail fold. In fact, the size of the in-vivo microscope and the depth of the capillaries precluded its use elsewhere. Orthogonal polarization spectral imaging is based on reflectance spectroscopy, and in recent years it has become possible to examine mucosal sites in the clinical field. In critically ill patients, the usual sites available for noninvasive microcirculation imaging are the lips, sublingual region,
And sometimes it is the site of enterostomy. Although technically possible, examination of the rectal and vaginal mucosa is difficult. Orthogonal polarization spectrum imaging technology is CYTO
SCAN ™ A / R (Cytometrics, PA, Philadelphia)
) Was incorporated into. In this trial, CYTOSCAN ™ A for intensive care
Report the results using / R.

PARA-sublingual region: Access to the sublingual region is crucial, but the key question is whether this region represents a microcirculatory change in circulatory disorders. . In fact, there is some evidence to support that the sublingual region is involved in the early stages of shock. Recently Weli and co-workers have shown that the sublingual PCO ₂ (PslCO ₂ ), which represents the balance of oxygen supply and demand in the sublingual region, is
It was reported that the shock level increased markedly in the shock state (Nakagawa Y et al., Am.
J Respir Crit Care Med. 157: (6 Pt 1) 1
838-1843 (1998)). PslCO ₂ also affects blood pressure and is directly involved in arterial lactate, a marker of tissue hypoxia. Psl
CO ₂ was also very sensitive to therapeutic intervention.

Imaging the sublingual microcirculation is very effective. Quantitative estimation is not practically feasible in critically ill patients due to motion artifacts, as compared to animal samples, where quantitative measurement of blood flow in the microcirculation is easy. Therefore, a semi-quantitative analysis was developed and a capillary index was calculated using the number of blood vessels that intersect the three vertical and three horizontal lines traced on the screen. Such blood vessels are large blood vessels (> 20 μm, mainly small veins) and small blood vessels (<20 μm).
, Mainly capillaries). 20-second continuous image (video s)
sequence) to estimate the blood flow velocity, and normal or fast blood flow, low blood flow,
It was classified into 4 groups with intermediate blood flow and no blood flow. In 3 healthy volunteers, sublingual neovascularization consisted of large and small vessels at a rate of 40/60% (
(Fig. 17). Almost all blood vessels (95%) were well perfused, but no blood flow was observed in only a few capillaries (5%).

A very different pattern was observed in 21 patients with septic shock. The number of perfused vessels was significantly reduced (78%, p <0.05), especially in small vessels (39%, p <0.01). In such vessels, it was observed that the number of capillary results was increased and the blood flow was either transient or absent. A typical example is shown in FIG. In 8 cases where blood flow was low due to cardiac shock, the index of the blood vessels of the capillaries decreased, and erythrocyte clusters were observed in the venules (FIG. 19). It was also observed that in such patients the number of perfused vessels was reduced, especially in small vessels (7).
5%, p <0.05).

These preliminary results suggest that large changes in microcirculation can be observed in various shock conditions. These changes are due to other groups (N
akagawa Y. It may be responsible for the elevated PslCO ₂ reported by et al., But further testing is needed to confirm this hypothesis.

Other Areas of Interest: The inside of the mouth, such as the lips and cheeks, can also be examined. In many cases, the lip microcirculation is better preserved, even in severe shock conditions. In 11 patients with septic shock and cardiac shock,
It was observed that 95% of all small vessels and all large vessels were perfused in the lip area, whereas only 32% of all small vessels and all large vessels were perfused in the sublingual area (
p <0.05). Thus, in shock, sublingual microcirculation testing may be more appropriate than lip microcirculation.

It may be more promising to study enterostomy, as the application of orthogonal polarization spectral imaging to the intestinal mucosa allows for direct imaging and characterization of the intestinal mucosa. FIG. 20 shows an orthogonal polarization spectrum imaging image of a patient with recurrent intestinal necrosis. In this example, the number of capillaries observed in the intestinal mucosa was very low, and blood flow was almost completely stopped in these capillaries. This report supports the utility of using orthogonal polarization spectral imaging for mesenteric ischemia and infarction. Examples of uses for orthogonal polarization spectral imaging include early detection of ischemia, quantification of its lesions, and visualization of ischemic areas with some potential for recovery.

Conclusions: Introducing orthogonal polarization spectral imaging into intensive care using CYTOSCAN ™ A / R allows direct and non-invasive imaging of the microcirculation to examine new areas at the bedside. there is a possibility.

Drug Application In this embodiment of the invention, orthogonal polarization spectral imaging probes are used in drug development.

[0413] In one aspect of this embodiment, a tumor or anti-neoplastic agent (tumor anti-a) is administered using a standard or high contrast orthogonal polarization spectral imaging probe.
The effects of various agents, such as N. geniogenesis drug) on the microcirculation are tested to determine if circulation to the tumor is blocked. The effects of cardioangiogenic drugs are also examined to determine if blood vessel growth and thus circulation (eg to the heart) has improved. Alternatively, the effects of antihypertensive agents are tested to elucidate the mechanism of action of new therapies at the microcirculatory or cellular level or the etiology of hypertension. This test is measured on the sublingual or direct tissues. It is possible to obtain continuous images and measurements in real time.

Capillary density, blood vessel (and microvessel) morphology, blood vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, blood vessel diameter, leukocyte-endothelial cell interaction, vascular dynamics (
One or more parameters such as vascular motion), functional blood vessel density, functional capillary density, blood flow, area-to-surround ratio, hemoglobin concentration, and hematocrit value may be quantitatively measured. Preferably two or more parameters are measured.

Tumor angiogenesis plays an important role in tumor growth, metastasis formation, malignant tumor detection and therapy. Recent studies have provided much evidence that quantitative analysis of tumor angiogenesis is essential for developing new therapeutic strategies such as options for anti-angiogenic and anti-vascular treatment.
Orthogonal polarization spectral imaging was validated for non-invasive visualization of tumor angiogenesis in vivo and used to evaluate anti-angiogenic tumor therapy in vivo.

An experiment was carried out on hamsters in which a melanin-deficient melanoma A-MEL3 was implanted in the dorsal skin chamber (skinfold chamber). The experimental animals were treated daily with only the anti-angiogenic agent SU5416 (25 mg / kg / body weight) or the vehicle (control) starting from day 0 after tumor cell implantation. Functional blood vessel density (f
vd), microvessel diameter (d), and red blood cell velocity ( _VRBC ) were imaged using both orthogonal polarization spectral imaging and fluorescence microscopy and analyzed using a digital imaging system.

The results were as follows. The morphological and functional characteristics of tumor microcirculation were clearly identified by orthogonal polarization spectral imaging. The value of fvd correlated well with the data obtained by fluorescence microscopy (y = 0.99x + 0.4.
_{^{8, r 2 = 0.97, R}} S = 0.98, absolute accuracy: 8.22 / cm and bias: -0.32 / cm). The correlation parameters d and _VRBC were similar (d
And _VRBC are r ² = 0.97, R _S = 0.99 and r ² = 0.
93, R _S = 0.94). Tumor angiogenesis was suppressed by treatment with SU5416. The fvd was 4.8 ± 2.1 / cm and 87.2 ± 10.2 / cm at 3 and 6 days after tumor cell implantation, respectively, 66.6 ± 10. 1 / cm and 147.4 ± 13.2 / cm.
In the treatment group using SU5416, in addition to the inhibition of tumor angiogenesis, the growth of tumor and the development of metastasis were significantly suppressed.

Orthogonal polarization spectral imaging allows non-invasive, repetitive and quantitative assessment of the effects of tumor angiogenesis and anti-angiogenic treatments on tumor vasculature. Tumor angiogenesis can be used to more accurately classify and monitor the biological properties of tumors and to determine the degree of tumor invasion in vivo.

In a related aspect of this embodiment, the effect of agents believed to improve perfusion using orthogonal polarization spectral imaging probes is observed. This includes
For example, the vasoactive class of drugs such as naftidrofuryl, pentoxifylline, and buflomedil.
lass) is included. That is, since it is possible to observe and quantify perfusion after drug administration using an orthogonal polarization spectrum imaging probe, it is used to solve the problem of perfusion using orthogonal polarization spectrum imaging in animal models or humans. The drug can be tested more accurately. Moreover, orthogonal polarization spectral imaging is practically useful in elucidating the mechanism of effect of these agents on humans.

In another aspect of this embodiment, a hemoglobin-based (hemoglobin) using a standard or high contrast orthogonal polarization spectral imaging probe.
-Based) oxygen carriers (ie, DCL Hb, synthetic hemoglobin) are observed to determine their effect on the microcirculation, eg, whether RBC flow was increased as a result of using the drug.

In another aspect of this embodiment, orthogonal polarization spectral imaging probes can be used to test the effect of ultrasound enhancers (ie, injectable dyes) on microcirculation.

In another aspect of this embodiment, an orthogonally polarized spectral imaging probe is used to injectable dyes or other injectable contrast-producing agents (co
It is possible to visualize and detect the leakage of blood from the blood vessels of the trust-generating agent.

Comparison to Capillary Microscope In this embodiment of the invention, a standard or high contrast orthogonal polarization spectral imaging probe was used, as was previously done with a standard capillary microscope. The capillary bed of the nail fold is imaged and detected.

In one aspect of this embodiment, a circulatory disorder patient such as Raynaud's disease, osteoarthritis, or systemic sclerosis is tested and diagnosed using a standard or high contrast orthogonal polarization spectral imaging probe. And evaluated.

Good results are obtained with orthogonal polarization spectral imaging probes as compared to standard capillary microscopes used to image and characterize skin capillaries. As a result of the comparison, it was found that when observing healthy and diseased humans, this method was worthy of comparison (and often gave excellent results).
The site where the orthogonal polarization spectral imaging probe is used is above the nail fold. The measurements obtained were RBC velocity and diameter. Capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology,
One or more of vessel diameter, leukocyte-endothelial cell interaction, vasodynamics (vasomotor etc.), functional vessel density, functional capillary density, blood flow, area-to-surround ratio, hemoglobin concentration, and hematocrit value The parameters may be quantitatively measured. Preferably two or more parameters are measured. In the next study, orthogonal polarization spectral imaging was validated using a conventional capillary microscope. To this end, a capillary microscope (CAP) was compared with orthogonal polarization spectral imaging for nail fold microcirculation. Three capillaries of 10 healthy male volunteers aged 23.5 ± 0.8 years (mean ± standard deviation) were visualized using CAP and O orthogonal polarization spectral imaging and videotaped for offline analysis. saved. Cap
Resting red blood cell velocity (rCBV) was measured using Image ™ software. Contrast ratios were calculated for both devices to evaluate the quality of each image. In Wilcoxon matched pairs test, P =
It was 0.32, and there was no significant difference in rCBV between OPS and CAP. The rCBV measured using OPS was 0.737 ± 0.237 mm / sec, the rCBV measured using CAP was 0.782 ± 0.196 mm / sec, and the average difference between the measured values was 0.071 ±. It was 0.130 mm / sec. The contrast ratio was significantly higher in OPS images (OPS 0.34 ± 0.08, CA
P 0.17 ± 0.06, P = 0.0039). OPS is C even when it is not good
It is concluded that nail fold microcirculation can be recorded with the same accuracy and resolution as AP. Also, an advantage of orthogonal polarization spectral imaging is that it is simple to use and can be applied to many other organ beds.

Anesthesia In this embodiment of the invention, orthogonal polarization spectral imaging is used in the area of anesthesia.

Each of the following features of this embodiment include: capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter,
One or more parameters such as leukocyte-endothelial cell interaction, vasodynamics (such as vasomotor movements), functional blood vessel density, functional capillary density, blood flow, area-to-ambient ratio, hemoglobin concentration, and hematocrit value It may be measured quantitatively. Preferably two or more parameters are measured.

In one aspect of this embodiment, standard or high contrast orthogonal polarization spectral imaging probes were used to monitor intraoperative blood loss. For example, orthogonal polarization spectral imaging probes can be used to monitor hemodynamic parameters such as hemoglobin concentration and hematocrit in non-invasively and continuously anesthetized patients.

In another aspect of this embodiment, a standard or high contrast orthogonal polarization spectral imaging probe is used in anesthetized patients to monitor red blood cell clamping and the initial formation of microemboli during the surgical procedure. It

Using high contrast orthogonal polarization spectral imaging probes, for example, the use of bubble oxygenators during cardiac bypass surgery, dialysis circuits.
introduction of air from a circuit or decompression during laparoscopy
Air bubbles or fat bubbles in the blood caused by any of the clinical conditions such as pressure) are monitored and detected. The ability to directly detect air bubbles in the blood can facilitate recovery of the early stages of pulmonary or cerebral infarction, which would result in death if untreated.

Positive end expiratory pressure breathing (PEEP) was tested using orthogonal polarization spectral imaging. This PEEE contributes to intestinal-derived sepsis and reduces visceral blood flow. The effects of PEEP on capillary levels are largely unknown. Since genetically modified mouse and mouse antibody markers are widely available on the market, studies on this problem in mouse models can provide insights into the molecular mechanism of PEEP. Therefore, we established a mouse model using orthogonal polarization spectral imaging to image the intestinal microcirculation. Anesthetized mice (n = 14, c57bl6, male) were mechanically ventilated. After laparotomy, the distal 2 cm of the ileum was exposed and continuously superfused with Tyrode's solution. An orthogonal polarization spectrum imaging probe was placed on the exposed intestinal mucosa. Mice were randomly assigned to PEEP or control groups. In the control group, recording was performed at three levels (steady state) at baseline, at PEEP of 3 mmHg, and at PEEP of 7 mmHg. In the control group, no PEEP was applied and the same number of recordings were made. Functional capillary density (FCD) was measured as the number of visible capillaries per area (
CapImage ™ software). In PEEP group, FCD is 56 ± 1
Up to 8% (PEEP = 3 mmHg, baseline mean ± standard deviation (%)) significant (p <
It decreased to 0.05, the baseline and the control group, and again 39 ± 14% (PEEP = 7mmHg
(P <0.05, PEEP3 and control group), but was stable in the control group (96 ± 23% and 80 ± 23%). From this study, PEEP was found to reduce intestinal capillary perfusion. Also, the establishment of a mouse model for testing mechanical ventilation was successful.

(Disseminated Intravascular Coagulation (DIC)) As used herein, “DIC” is a complication of fibrin production in blood and obstetrics (eg, abruptio placenta premature ablation). ,infection(
Gram-negative bacteria), malignant tumors, and other serious diseases, and consumption of procoagulants and platelets. Other causes of DIC include intravascular hemolysis, vascular disorders, thrombosis, snakebite, extensive tissue damage, trauma (
Head trauma, especially in children), hypoxia, liver disease, infant and adult respiratory distress syndrome (R
DS), lightning purpura and burns.

The most severe clinical forms of DIC manifest as excessive consumption of coagulation proteins, marked fibrin deposition, and bleeding. Moderate forms of DIC have an endogenous marker for thrombin generation, with or without apparent coagulopathy.

In this embodiment of the invention, a patient's DIC is imaged, characterized and / or monitored using a standard or high contrast orthogonal polarization spectral imaging probe.

In each of the following aspects of this embodiment, capillary density, vessel (and microvessel) morphology, vessel density, vasospasm, red blood cell (RBC) velocity, cell morphology, vessel diameter,
One or more parameters such as leukocyte-endothelial cell interaction, vasodynamics (such as vasomotor movements), functional blood vessel density, functional capillary density, blood flow, area-to-ambient ratio, hemoglobin concentration, and hematocrit value It may be measured quantitatively. Preferably two or more parameters are measured.

In one aspect of this embodiment, a standard or high-contrast orthogonal polarization spectral imaging probe is used to image and characterize the DIC due to infection, and more specifically due to meningitis. And / or is monitored.

Leukocyte Kinetics / Leukocyte Endothelial Cell Interactions In another embodiment of the invention, standard or high contrast orthogonal polarization spectral imaging probes are used to affect inflammation and infection, or other diseases or leukocytes. Changes in leukocyte dynamics, such as those that occur during the effecting treatment, are imaged, characterized and / or monitored. This helps doctors identify the cause of "unexplained fever". Leukocyte-endothelial cell interactions (eg, leukocyte adhesion or leukocyte rotation, etc.) are imaged, characterized, and monitored.

As used herein, “white blood cell adhesion” refers to a type of leukocyte-endothelial cell-cell interaction in which white blood cells adhere to a certain place for a certain period of time.

As used herein, “leukocyte rotation” refers to a type of leukocyte-endothelial cell-cell interaction in which leukocytes rotate along the blood vessel wall at a velocity slower than the RBC velocity.

The effect of TNF-α on leukocyte rotation in iNOS-deficient mice was examined using orthogonal polarization spectral imaging.

Nitric oxide (NO) is an important endogenous modulator of leukocyte-endothelial cell interaction. The purpose of this study was to determine how NO affects TNF-α-induced leukocyte rotation and adhesion. iNOS knockout mice and their wild-type hindlimb muscles were surface perfused with Tyrode's solution containing 96% N ₂ and 5% CO ₂ , followed by 3 hours of surface perfusion with TNF-α (100 ng / ml) under baseline conditions. The postcapillary venules were examined using orthogonal polarization spectral imaging at. Leukocyte rotation and adhesion were quantified offline from 30 minute interval video recordings.

3 by baseline and Tyrode's solution in iNOS-deficient and wild-type mice
No difference was observed in leukocyte rotation and adhesion between time and surface perfusion. When TNF-α was included in the superfusate, the total number of leukocytes was increased in both groups. However, since the number of attached leukocytes was increased in the iNOS knockout mouse, the number of rotating leukocytes in the iNOS knockout mouse was smaller in the iNOS-deficient mouse than in the wild-type mouse. In some venules of iNOS knockout mice, leukocytes were so abundantly accumulated that vascular occlusion occurred. In wild-type mice, adhesion was not often observed, and most of white blood cells were in rotary motion. This in-vivo test was supported by post-mortem histological analysis.

Application to Basic and Clinical Research In this embodiment of the invention, in the areas previously described (eg, cardiology, cardiology,
Wound healing, diabetes, hypertension, ophthalmology, neurosurgery, orthopedic / reconstructive surgery, surgery, transplantation,
Orthogonal Polarization Spectral Imaging Probes for basic or clinical in vitro or in vivo any and all anesthesia, and pharmacology, especially drugs that inhibit or promote angiogenesis, or antihypertensive drugs) Is used.

Experimental animal models of numerous diseases related to microvascular pathology (ie, diabetes, hypertension, Raynaud's disease) have been developed, and the etiology of the disease, improved or early diagnosis, disease progression, and new therapies. Orthogonal polarization spectral imaging is used to study

Teaching Tool In a final embodiment of the invention, the orthogonal polarization spectral imaging probe is
For example, it is used as a teaching tool for medical and / or science students studying physiology, anatomy, pharmacology, microcirculation, and disease states affecting the microcirculation.

Although various embodiments of the present invention have been described above, it should be understood that they are shown by way of example only, and not by way of limitation. The illumination technique of the present invention is applicable to analytical in-vivo and in-vitro medical (clinical or research) applications that require optical measurement and visual observation of the properties of objects. It is also possible to measure the spectral absorption and scattering properties of the object using orthogonal polarization spectral imaging. Accordingly, the scope and scope of the invention is not limited by any of the above-described exemplary embodiments, but is within the spirit and scope of the invention as defined by the following claims and equivalents thereof. It is intended to cover all modifications and variations in.

All publications and patents mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patents are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually intended to be incorporated by reference.

[Brief description of drawings]

The accompanying drawings, which are incorporated in and constitute a part of the specification of the present application, illustrate embodiments of the invention and, in conjunction with the text, explain the principles of the invention and enable those skilled in the art to embody the invention. Play an additional role in enabling it to be used. In the drawings, like numbers indicate identical or functionally similar elements.

FIG. 1 is a block diagram showing one specific embodiment of an OPS imaging probe for non-invasive in vivo analysis of a vasculature of interest. In this particular embodiment,
The length of the objective lens is 8 inches. This embodiment is particularly useful for imaging cervical tissue.

FIG. 2 shows OPS images of small arteries (A) and small veins (V) in normal brain tissue after dural incision during neurosurgery. The bar indicates 100 μm.

FIG. 3 is an OPS image showing a target region of the brain where capillaries are dominant. The bar indicates 100 μm.

FIG. 4 is a diagram showing an OPS image of cortical microvessels of a patient with subarachnoid hemorrhage. Extravasation of red blood cells is seen as black spots in the extravascular space. Small arteries (A) show pearlstring evidence of multiple partial microvasospasms (arrows). No microvasospasm is seen in the venule (V). The bar indicates 100 μm.

FIG. 5 shows an OPS image of an opaque endothelial layer within some blood vessels near the brain tumor incision. The underlying pathophysiology is unknown. Endothelial swelling or plasma layer flow along the endothelium can be observed. The bar indicates 100 μm.

FIG. 6 is an OPS image showing tumor defect formation in a glioblastoma pleomorphic WHO IV patient. It is a typical tortuous irregularly shaped clot of newly formed tumor blood vessels. 100 bars
μm is shown.

FIG. 7 is a bar graph showing functional capillary density before and after tumor incision / excision of an aneurysm. Mean ± SEM.

FIG. 8 is a bar graph showing the distribution of small artery and small vein diameters before and after tumor dissection / resection of an aneurysm. Boxes and bars indicate the median and the 10, 25, 75 and 90th percentiles.

FIG. 9 is a bar graph showing the frequency distribution of red blood cell velocities in arterioles and venules before and after tumor ablation / ablation of aneurysms, given as a percentage of the total number of vessels. nm is not measured.

FIG. 10 shows changes in portal vein blood flow in mimetic and endotoxemia (ETX) animals during the study period. All data are presented as box plots containing the median, the 10, 25, 75 and 90th percentiles, as well as the maximum and minimum values. # Indicates the difference from the baseline, and § indicates the difference between the two groups.

[11] Mucosal gastrointestinal in擬体and endotoxemia animal during the study period - is a graph showing changes in arterial pCO ₂ gap (r-aPCO _2). All data are median, 1
Presented as a boxplot containing the 0th, 25th, 75th and 90th percentiles, as well as the maximum and minimum. # Indicates the difference from the baseline, and § indicates the difference between the two groups.

FIG. 12: Changes in the number of perfused / heteroperfused / non-perfused villi in mimetic and endotoxic animals during the study period.

FIG. 13: Relative changes in perfused / heteroperfused / non-perfused villus counts in mimetic and endotoxic animals during the study period. Data are shown as percentage of villi counted.

FIG. 14   FIG. 3 is a schematic diagram showing an image of an OPS imaging device and a capillary of a nailfold.

FIG. 15A is a diagram showing a change (%) (peak bundle density) in bundle density during venous occlusion and after arterial occlusion with respect to palm-side normal bundle density. One subject in the pre-eclampsia group had a peak flux density increase of 178% over normal values.

FIG. 15B is a diagram showing changes (%) (peak bundle density) in bundle density at the time of vein occlusion and after arterial occlusion with respect to the bundle density in the normal state on the back side. One target in the target group is
The bundle density at the time of vein occlusion was increased by 411%.

FIG. 16   It is a figure which shows the change (%) of the speed at the time of blockage with respect to the speed at normal times.

FIG. 17 shows an OPS image of the lower tongue of a healthy volunteer. Note the high density of venules and capillary networks.

FIG. 18 is a diagram showing an OPS image of the lower tongue of a septic shock patient (mean arterial pressure 68 m).
mHg, lactic acid 3.8 mEq / l, dopamine 20 mcg / kg. min, norebinephrine 0.13 mcg / kg / min). Note that many capillaries have reduced capillary density due to stop and transition flow.

FIG. 19 is a diagram showing an OPS image of the lower tongue of a patient with severe cardiac shock (mean arterial pressure 50 mmHg, lactate 10.5 mEq / l, dobutamine 20 mcg / kg.min).
, Dopamine 20 mcg / kg. min, norebinephrine 3 mcg / kg. m
in). Note the reduced density of capillaries and the presence of red blood cell clots within the large vessels that represent depressive blood flow.

FIG. 20 shows necrotic ileal fistula formation. Note that the number of intestinal mucosal capillaries is reduced, many of which are not perfused.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 21/21 A61B 3/12 E 33/483 3/10 R 33/49 5/14 310 (81) designation Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW) ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor James W. Winkelman United States 02167 Chestnut Hill Rangery Road, Massachusetts 62 F Term (reference) 2G045 AA02 AA06 AA26 DA51 FA19 FA29 GB00 JA01 2G059 AA05 BB12 BB13 CC16 CC18 EE02 EE05 KK01 JJ05 JJ01 JJ01 JJ01 JJ01 JJ01 JJ01 JJ02 GG01 JJ02 GG02 H0202 H02 02 H02 02 H02 02 H02 02 PP10 4C038 KK00 KL05 KL07 KX01 4C061 AA05 CC06 DD01 HH51 LL03

Claims (28)

[Claims] 1. A method for detecting circulatory disorders, comprising: (a) using an orthogonal polarization spectrum (“OPS”) imaging probe,
In obtaining a single captured image or sequence of images of a microcirculation of an individual with or suspected of having a circulatory disorder, (i) Irradiating a first plane of polarization with polarized light; and (ii) capturing at least one image or sequence of images reflected from the tissue, substantially orthogonal to the first plane of polarization. Obtaining a captured image by passing the reflected image through an analyzer having a polarization plane to generate a raw reflected image, and (b) analyzing the captured image to determine characteristics of microcirculation. A method comprising identifying the circulatory disorder by identifying. 2. The OPS imaging probe is a high contrast OPS.
The method of claim 1, wherein the method is an imaging probe. 3. The method of claim 1, further comprising performing imaging and characterization of blood cell components. 4. The method according to claim 3, wherein the blood cell component is red blood cells, white blood cells, or plasma. 5. The method according to claim 4, wherein the blood cell component is red blood cells. 6. The method of claim 1, further comprising imaging extravascular red blood cells. 7. The method of claim 6, wherein the extravascular red blood cells are caused by bleeding. 8. Hemoglobin concentration, hematocrit, tube morphology, area margin ratio,
Selected from the group consisting of tube density, capillary density, tube diameter, functional tube density, vasospasm, functional capillary density, red blood cell velocity, cell morphology, blood flow, leukocyte-endothelial cell interaction, and vasodynamics 1 The method of claim 1, further comprising analyzing one or more features. 9. The method of claim 8, wherein more than one of the features is analyzed. 10. The method of claim 1, wherein the image is a single captured image. 11. The method of claim 10, wherein the characteristic of the microcirculation is tube diameter, tube morphology, cell morphology, capillary density, tube density, area marginal ratio, or vasospasm. . 12. The method of claim 1, wherein the image is a sequence of images. 13. The microcirculation characteristic is erythrocyte velocity, functional capillary density, functional tube density, blood flow, leukocyte-endothelial cell interaction, or vasodynamics. The method described in. 14. A method of monitoring the microcirculation of an individual before, during, or after medical treatment, comprising: (a) using an OPS imaging probe before, during, or after medical treatment. , Later in obtaining a single captured image or sequence of images of the microcirculation of the individual, (i) illuminating tissue in the microcirculation of the individual with light polarized in a first plane of polarization; ii) capturing at least one image or sequence of images reflected from the tissue, the reflected image being passed through an analyzer having a plane of polarization substantially orthogonal to the first plane of polarization. Obtaining a captured image by generating a reflection image of the individual, and (b) analyzing the captured image to determine the fine image of the individual before, during, or after medical treatment. Method characterized by comprising monitoring circulation. 15. The OPS imaging probe is a high contrast OP.
The method according to claim 14, wherein the method is an S imaging probe. 16. The medical treatment according to claim 1, wherein the medical treatment is surgery.
The method according to 4. 17. Hemoglobin concentration, hematocrit, tube morphology, cell morphology,
From the group consisting of tube density, capillary density, tube diameter, functional tube density, vasospasm, functional capillary density, erythrocyte velocity, cell morphology, blood flow, leukocyte-endothelial cell interaction, area margin ratio, and vasodynamics 15. The method of claim 14, further comprising analyzing one or more selected features. 18. The method of claim 1, wherein more than one of the features is analyzed.
7. The method according to 7. 19. The method of claim 14, wherein the image is a single captured image. 20. The method of claim 19, wherein the characteristic of the microcirculation is tube diameter, tube morphology, cell morphology, capillary density, tube density, area marginal ratio, or vasospasm. . 21. The method of claim 14, wherein the image is a sequence of images. 22. The characteristic of the microcirculation is erythrocyte velocity, functional capillary density, functional tube density, blood flow, leukocyte-endothelial cell interaction, or vasodynamics. The method described in. 23. A method for diagnosing epithelial or epithelial cell-to-epithelial cell lesions, comprising: (a) capturing an uptake image of epithelial or epithelial-intercellular cells in a tissue by using an OPS imaging probe; Irradiating the tissue with light polarized in a first plane of polarization; and (ii) capturing a reflection image reflected from the tissue, the plane of polarization being substantially orthogonal to the first plane of polarization. Acquiring the captured image by passing the reflected image through an analyzer having the same to generate a raw reflected image, and (b) analyzing the captured image to diagnose the epithelial or interepithelial cell lesion. A method comprising: 24. The OPS imaging probe is a high contrast OP.
24. The method of claim 23, which is an S imaging probe. 25. The method of claim 23, wherein the epithelial or interepithelial cell lesion is a premalignant epithelial or epithelial intercellular tumor. 26. The method of claim 23, wherein the epithelial or epithelial cell-to-epithelial lesion is a malignant epithelial or epithelial cell-to-epithelial tumor. 27. The method of claim 25, wherein the epithelial or interepithelial cell lesion is a lesion from the neck. 28. The method of claim 26, wherein the epithelial or interepithelial cell lesion is a lesion from the neck.