Abstract:
The present application discloses systems and methods for the comprehensive monitoring of the microcirculation in order to assess the ultimate efficacy of the cardiovascular system in delivering adequate amounts of oxygen to the organ cells. In some cases, system embodiments may utilize reflectance avoidance by reflectance filtering, such as OPS imaging or Mainstream Dark Field imaging, or by Sidestream Dark Field imaging, which utilizes external direct light on the tip of the light guide to achieve reflectance avoidance whereby incident and reflected light do not travel down the same pathway.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a continuation of U.S. patent application Ser. No. 10/956,610, filed on Oct. 1, 2004, and naming Can Ince as inventor, which claims priority to U.S. Provisional Patent Application Ser. No. 60/508,347, filed on Oct. 3, 2003, and naming Can Ince as inventor, and which also claims priority to U.S. Provisional Patent Application Ser. No. 60/557,792, filed on Mar. 29, 2004, and naming Can Ince as inventor, all of which are incorporated by reference herein in their entirety. 
     
    
     BACKGROUND 
       [0002]    Currently, physicians typically monitor a number of systemic (e.g. the macrocirculation) hemodynamic parameters when diagnosing and monitoring of the hemodynamic condition of patients. For example, blood flow and pressure are regularly monitored. In addition, a blood sample may be withdrawn from the patient to determine the oxygenation of the red blood cells as well as the oxygen carrying capacity of the circulating blood. Furthermore, a biopsy may be required to determine the functional state of tissue cells (e.g. the oxygenation and viability of tissue cells) of the organ system. 
         [0003]    While monitoring these macrohemodynamic parameters has proven successful in diagnosing and monitoring a number of conditions, several shortcomings have been identified. For example, examining macrocirculatory parameters provides little or no information relative to the microcirculatory (i.e. hemodynamics and structure of blood vessels smaller than 250 microns) characteristics of patients. Current research has shown that distress at the microcirculatory level involved in a large number of disease states is not discoverable by monitoring macrocirculation. As such, diseases or other complications evident through microcirculatory monitoring may go undetected and untreated. 
         [0004]    It is believed, for example, that improved clinical observation of the microcirculation of human organs would be extremely useful in assessing states of shock such as septic, hypovolemic, cardiogenic and obstructive shock in patients and in guiding resuscitation therapies aimed at correcting this condition. In particular, it has been found that the active recruitment of the microcirculation maybe an important component of resuscitation. Additionally, improved clinical observation of the microcirculation would be helpful in observing gross circulatory abnormalities in pathologies such as tumors and cardiovascular disease. 
         [0005]    To fully monitor the function of the microcirculation, that is the structure and perfusion of vessels smaller than 250 micrometers, in addition to measuring blood flow it is important to measure and asses whether the blood cells are successful in transporting their oxygen to the microcirculation and thereafter to the surrounding tissue cells. Of particular importance is the assessment of the perfusion of the capillaries, which are between approximately 5 to 10 micrometers, because it is at this level that oxygen is transported by the red blood cells to the tissue cells of the organ for the purposes of respiration and survival. Monitoring the functional state of the microcirculation can thus be regarded as monitoring the ultimate efficacy and function of the cardiovascular system to deliver adequate amounts of oxygen to the organ cells. 
         [0006]    It is believed, for example, that improved and comprehensive imaging of the properties of the microcirculation would be helpful in observing and assessing the beneficial effects of therapy during the resuscitation of shock patients. An accurate assessment of both blood flow and oxygen availability at the level of the microcirculation could thus provide a clinical tool with which to guide resuscitation. A comprehensive way to monitor the microcirculation could generally provide an improved clinical diagnostic tool for evaluating and monitoring the functional state of the microcirculation in the peri-operative phase of treatment. 
         [0007]    To date, there have been limits to a comprehensive monitoring of the microcirculation in order to provide the benefits discussed above. Specifically, several factors have limited the ability to evaluate the oxygen transport variables of the microcirculation comprehensively. For example, devices which contact the surface of the microcirculation inhibit their ability to obtain quantitative information about blood flow in the various categories of micro-vessels in the microcirculation by impeding flow due to exerted pressure. Furthermore, current devices and techniques for imaging the microcirculation do not provide the additional needed information about the oxygen availability in the microcirculation or about the adequacy of oxygenation of the tissue cells. This information would be very helpful in assessing the functional state of the microcirculation, specifically its function in allowing adequate transport of oxygen to the tissue cells. Thus, there is a need for an improved system and method for a more effective and a more comprehensive clinical observation of the microcirculation which includes these parameters. 
       SUMMARY 
       [0008]    The system and method disclosed herein provides comprehensive information about the microcirculation by providing multiple modes of optical spectroscopy and imaging in a manner which does not influence the microcirculation. In one aspect, the system avoids reflection of light from the tissue in the various imaging modes. This reflectance avoidance can be provided by reflectance filtering, such as orthogonal polarization or cross-polarization of light or dark field imaging, or by sidestream dark field imaging, wherein, for example, incident and reflected light may not travel down the same pathway. 
         [0009]    In order to image flowing cells in the microcirculation, light has to be illuminated on to the surface of the organs, which is the substrate, and a magnifying lens may be used. Use of a specific wavelength of light (e.g. green light) may allow for better observation of the contrasting red blood cells due to the absorption characteristics of the hemoglobin (hereinafter Hb) in the red blood cells. However, surface reflections from the substrate can interfere with the ability to clearly visualize the underlying microcirculation structures and the flowing blood cells therein. Filtering out of these surface reflection by various methods allows visualization of the blood flow in the underlying microcirculation on organ surfaces by measurement of the images of the moving cells. Reflectance filtering can be achieved by a number of techniques which are known to those of skill in the art. The system and method disclosed herein may utilize some of these known techniques, but some novel ones are disclosed as well. 
         [0010]    In some embodiments, the system and method utilizes reflectance avoidance by known techniques of reflectance filtering, such as: 1) OPS imaging, whereby illuminating light and reflected light travel down the same light guide; or 2) Mainstream Dark Field imaging, whereby illuminating light and reflected travel down the same light guide but peripheral illumination is achieved by directing the light through, for example, a hole in a 45° mirror or design of a lens in the illuminating pathway, which impedes transmission of the light through the middle, and/or a lens which poorly allows transmission of the light through the centre is put in the pathway of the light to achieve the same effect. 
         [0011]    In other embodiments, a novel method of reflectance avoidance is disclosed which is an alternative to reflectance filtering. This novel approach, referred to herein as Sidestream Dark Field imaging (hereinafter SDF), utilizes external direct light on the tip of the light guide to achieve reflectance avoidance whereby incident and reflected light do not travel down the same pathway. This form of imaging can be provided in combination with a hand-held microscope. A feature of SDF imaging is that illuminated light and reflected light travel via independent pathways. With this modality, the illumination can be placed directly on the tissue and the observations can be made adjacent to it without light crossing over between two paths. The illuminating light source is typically placed on or near contact with the tissue. The scattering of the reflected light is thus outside of the image as most light cross over is below the tissue surface. To date, Mainstream Dark Field imaging has been described as a way of improving contrast and lowering surface reflectance, but it typically utilizes illumination and reflectance light paths that travel up and back the same pathway. In the past, SDF illumination has been applied by ring illumination to improve epi-illumination. It is believed, however, that it has not been applied to achieve true dark field illumination by illuminating one segment of a substrate and observing in another segment images of the microcirculation and its flowing cells. It is believed that SDF imaging has characteristics which make it superior to other modes of imaging. 
         [0012]    The foregoing reflectance avoidance imaging systems, whether they utilize OPS, Mainstream Dark Field illumination, or SDF illumination, can be used to enable the comprehensive evaluation of the functional state of the microcirculation. This is achieved by an analysis of the moving cells in the images, which permits the quantitative measurement of red blood cell flow in the capillaries, as well as in the larger vessels of the microcirculation. This measurement is believed to represent a truly sensitive measurement which is indicative of cardiovascular disease and dysfunction. Laser Doppler measurements, for example, provide an over all flux of moving particles in an unidentified compartment of the circulation, but do not have the specificity for measurement of cellular perfusion of these smallest capillaries. 
         [0013]    The system and method disclosed herein, in providing reflectance avoidance in combination with optical magnification, provides a superior method of measurement of the functional state (e.g. perfusion/oxygenation) of the microcirculation. Next to the measurement of perfusion, morphological characteristics of the microcirculation, such as functional capillary density and micro-vessel morphology, can be measured using reflectance avoidance imaging. Homogeneous perfusion of the capillaries is a prerequisite for normal function of the microcirculation and abnormal perfusion or diminished capillary perfusion is considered an early and sensitive indicator of cardiovascular disease and failure. 
         [0014]    The present application thus relates to a variety of imaging systems for analyzing the reflectance of an examination substrate. While the imaging system disclosed herein may be used to analyze the reflectance characteristics of a variety of substrates, it is particularly well suited for non-invasively imaging the micro-circulation with a tissue sample. 
         [0015]    In one embodiment, the present application discloses a system for imaging the reflectance of a substrate and includes a light source, a light transport body configured to project light from the light source to an examination substrate and transmit light reflected and scattered by the examination substrate, an analysis section in optical communication with the light transport body and having an orthogonal polarization spectral imaging module or any other of the reflectance avoidance imaging systems, and at least one of a reflectance spectrophotometry module and a fluorescence imaging module. 
         [0016]    In an alternate embodiment, the present application discloses an orthogonal polarization imaging system and includes a light source configured to emit white light, a first polarizer to polarize the white light, a light transport body to transport the polarized light to an examination substrate and reflect light from an examination substrate, a second polarizer to filter the light reflected and scattered by the examination substrate, a filter bank containing at least one wavelength filter to filter the reflected light, and an image capture device in optical communication with the light transport body and configured to image the reflected light. 
         [0017]    In still yet another embodiment, the present application discloses a method of imaging the reflectance of a substrate and includes illuminating an examination substrate with light, transmitting a portion of light reflected by the examination substrate to a reflectance spectrophotometer, determining a concentration of hemoglobin within the examination substrate based on a spectral characteristic of the examination substrate with the reflectance spectrophotometer, transmitting a portion of the light reflected by the examination substrate to an orthogonal polarization spectral imaging module, and measuring a flow through a vessel within the examination substrate with an orthogonal polarization spectral imaging module. 
         [0018]    In one embodiment, the present application discloses a novel manner of applying dark field imaging on the tip of a light guide to provide clear images of the microcirculation on human organ surfaces. This can be accomplished by putting light emitting diodes (LED&#39;s) around the tip of the light guide in combination with a separator so that the illuminating light does not enter the reflection light guide directly by surface reflection, but via the internal structures inside the substrate. This modality of reflectance avoidance is a form of dark field imaging which we have called Sidestream Dark Field or SDF imaging and provides remarkably clear images of the microcirculation. 
         [0019]    In some embodiments, reflectance avoidance imaging is used to obtain a microcirculatory perfusion index as well as a heterogeneity of flow index in a device that does not impact flow patterns. This may be accomplished by using non-contact modes such as, for example, using a long focal length, immobilizing the device and substrate by suction at the tip, or utilizing a spacer between the tissue and the light emitting tip. 
         [0020]    In one such embodiment, a novel, “castle” type of spacer is utilized to provide distance from the examining substrate and to avoid pressure of the tip on the substrate. In another embodiment, a needle camera is utilized with a spacer to provide a dark field illumination device. In yet another embodiment, a suction device is used with reflectance avoidance imaging techniques. 
         [0021]    In another embodiment, a distance spacer is used to achieve reliable capillary perfusion measurements whereby the tip of the image guide does not impede flow in the microcirculation by pressure. In yet another embodiment, reflectance avoidance imaging is used in combination with a space through which fluid, drugs or gasses can be perfused. 
         [0022]    In one embodiment, a disposable tip attaches to the end of the device and is removed by a release mechanism so that it can be disposed of without having to touch the disposable. 
         [0023]    The utilization of reflectance avoidance in the present invention provides an improved method of observing microcirculatory hemodynamics and functional morphology. Image analysis can provide a plurality of clinical parameters which will have utility for various clinical conditions. The method and device will assist in providing a perfusion index such as a measure of functional capillary density, which is the number of perfused micro-vessels showing per field observed. Other parameters include the distribution and heterogeneity of micro-vascular flow, torsion and functional morphology of the blood vessels, the distribution of diameters of blood vessels, white blood cell kinetics, abnormal red blood cell kinetics (e.g. the presence of micro-vascular coagulation, sludging or adhesion). 
         [0024]    For a comprehensive assessment of the functional state of the microcirculation, it may be preferable to have more than just perfusion information. It would also be useful to have Information about the amount of oxygen bound to the Hb, which can be provided by reflectance spectrophotometry, and information as to whether the tissue cells are getting sufficient amount of oxygen, which can be provided by measuring tissue CO 2  by sensing the CO 2  in the inside of the disposable, using, for example, CO 2  sensitive fluorescence quenching dyes. The light guide can then be used to excite the dye with a pulse of light and a detector which measures the CO 2  dependent quenching of fluorescence life time would provide the measurement. Also, mitochondrial energy states by NADH via fluorescence imaging can be obtained. Information may be obtained about whether there is movement of the red blood cells in the microcirculation, whether the red blood cells are transporting oxygen (i.e. Hb saturation), and whether the tissue cells are getting enough oxygen (tissue CO 2  measurement and/or NADH fluorescence imaging). 
         [0025]    In some embodiments, reflectance spectrophotometry in conjunction with reflectance avoidance is used to assess the adequacy of oxygen availability. This may provide for the assessment of microcirculatory oxygen transport. In some embodiments this can be accomplished by an analysis of the full reflected spectrum of light (e.g. 400-700 nm). In other embodiments it is accomplished by an analysis of discrete wavelengths outputs of a color sensitive imaging device. Microcirculatory Hb saturation, microcirculatory Hb concentration, and microcirculatory hematocrit can all be measured. 
         [0026]    In some embodiments, the SDF imaging technique is combined with the use of different wavelengths LED&#39;s wherein the images are normalized and Beer Lambert equations are applied. 
         [0027]    In some embodiments, NADH fluorescence imaging is used to measure the adequacy of the need for mitochondrial oxygen. This can be used to assess tissue cell dysoxia. 
         [0028]    In some embodiments, fluorescence spectroscopy is used for tissue cell diagnostics using endogenous molecules, reporter genes or external indicator dyes. With appropriate filters, apoptosis can be detected (e.g. via annexin fluorescence), green fluorescent labeled cells used in gene therapy could be located in terms of their efficacy in homing in on the target. 
         [0029]    In one embodiment, a method of imaging the microcirculation by avoiding surface reflections is combined with reflectance spectrophotometry, Raman spectroscopy, fluorescence spectroscopy and/or other types of spectroscopic modalities, such as light scatter measurements or optical coherence tomography. 
         [0030]    In some embodiments, the device is a light guide based system wherein emission and excitation light travels via light guides. In some embodiments, the images are detected at the tip with a tip camera. The device may have a fused silicon lens which will allow 360 nm to pass in order to enable NADH fluorescence imaging. The device can be either hand held or a flexible endoscopic type. 
         [0031]    In addition, to direct contact imaging, the reflectance avoidance imaging system disclosed herein may also be capable of operating in a non-contact mode which makes use of a spacer to avoid pressure in the tissue surface which may impede blood flow therethrough. Various spacer options exist, including; 
         [0032]    a. plastic upside down cup attached as disposable; 
         [0033]    b. a doughnut shaped spacer (which can be inflatable) with an upside down situation/cup; 
         [0034]    c. a device (e.g. a plug for around the scope end), such as a concentric ring with suction ports, for providing suction through little holes around the perimeter of the scope thereby immobilizing the perimeter but leaving the microcirculation in the field of view unstressed; or 
         [0035]    d. a transparent cushion either solid, air inflatable or filled with fluid. 
         [0036]    What is also disclosed is a non-contacting tip for endoscopic use. In one embodiment, long focus distance imaging can be used to observe retinal microcirculation. This modality can be used to monitor eye diseases and as a monitoring tool during surgery to monitor brain function non-invasively. In the retinal application imaging light can be pulsed and small clips of moving images used for monitoring, thus minimizing retinal light exposure. 
         [0037]    In one embodiment, the system is configured to operate in a no contact mode without use of a spacer. Thus, the system may be used during brain surgery or heart surgery. Any movement of the object surface can be corrected by image processing either on-line or after a delay. 
         [0038]    In one embodiment the light guide system has an L-shape at the end. Here a 45° mirror creates the bend and LED illumination, using SDF, imaging is present at the tip, with or without a spacer and/or suction module. This embodiment may be used to inspect the sides of hollow spaces such as is present in the digestive track. 
         [0039]    In another embodiment, large objective magnification may be used. For example, image processing software may be used to immobilize or stabilize the images, thereby allowing for better image processing of the movements. 
         [0040]    In still another embodiment, magnification of the substrate image can be influenced in several ways. For example, different lenses may be used (different spacer on the tip), or movement of exiting lenses by an opto-mechanical system, or in the electronic mode a larger number of pixel CCD or CMOS chips, which are known to those of skill in the art, or a larger density of pixels in the chip can be utilized. Movement of the CCD or CMOS can also be used to influence magnification. 
         [0041]    In still another embodiment, any number of specified color cameras may be used with the present system. For example, a choice of color or combination of colors would allow images to be generated of the saturation of the Hb of the red blood cells in the microcirculation. A further embodiment involves looking at only the red output of a color camera and to filter out of the rest of the image. This would result in red cells moving in a white background. 
         [0042]    Use of a high speed rate (i.e. higher than video rate) can be used for obtaining a proper velocity measurement in conditions in which red blood cells are moving faster than the video rate. 
         [0043]    In some embodiments, a CO 2  measurement of the tissue in the field of view can be made simultaneously with a reflectance avoidance flow measurement and an oxygen availability measurement, such as with spectrophotometry, as a measure of tissue wellness. 
         [0044]    In one embodiment, a disposable spacer (e.g. upside down cup) may be employed. In this embodiment, a CO 2  sensing dye can be impregnated with which CO 2  can be sensed within the cup environment. The dye works to provide a fluorescence decay measurement and the excitation and emission light of this dye in the disposable tip can be measured through the light guide. The CO 2  measurement can be combined with a reflectance avoidance flow measurement, such as an OPS or SDF imaging based perfusion measurement. Furthermore, a CO 2  probe may be inserted into the nose of a patient to assess tissue pCO 2  and combine this information with simultaneously measured perfusion (e.g. by OPS or SDF imaging) and oxygen availability (spectrophotometry) measured sublingually. In another embodiment, the CO 2  probe may be used rectally. These measurements may be made continuously. The sensor may be embedded within a pliable of cushioning material. For example, the sensor may be positioned within a sponge so as to trap and sense the CO 2  sufficiently. 
         [0045]    The CO 2  sensor can be used in the nose and/or rectally as alternative locations for a separate sensor which is then integrated in the measurement. This can be in single or in multi mode. The latter technique, which makes use of more than one CO 2  sensor, will give information about regional heterogeneity. Using multi locations is believed to be a new use of a CO 2  measurement. 
         [0046]    In some embodiments, a laser can be included as a therapeutic modality. This can be accomplished, for example, by the use of dark field illumination in which the laser goes through the hole in the slanted mirror. In this embodiment, reflectance avoidance imaging is combined with the use of the laser for photodynamic therapy (e.g. for cancer) or to coagulate micro-vessels in port wine stains or other cosmetic corrective procedures. 
         [0047]    In another embodiment, reflectance avoidance imaging is used to observe the microstructure of the wound, and temperature is sensed by a solid state or thermo-sensitive color sensor as well as by optical spectroscopy to measure the water content. It is thereby that wound perfusion (via e.g. OPS or SDF imaging), wound temperature and edema (water content) will give a comprehensive measurement of the phase of wound healing and allow assessment of the response to therapy. 
         [0048]    In the photodynamic embodiment (where the patient receives a photosensitive drug) it is possible to apply fluorescence in combination with reflectance avoidance for detection of the drug (which accumulates in tumors) or for enhanced fluorescence in ALA induced protoporphyring fluorescence. Combining a therapeutic laser in the device would make it possible to deliver photodynamic therapy directly to the area of high fluorescence. 
         [0049]    Alternative illumination modalities may include pulsing the LED illumination in combination with synchronization with a camera for the measurement of high blood flow velocities. Another alternative includes the use of an optical foil, acting as a light guide, or other material which may be wrapped around the tip of the probe providing illumination from the side of the tip as an alternative way of illuminating the object and accomplishing reflectance avoidance. This is similar to the method which is accomplished by the use of optical fibers placed around the out side of the scope. 
         [0050]    Other embodiments which include laser therapies include the use of reflectance avoidance imaging to verify the effectiveness and allow for the accurate titration of laser doses. A second example is the use of photodynamic therapy for on-line treatment of photosensitized tumors. 
         [0051]    In another embodiment, a custom spacer is disclosed in which it is possible to introduce a drug or gas to the field of observation and measure the reactivity of the blood vessels (i.e. losses of which are an indication of poor function). This spacer could be a suction spacer which would provide space in the field of view to ensure that there is no contact with the tip and also provide space to inject a drug (for microcirculatory responsiveness) or for calibration that may be needed for the embodiment which utilizes a CO 2  sensor placed in the probe. Drugs which can be considered challenges to the microcirculation are vasodilators acting on specific locations of the microcirculation e.g. acetyl choline, lidocaine or nitrate. Others include vasopressors, such as noradrenaline or dobutamine. This modality can also be used in local treatment of tumors by application of a topical administration of a chemotherapeutic drug. 
         [0052]    Measuring the reactivity of the blood circulation to challenges (also given systemically) via, for example, trend measurements, yield parameters which give additional information than a snap shot analysis. Response to therapy of the microcirculation can be monitored continuously providing on-line information about the functional state of the microcirculation during illness. 
         [0053]    A further challenge can be induced through a specialized spacer which applies a momentary suction pulse and measures the time of microcirculatory refill. 
         [0054]    In some embodiments multi-wavelength imaging can be used for the measurement and analysis of Hb saturation images. The object is sequentially or simultaneously illuminated by specific colored LED&#39;s, placed in SDF mode, which are chosen at specific wavelengths along the absorption spectrum of Hb, such that when combined in a composite image they provide an image of the distribution of Hb saturation (or Hb concentration or Hematocrit) of the cells of the microcirculation. A second embodiment for achieving the same objective utilizes white light. The reflected light is then split by a multi-wavelength optical member which may consist of mirrors and filters which project two or more images each at a different wavelength onto the imaging device to allow reconstituted saturation images to be made. 
         [0055]    In one embodiment the use of fluorescence SDF imaging (endogenous leucocyte fluorescence), or observing light scatter, to view differences between cells moving in the circulation (i.e. leucocytes scatter more light than red blood cells) and combining such imaging, with or without filtering of special wavelengths, optical conditions permit the observation and quantification of the amount of leucocytes flowing in the microcirculation. Such a measurement would allow quantification of the immune status of the observed field of view by counting the amount of leucocytes and or observing the kinetics of cell sticking or rolling. 
         [0056]    In one embodiment, annexin fluorescence can be used for the detection of apoptotic cells. A combination of fluorescence techniques includes but is not limited to annexin-labeled cells which will allow for the visualization of apoptotic cells which are directed to programmed cell death, a precursor to necrosis and cell death. These measurements may be important in assessing cell failure in cardiovascular disease, sepsis and in identification and staging of the severity of cancer, or other stages of diseases such as inflammatory bowel disease. In this application fluorescence labeled annexin is administered to the patient, or applied topically to the site of interest and utilizes the fluorescence mode of the scope. In the fluorescence mode of the scope we describe a hand tool (a fluorescence boroscope) such as described for the reflectance avoidance imaging but in which fluorescence modality is utilized. Reflectance avoidance imaging can be used to improve fluorescence imaging, by filtering or avoiding surface reflections, and can be applied in the boroscope application or also in fluorescence endoscopy where, to date, the combination of fluorescence and reflectance avoidance imaging has not been disclosed. 
         [0057]    In this embodiment, the appropriate choice of filters can be used to image mitochondrial energy states (NADH levels) through the use of fluorescence. NADH in vivo fluorescence imaging involves dual wavelength fluorescence combined with reflectance avoidance imaging to correct for changes in absorption in the image, which can be caused by variation in Hb (which is an absorber) in the vessels in the image (results in heterogeneous images). In addition, fluorescence spectrophotometry may be combined with reflectance avoidance imaging to allow cell diagnostics during surgery directly at the bedside. Tissue cell diagnostics will target the functional state of the mitochondria by measurement of the energy of the mitochondria by NADH fluorescence, the gold standard for assessment of tissue dysoxia. Such fluorescence imaging can also be used in conjunction with diagnostic dyes for identification of apoptosis or tumor cells and reporter genes during gene therapy. Combination of fluorescence dyes and cell labeling techniques can be used by this modality (with appropriate filters) to observe and quantify the degree of degradation of the glycocalix lining of the endothelia cells. This observation provides a microcirculatory indication of the severity of cardiovascular disease. Finally measurement of the time course of transport through the microcirculation of a pulse of fluorescent dye allows microcirculatory flow at the capillary level to be quantified when detected by fluorescence. 
         [0058]    In some embodiments, reflectance avoidance imaging will be combined with Raman spectroscopy, thereby combining microcirculatory reflectance avoidance imaging with information about the constituents of the tissues. 
         [0059]    The above embodiments can be used in an endoscopy mode. For example, dark field endoscopy, OPS imaging, and\or side illumination can be used to make observations in the gastric tract, with for example, the L-tip device discussed above. Polarization can be achieved at the tip of a flexible endoscope. Dark field illumination can be used in the same way by concentric illumination. A light conducting foil can be used at the outside. A 45° mirror can be included at the tip for observation of the sides of the gastric tubes. Thin scopes can be made for pediatrics. 
         [0060]    In some embodiments, optical coherence tomography can be used for measurement of optical path-length using Beer Lambert as a quantitative measurement. 
         [0061]    Sublingual Near Infra-red Spectroscopy can be used in the transmission mode or in the reflectance mode to measure total oxygenation of the tongue. 
         [0062]    The foregoing methodologies for comprehensive imaging of the microcirculation provide a useful clinical tool in assessing states of shock such as septic, hypovolemic, cardiogenic, and obstructive shock in patients and in guiding resuscitation therapies. 
         [0063]    Other objects, features, and advantages of the imaging system and method disclosed herein will become apparent from a consideration of the following detailed description. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0064]    The imaging system of the present application will be explained in more detail by way of the accompanying drawings, wherein: 
           [0065]      FIG. 1  shows a block diagram of an embodiment of an imaging system for analyzing light reflected from an examination substrate; 
           [0066]      FIG. 2  shows a block diagram of an embodiment of an analyzing section of an imaging system; 
           [0067]      FIG. 3  shows a schematic diagram of an embodiment of a light transport section configured to project light on and receive reflected light from an examination substrate; 
           [0068]      FIG. 4A  shows a perspective view of an embodiment of a light transport body of a light transport section; 
           [0069]      FIG. 4B  shows a perspective view of an alternate embodiment of a light transport body of a light transport section; 
           [0070]      FIG. 4C  shows a perspective view of another embodiment of a light transport body of a light transport section; 
           [0071]      FIG. 4D  shows a perspective view of still another embodiment of a light transport body of a light transport section; 
           [0072]      FIG. 5  shows a schematic diagram of another embodiment of a light transport section configured to project light on and receive reflected light from an examination substrate; 
           [0073]      FIG. 6  shows a side view of an alternate embodiment of a light transport body of a light transport section; 
           [0074]      FIG. 7  shows side view of an embodiment of a spacer device coupled to an embodiment of a light transport body; 
           [0075]      FIG. 8  shows a side view of another embodiment of a spacer device coupled to an embodiment of a light transport body; 
           [0076]      FIG. 9  shows a side view of an embodiment of a spacer device configured to couple to an examination substrate coupled to an embodiment of a light transport body; 
           [0077]      FIG. 10  shows a bottom view of an embodiment of the spacer device shown in  FIG. 9 ; 
           [0078]      FIG. 11  shows a cross sectional view of an embodiment of an imaging system for analyzing reflected light; 
           [0079]      FIG. 12  shows a side view of an optical system for use in the an imaging system for analyzing reflected light shown in  FIG. 10 ; 
           [0080]      FIG. 13  shows a cross sectional view of an embodiment of an imaging system for analyzing reflected light having an internal light source positioned therein; 
           [0081]      FIG. 14  shows a side cross-sectional view of an embodiment of an imaging system configured to permit side stream dark field imaging of an area; 
           [0082]      FIG. 15  shows a perspective view of the distal portion of an embodiment of the imaging system shown in  FIG. 14 ; 
           [0083]      FIG. 16  shows a cross sectional view of an embodiment of an imaging system having one or more illumination sources located within illumination passages formed in a body; 
           [0084]      FIG. 17  shows a cross sectional view of an embodiment of an imaging system having a body coupled to handle portion; 
           [0085]      FIG. 18  shows a schematic diagram of an embodiment of an imaging system for projecting light to a substrate and collecting light therefrom for analysis; 
           [0086]      FIG. 19  shows a perspective view of the distal portion of the imaging system shown in  FIG. 18 ; 
           [0087]      FIG. 20  shows a perspective view of the distal portion of another embodiment of imaging system shown in  FIG. 18 ; 
           [0088]      FIG. 21  shows a side cross sectional view of an embodiment of an imaging system wherein the distal portion thereof is in contact with an examination substrate; 
           [0089]      FIG. 22  shows a side cross sectional view of an embodiment of an imaging system wherein the distal portion includes an engaging device thereon; 
           [0090]      FIG. 23  shows a side cross sectional view of an embodiment of an imaging system wherein the distal portion is not in contact with the examination substrate; 
           [0091]      FIG. 24  shows a block diagram of diagram of an embodiment of an imaging system for imaging microcirculation within a structure and analyzing light reflected from an examination substrate; 
           [0092]      FIG. 25  shows a cross sectional view of an embodiment of a cap device which may be affixed to a body of an imaging system; 
           [0093]      FIG. 26  shows a perspective view of embodiment of an imaging system configured for sub-surface imaging of an area; and 
           [0094]      FIG. 27  shows a side cross sectional view of the imaging system shown in  FIG. 26 . 
       
    
    
     DETAILED DESCRIPTION 
       [0095]      FIG. 1  shows a block diagram of an embodiment of a reflectance imaging system. The imaging system  10  includes an analyzing section  12  and a light transport section  14  configured to project light on and/or receive reflected light from an examination substrate  16 . In one embodiment the light transport section  14  may include an internal light source  18  therein configured to provide light of at least one selected wavelength and/or polarization to the examination substrate  16 . Optionally, the internal light source  18  may be used with or may comprise a source of white or full spectral light thereby enabling spectral analysis of light reflected by the examination substrate  16 . In an alternate embodiment, an external light source  20  may be in optical communication with the light transport section  14  and configured to illuminate the examination substrate  16 . Optionally, the imaging system  10  may include both an internal light source  18  and an external light source  20 . As such, the internal and external light sources may have the same or different wavelengths and/or polarizations. In another embodiment, an ancillary illuminator  22  may be used to illuminate the examination substrate  16 . As shown, the ancillary illuminator  22  directly illuminates the examination substrate thereby foregoing the light transport section  14 . The various components of the analyzing section  12  and the light transport section  14  will be described in greater detail below. 
         [0096]    Referring again to  FIG. 1 , in one embodiment the analyzing section  12  includes any number of modules configured to analyze light reflected from the examination substrate  16  and transported to the analyzing section  12  by the light transport section  14 . In the illustrated embodiment, the analyzing section  12  includes an orthogonal polarization spectral (OPS) imaging module  30 , a reflectance spectrophotometry (RFS) module  32 , and a fluorescence (FLS) imaging module  34 . Any number of additional modules  36  may be included in the analyzing section  12 . Exemplary additional modules include, without limitation, Raman spectroscopy modules, optical coherence tomography modules, dark field imaging including side stream dark field imaging (See below), and various light scattering measurement modules. 
         [0097]    As shown in  FIGS. 1 and 2 , the OPS imaging module  30  receives a light sample  40  from a beam director  98 . The light sample comprises light reflected from the examination substrate  16  and transmitted to the beam director  98  by the light transport section  14 . As such, the OPS imaging module  30  is configured to image the examination substrate  16  using wither dark field or non-dark filed illumination. Thereafter, the light sample  40  may encounter a polarizing section  42  having one or more optical polarizers therein. The polarizing section  42  permits only light of a selected or desired polarization to transmit therethrough, thereby filtering the light reflected by the examination substrate  16  and improving image quality. IN an alternate embodiment, the OPS imaging module  30  may incorporate a variety of other optical devices or methodologies to optimize image quality. The polarized light  44  is then incident upon a filtering section  46  having one or more optical filters therein. For example, in one embodiment the filtering section  46  contains at least one narrow band pass filter therein configured to permit light within a desired wavelength range to be transmitted therethrough. Exemplary narrow band pass filters include, without limitation, from about 380 nm to about 450 nm (violet filter), from about 445 nm to about 510 nm (blue filter), from about 495 nm to about 580 nm (green filter), from about 575 nm to about 595 nm (yellow filter), from about 590 nm to about 625 nm (orange filter), from about 615 nm to about 710 nm (red filter), and from about 690 nm to about 910 nm (color or photo infrared filter). Optionally, the OPS imaging section  30  may include filters enabling ultraviolet radiation to transmit therethrough. In an alternate embodiment, the filtering section  46  receives light from the light transport section  14  prior to the light sample  40  being polarized. 
         [0098]    Referring again to  FIG. 2 , the filtered light  48  is then transmitted from the filtering section  46  to an image capture device  50 . Exemplary image capture devices  46  include, without limitations, charge coupled devices (CCD) and photomultiplier devices. For example, in one embodiment a CCD chip having about 1000 by 1000 pixel resolution or higher may be used. Optionally, images captured at various wavelengths may be captured and compared to permit image normalization. In an alternate embodiment, an image capture device  50  may be utilized to correct for motion effects and aberrations. The image capture device  50  forms an image of light reflected from the examination substrate  16  and transmitted to the OPS imaging section  30  by the light transport section  14 . (See  FIG. 1 ). In the illustrated embodiment, the image capture device  46  is in communication with a processor and display device  52 . The processor and display device  52  may be used to process information from the image capture device  50  and display the information in any number of ways. Exemplary processor and display devices include, without limitations, computers and display terminals. 
         [0099]    As shown in  FIG. 2 , the OPS section  30  may include a light modulator  54  and/or an OPS optics suite  56 . The light modulator  54  may be used to segment the sample light  40 , thereby providing a stroboscopic effect thereto. Exemplary light modulators  54  include, without limitations, light choppers, shutters, and light valves including liquid crystal light valves. An OPS optics suite  56  may be used to focus, defocus, collimate, or otherwise refine the light sample  40  transmitting through the OPS imaging section  30 . Exemplary components which may be used within the OPS optics suite  56  include, without limitations, mirrors, positive lenses, negative lenses, acromats, compound lenses, astigmats, windows, flats, adaptive optics, holographical optical elements, spatial filters, pinholes, collimators, stages, and beam splitters. The light modulator  54  and the OPS optics suite  56  may be positioned at various locations within the OPS imaging section  30 . 
         [0100]    Referring again to  FIG. 2 , the reflectance spectrophotometry module  32  includes a spectrophotometer  70  coupled to a RFS image processor  72  for computing and displaying spectral characteristics of the light reflected from the examination substrate  16 . (See  FIG. 1 ). For example, full spectrum (e.g. white) light is used to illuminate an examination substrate. Thereafter, the light reflected by the examination substrate  16  may be captured and the spectral characteristics thereof may be examined to measure a variety of characteristics of the examination substrate  16 , including, without limitation, hemoglobin saturation and hematocrit concentration. Exemplary RFS image processors  72  include, without limitation, CCD and CMOS chips and photo-multiplier devices coupled to processors and display monitors. As such, the spectrophotometer  70  is in optical communication with the light transport section  14 . In one embodiment, an RFS optics suite  74  may be used to process and refine the light received from the light transport section  14 . Exemplary components which may be used within the RFS optics suite  74  include, without limitations, mirrors, positive lenses, negative lenses, acromats, compound lenses, astigmats, windows, flats, adaptive optics, holographical optical elements, spatial filters, pinholes, collimators, stages, wavelength filters, emission filters, and beam splitters. 
         [0101]    As shown in  FIG. 2 , the fluorescence imaging module  34  includes a fluorescence imaging system  90  and a fluorescence image capture device  92 . Exemplary fluorescence imaging systems  90  may include variety of optical components including, without limitation, microscopes, filter wheels, shutters, and optical filters. For example, green, yellow, and clear optical filters may be included. In one embodiment, the fluorescence imaging system  90  is configured to detect fluorescence from ultraviolet (UV) to infrared (IR) wavelengths. The fluorescence image capture device  92  may include a variety of devices including, without limitation, CCD chips and photomultiplier devices. Optionally, the fluorescence imaging module  34  may include a fluorescence optical suite  94  to refine or otherwise alter the light entering the fluorescence module  34 . Exemplary components which may be used within the fluorescence optical suite  94  include, without limitations, mirrors, positive lenses, negative lenses, acromats, compound lenses, astigmats, windows, flats, adaptive optics, holographical optical elements, spatial filters, pinholes, collimators, stages, wavelength filters, emission filters, and beam splitters. 
         [0102]    Referring again to  FIG. 2 , a beam director  98  may be included within or proximate to the analyzing section  12  and configured to direct light from the light transport section  14  to the OPS imaging module  30 , the reflectance spectrophotometry module  32 , and/or the fluorescence imaging module  34 . Exemplary beam directors  98  include, without limitation, mirrors including dichroic mirror or elements and dark field mirrors, beam splitters, optical switches, movable or spinning geometric mirrors, corner cubes, prisms, and optical gratings. For example, in one embodiment the beam director  98  comprises a beam splitter directing fifty percent of the incoming light to the OPS imaging module  30  and 50 percent of the incoming light to the reflectance spectrophotometry module  32 . In an alternate embodiment, the beam director  98  comprises a mirror having a non-reflecting area formed thereon, thereby reflecting a portion of light to the spectrophotometer and permitting dark field illumination to the OPS imaging module  30  and/or fluorescence imaging module  34 . Optionally, the beam director  98  may comprise a spinning or moving mirrored polygon configured to reflect light from the light transport section  14  to the OPS imaging module  30 , the reflectance spectrophotometer module  32 , and/or the fluorescence imaging module  34 . In another embodiment, the beam director  98  may be selectively actuated by the user to direct light to at least one of the OPS imaging module  30 , the reflectance spectrophotometer module  32 , the fluorescence imaging module  34 , and/or any additional modules  34  coupled to or in optical communication with the analyzing section  12 . 
         [0103]    In one embodiment of the imaging system  10 , the OPS imaging module  30  is coupled to the light transport section  14 , while the reflectance spectrophotometer module  32  and/or the fluorescence imaging module  34  are positioned external to the imaging system  10  in optical communication therewith. A beam director  98  is positioned within the OPS module  30  and configured to direct a percentage (e.g. fifty percent) of the light received by the analyzing section  12  along an optical path to the reflectance spectrophotometer module  32  and the fluorescence imaging module  34 , while the remaining light is directed to the OPS imaging module  30 . An external beam director (not shown) may be used to further divide the directed light between the reflectance spectrophotometer module  32  and the fluorescence imaging module  34 . 
         [0104]      FIGS. 1 and 3  show an embodiment of a light transport section  14  of an imaging system  10 . In the illustrated embodiment, the light source  20  is positioned proximate to a first lens  100 . A variety of light sources may be used to illuminate the examination substrate  16 , including, without limitation, incandescent lamps, gas discharge lamps, dye lasers, solid state devices such as light emitting diodes, laser diodes, gas lasers, excimer lasers, solid states lasers, and chemical lasers. For example, in one embodiment the external light source  20  comprises an incandescent lamp configured to irradiate the examination surface  16  with white light. In an alternate embodiment, the external light source  20  comprises a mercury lamp thereby stimulating fluorescence in the tissue of the examination substrate  16 . In still another embodiment, the light source  20  comprises one or more LEDs configured to illuminate the examination substrate  16  with light of a discreet wavelength. In still another embodiment, the light transport section  14  may include a number of light sources. For example, a white light source and a UV light source could be used simultaneously. When using multiple light sources a shutter or beam splitter may be used to operate the system with a desired light source. For example, to operate the system using the white light source a shutter could be positioned to prevent the UV light from entering the light transport section  14 . Thereafter, the user may actuate the shutter to illuminate the examination substrate  16  with the UV light rather than white light. In an alternate embodiment, a laser source may be coupled to or in optical communication with the imaging system  10  to treat the examination substrate  16 . For example, the laser source may be used to treat microcirculatory disorders including, without limitation, cancerous tissue, skin discolorations, and/or tissue lesions. Optionally, the imaging system  10  may be operated without a first lens  100 . 
         [0105]    Referring again to  FIGS. 1 and 3 , light emitted by the external light source  20  is incident on a polarizer  102  configured to polarize light to a desired orientation. Thereafter, polarized light rays  104 A,  104 B, and  104 C are incident on a light director  106  configured to direct light rays  104 A′, and  104 B′ to the examination substrate  16 . In the illustrated embodiment, the light director  106  includes a non-reflective or dark field spot  108  formed thereon, thereby permitting light ray  104 C to proceed therethrough and be absorbed by a beam dump or absorber  110 . Exemplary light directors include, without limitation, beam splitters, dichroic junctions, and mirrors. 
         [0106]    A light guide  112  in optical communication with the light source  20  receives and transmits light rays  104 ′A,  104 B′ to the examination substrate  16 . In the embodiment illustrated in  FIG. 3 , the light guide  112  includes an illumination segment  114  and a reflectance segment  116 . The illumination segment  114  transmits light to the examination substrate  16  for illumination, while the reflectance segment  116  transmits reflected light from the examination substrate  16  to the beam director  98  of the analyzing section  12 . Exemplary light guides include, for example, boroscopes, endoscopes, liquid light guides, polymer light guides, glass light guides, tubular bodies, and single or bundled optical fibers. For example,  FIGS. 4A-4D  show several embodiments of light guides  112  which may be used with in the light transport section  14 . As shown in  FIG. 4A , the light guide  112  may include polymer illumination and reflectance segments  114 ,  116 , respectively. The reflectance segment  116  may be optically isolated from the illumination segment  114 , for example, by an internal cladding  115 . Similarly, the illumination segment  114  may include an external cladding  117  thereon. As shown in  FIG. 4B , the reflectance segment  116  may be comprised of a bundle of optical fibers while the illumination segment  114  comprises a polymer light guide. In the alternative,  FIG. 4C  shows a light guide  112  having an illumination segment  114  constructed of a bundle of optical fibers and having a polymer reflectance segment  116  therein.  FIG. 4D  shows another embodiment wherein the illumination segment  114  and the reflectance segment  116  are constructed from a bundle of optical fibers. 
         [0107]    As shown in  FIG. 3 , a lens or lens system  118  may be included within the light transport section  14  to focus the light rays  104 A′,  104 B′. The focal point  120  of the lens system  118  may be located above, at the surface of, or below the surface of the examination substrate  16 . Optionally, the lens system  118  may include a reflector or other device configured to project illuminating light at any angle relative to the longitudinal axis of the light transport body  14 . For example, the lens system  118  may permit a user to project light at an angle of about 90 degrees relative to the longitudinal axis of the light transport body  14 . As shown, the distal tip  137  of the light transport body  14  is positioned a distance D from the examination substrate  16 . As a result, the light transport body  14  does not contact the examination substrate  16  thereby permitting the unimpeded flow of material through the examination substrate  16 . As such, the imaging system  10  permits the user to measure the flow of a material through the examination substrate  16  in real time. Light  122  reflected from the examination substrate  16  is captured by the lens  118  and transmitted through the reflectance segment  116  and the dark field spot  108  of the light director  106  to the beam director  98  analyzing section  12 . Optionally, a polarizer (not shown) may be positioned proximate to the distal tip  137  of the light transport body  14  and configured to polarize light prior to illuminating the examination substrate  16 . 
         [0108]      FIG. 5  shows an alternate embodiment of a light transport section  14 . As shown, an internal light source  18  may be used to illuminate the examination substrate  16 . For example, one or more LEDs may be used to illuminate an examination substrate  16  with a discreet wavelength of light. In an alternate embodiment, the internal light source  18  may comprises LEDs of different color, thereby illuminating the examination substrate  16  with light of multiple discreet wavelengths or with full spectrum light for additional treatment (e.g. laser ablation). Multiple wavelength LED&#39;s can also be used to generate images of the distribution of Hb saturation in an SDF imaging modality. Those skilled in the art will appreciate that the use of LEDs as a light source enables the imaging system  10  to be powered by a battery or other low-power power supply relative to previous systems. For example, the imaging system  10  may be powered by coupling the imaging system  10  to a universal serial port of a personal computer. One or more internal lenses  130  may, but need not be, included within the light transport section  14  and positioned proximate to the internal light source  18 . Similarly, one or more optical polarizers or filters  132  may be positioned proximate to the internal lenses  130 . The internal light sources  18  emit rays  134 A,  134 B which are transmitted to the examination substrate  16  by the illumination segment  136  of the light guide  138 . An examination lens system  140  may be used to focus the light rays  134 A,  134 B to the examination substrate  16 . A focal point  142  of the lens system  140  may be located above, at the surface of, or below the surface of the examination substrate  16 . Thereafter, light rays  144  reflected by the examination substrate  16  are collected by the lens system  140  and transmitted to the beam director  98  of the analyzing section  12  by the reflectance segment  146  formed within the light guide  138 . 
         [0109]      FIG. 6  shows an alternate embodiment of a light guide  150 . As shown, the light guide  150  includes an illuminating segment  152  having a focused or curved distal tip  154 , thereby directing light rays to a focal point within an examination substrate (not shown). The reflectance segment  156  is configured to transmit light from the examination substrate  16  to the analyzing section  12  (See  FIG. 1 ). 
         [0110]      FIGS. 7 and 8  show embodiments of spacer devices which may be affixed to the distal end or distal section of the light guide.  FIG. 7  shows a light guide  160  having a spacer  162  attached thereto. In the illustrated embodiment, the distal section of the light guide  160  may include one or more lock members  164  thereon to securely couple the spacer  164  to the light guide  160 . As such, the spacer  160  may include a locking member recess  166  to accommodate the locking members  164 . The spacer  162  ensures that the light guide  160  remains at least a distance d from the examination substrate  16 .  FIG. 8  shows an alternate embodiment of a spacer  172  coupled to a light guide  170 . The spacers  162 ,  172  may be manufactured from a variety of materials including, without limitation, plastic, rubber, elastomer, silicon, or any other biologically compatible material. In one embodiment, the spacer  162 ,  172  are disposable. 
         [0111]      FIGS. 9 and 10  show an embodiment of a light guide  180  having an alternate embodiment of a spacer  182  attached thereto. The spacer  182  includes a vacuum port  184  attachable to a source of vacuum (not shown). The spacer  182  includes a spacer aperture  186  for irradiating the examination substrate (not shown). The spacer  182  includes one or more attachment orifices  188  thereon which are in communication with the vacuum port  184 . The attachment orifices  188  are formed between an exterior wall  190  and an interior wall  192  of the spacer body  194  and are isolated from the spacer aperture  186 . As such, the spacer  180  is configured to couple to the examination substrate (not shown) when the vacuum source is actuated without adversely effecting the irradiation of the examination surface. As such, the spacer  180  may be rigid or, in the alternative, may be constructed of a compliant material for use within or on compliant organs or structures. Like the embodiments described above, the spacer  182  may be manufactured from a variety of materials and may be disposable. One or more additional ports may be formed on the spacer body  194  for the administration of medicinal or therapeutic agents. 
         [0112]      FIGS. 11 and 12  show an embodiment of an imaging system  200 . As shown, the imaging system  200  includes an illumination body  202  and a reflectance body  204 . The illumination body  202  defines an optics recess  206  configured to receive an optical system  208  therein. The optical system  208  includes a first spacer  210 , a first dark field mirror  212 , a filter spacer  213 , and a filter bank  214 . In the illustrated embodiment, the filter bank  214  includes a clear filter  216 , a yellow filter,  218 , a green filter  220 , and a white filter  222 . A second spacer  224  is positioned proximate to the filter bank  214 . A third spacer  226  is positioned between the second spacer  224  and a lens  228 . A fourth, fifth, and sixth spacers  230 ,  232 , and  234 , respectively, are positioned proximate thereto. A second dark field filter  236  is positioned between the sixth spacer  234  and the seventh spacer  238 . 
         [0113]    Referring again to  FIG. 11 , the reflectance body  204  includes a light director  240  therein. The light reflector  240  includes a non-reflective area  242  formed thereon. In addition, the reflectance body  204  includes an examination tip  244  which is configured to be positioned proximate to the examination substrate (not shown). A polarizer and/or filter  246  and an image capture device  248  may be positioned within the analyzing section  250  of the reflectance body  204 . During use, a light source  252  projects light which is filtered and focused by the optical system  208  located within the illumination body  202 . The light from the light source  252  is directed by the light director  240  to the examination substrate (not shown) located proximate to the examination tip  244 . Light reflected by the examination substrate (not shown) is transmitted to the analyzing section  250  by the light guide  256 , where the light is depolarized and analyzed. 
         [0114]      FIG. 13  shows another embodiment of an imaging system. As shown, the imaging system  300  includes an illumination body  302  and a reflectance body  304 . The illumination body  302  defines an optics recess  306  configured to receive an optical system  308  therein. The optical system  308  includes a first lens  310  and a second lens  312 . Positioned proximate to the first lens  310  is an internal light source  318 . In the illustrated embodiment, the internal light source  318  comprises a number of LEDs configured to project light through the optical system  308 . One or more reflectors  316  may be used to ensure that the light is transmitted through the illumination body  302 . 
         [0115]    As shown in  FIG. 13 , the reflectance body  304  includes a light director  340  therein. The light reflector  240  includes a non-reflective area  342  formed thereon. In addition, the reflectance body  304  includes an examination tip  344  which is configured to be positioned proximate to the examination substrate (not shown). A beam director  398  and an image capture device  348  may be positioned within the analyzing section  350  of the reflectance body  304 . During use, the light source  318  projects light which is focused by the optical system  308  located within the illumination body  302 . The light from the light source  318  is directed by the light director  340  to the examination substrate (not shown) located proximate to the examination tip  344 . Light reflected by the examination substrate (not shown) is transmitted to the analyzing section  350  by the light guide  356 , where the light is analyzed. As shown, the analyzing section  350  may include one or more filters or polarizers  360  therein. 
         [0116]    As shown in  FIGS. 3-5 , at least one light source may be used to illuminate structures located below the surface of a substrate.  FIGS. 14 and 15  show alternate embodiments of imaging systems useful in imaging sub-surface structures while avoiding or reducing the effects of surface reflection.  FIG. 14  shows an imaging system  400  comprising a body  402  having one or more imaging passages  404  formed therein. One or more illumination passages  406  may be formed within the body  402  and may be optically isolated from the imaging passage  404 . In one embodiment, the body is rigid. In an alternate embodiment, the body  402  is flexible. For example, the body  402  may comprise a catheter body. Optionally, the body  402  may include an additional lumen formed therein. For example, an additional lumen may be positioned within the body  402  and may be used to deliver therapeutic agents to a treatment site. In another embodiment, an additional lumen may be used to deliver a vacuum force to a treatment site. In the illustrated embodiment, the illumination passage  406  is positioned radially about imaging passage  404 . In the illustrated embodiment, the illumination passage  406  encircles the imaging passage  404 . In an alternate embodiment, the illumination passage  406  may be positioned anywhere within the body  402 . As shown, the illumination passage  406  is optically isolated from the imaging passage  404 . Therefore, illuminating energy transported through the illumination passage  406  is prevented from entering the imaging passage  404 . As such, the present systems permits side stream dark field imaging (hereinafter SDF). As shown in  FIG. 14 , a feature of SDF imaging is that the illuminated light  412 A and  412 B and the reflected light  414  travel via independent pathways. Thus, the illumination can be placed directly on the tissue and the observations can be made adjacent to it without light crossing over between two paths. 
         [0117]    Referring again to  FIG. 14 , at least one illumination source may be positioned within the illumination passage  406 . In one embodiment, the illumination source  410  comprises one or more LED&#39;s configured to project a selected wavelength to the substrate  420 . In an alternate embodiment, the illumination source  410  comprises a plurality of LED&#39;s configured to project multiple wavelengths to the substrate  420 . For example, as shown in  FIG. 14  a first illumination source  410 A configured to project light to the substrate  420  is positioned at the distal portion  418  of the body  402 . Similarly, a second illumination source  410 B is positioned at the distal portion  418  of the body  402 . As such, the first and second illumination sources  410 A,  410 B are positioned proximate to the substrate  420  under examination. Optionally, any number of illumination sources may be positioned within the body  402 . Exemplary illumination sources include, without limitation, LED&#39;s, LLED&#39;s, incandescent bulbs, laser light sources, etc. 
         [0118]      FIG. 15  shows a perspective view of the distal portion of an alternate embodiment of the imaging device  400  shown in  FIG. 14 . As shown, the body  402  includes an imaging passage  404  and at least one illumination passage  406  optically isolated from the imaging passage  404 . One or more illumination devices  410  are located within the illumination passage  406  and positioned proximate to the distal portion  418  of the body  402 . As such, during use the illumination source are positioned proximate to the substrate  420 . 
         [0119]      FIG. 16  shows a cross sectional view of the distal portion of an embodiment of an imaging device. As shown, The body  402  defines an imaging passage  404  and an illumination passage  406  therein. Like the previous embodiments, the illumination passage  406  is optically isolated from the imaging passage  404 . In the illustrated embodiment, the illumination passage  406  terminates proximate to the distal portion  418  of the body  402 . Optionally, the illumination passage  406  may continue through the length of the body  402 . As such, the illumination passage  406  may include one or more optical fibers configured to deliver illuminating energy to the substrate  420  from a remote location. In the illustrated embodiment, one or more illumination sources  410  are positioned within the illumination passage  406 . For example, one or more LED&#39;s may be positioned within the illumination passage  406 . Like the previous embodiments shown in  FIGS. 14 and 15 , the illumination passage  406  is optically isolated from the imaging passage  404 . Optionally, at least one conduit  424  may traverse through the body  402  thereby coupling the illumination source  410  to a source of power. In the illustrated embodiment at least one lens  422  is positioned within the imaging passage  404  thereby transmitting an image received from a substrate  420  to an image capture device  416 . (See  FIG. 14 ). Optionally, the imaging system shown in  FIGS. 14-16  may be used without a lens  422 . 
         [0120]    With reference to  FIG. 14 , during use, the first illumination source  410 A projects illuminating energy  412 A to the substrate  420 . Similarly, the second illumination source  410 B projects illuminating energy  412 B to the substrate  420 . As shown in  FIG. 14 , the illumination energies  412 A,  412 B are optically isolated from the imaging passage  404 . The first and second illumination energies  412 A,  412 B may be the same or differing wavelengths. Further, as the first and second illumination sources  410 A,  410 B are positioned at the distal portion of the body  402  proximate to the substrate  420 , surface reflections therefrom are reduced or eliminated. As shown, a sub-surface image  414  is transported by the imaging passage  404  from the substrate  420  to an image capture device  416 . Exemplary image capture devices include, without limitation, CCD devices, cameras, spectrophotometers, photomultiplier devices, analyzers, computers, etc. Optionally, one or more lenses  422  may be positioned within the image passage  404  or body  402  to focus illumination energy  412  to the substrate  420  or to assist in the transport of an image  414  from the substrate  420  to the image capture device  420416 , or both. As stated above, the optical isolation of the illumination energy from the image received from the substrate reduces or eliminates the effects of surface reflections while enabling SDF imaging in addition to a variety of alternate imaging modalities or spectroscopic examination of an area. 
         [0121]      FIG. 17  shows an alternate embodiment of an SDF imaging system. As shown, the SDF imaging system  450  comprises a body  452  defining an imaging passage  454  and an illumination passage  456  optically isolated from the imaging passage  454 . The illumination passage  456  includes one or more illumination sources  460  therein. Exemplary illumination sources  460  include, without limitation, LED&#39;s, LLEDs, and incandescent bulbs. As shown, the illumination sources  460  are located proximate to the distal portion  462  of the body  452 . Optionally, the illumination sources  460  may be located some distance from the examination area. As such, illuminating energy may be transported to the examination area through fiber optic conduits positioned within the body  452 . Like the previous embodiments, the body  402  may be rigid or flexible. In the illustrated embodiment, a cap device  464  is positioned over the body  452 . In one embodiment, the cap device  464  may comprise an optically transparent disposable cap device  464  configured to be detachably coupled to the body  452 . During use the cap device  464  may protect the body  402  from biological materials and contaminants. As such, the cap device  464  may be sterile. 
         [0122]    Referring again to  FIG. 17 , at least one lens  466  may be positioned within the imaging passage  454 . The imaging passage  454  is in optical communication with an imaging capture device  468 . The image capture device  468  may comprise any of devices useful in capturing and analyzing an image received from a substrate. For example, the image capture device  468  may comprise a CCD device, photomultiplier, computer, spectrophotometer, and the like. Further, a focusing device  470  may be included within the body  452  or the image capture device  468 . Exemplary focusing devices include, without limitation, additional lenses, mechanical drives or positioners, and the like. Optionally, the SDF imaging system  450  may further include a handle  472  to assist a user in positioning the device. Further, the SDF imaging system  450  may be configured to be coupled to a computer, power source, etc. 
         [0123]      FIG. 18  shows an alternate embodiment of an imaging system. As shown, the imaging system  500  includes a body  502  defining an imaging passage  504  and at least one illumination passage  506  optically isolated from the imaging passage  504 . The illumination passage  506  includes one or more illumination sources  510  therein. As shown, the illumination sources  510  are located proximate to the distal portion  518  of the body  502 , however, the illumination source may be located anywhere on the body  502 . Optionally, a cap device (not shown) may be positioned over the body  502 . For example, the cap device (not shown) may comprise an optically transparent disposable device configured to be detachably coupled to the body  502 . 
         [0124]    Referring again to  FIG. 18 , at least one lens  522  may be positioned within the imaging passage  504 . The imaging passage  504  is in optical communication with at least one image capture device  516 . In the illustrated embodiment, a first image capture device  516 A and a second image capture device  516 B may be used with the system. Further, one or more optical modulators  526  may be positioned within the image passage  504  and configured to modulate imaging signals from the substrate  520 . exemplary optical modulators  526  include, without limitation, mirrors, band pass plates, polarizers, gratings, and the like. The image capture devices  516 A,  516 B may comprise any number of devices useful in capturing and analyzing an image received from a substrate. For example, the image capture devices  516 A,  516 B may comprise CCD devices, spectrophotometers, spectrum analyzers, and the like. 
         [0125]    As shown in the  FIGS. 18 and 19 , the illumination sources  510  may comprise LED&#39;s of a single wavelength. In the alternative, the illumination sources  510  may be configured to irradiate light of multiple wavelengths. For example,  FIG. 19  shows a device having a first illumination source  510 A irradiating at a first wavelength and a second illumination source  510 B irradiating at a second wavelength.  FIG. 20  shows a device having a first illumination source  510 A, a second illumination source  510 B, and a third illumination source  510 C, each illumination source irradiating at a different wavelength. As such, the system may be configured to perform a number of imaging and analyzing procedures with a single device. For example, a first wavelength may be projected to the substrate and used for SDF microcirculation imaging within the underlying vasculature, while a second wavelength may be projected to the substrate and used for detecting oxygen saturation within a blood flow. In short any number of wavelengths of illuminating energy may be projected from the illumination sources  510  and used for any number of analytical processes. For example, the imaging system  500  may be configured to permit imaging of the microcirculation and spectroscopic examination of an area with a single device. 
         [0126]    Referring to  FIGS. 18 and 21 , during use the distal portion  518  of the body  502  may be in contact with the substrate  520  under examination. As such, the illumination source(s)  510  may be positioned in close proximity to the substrate  520 . Optionally, the distal portion  518  may include one or more engaging devices  528  coupled to the body  504  or the cap device (not shown). For example, as shown in  FIG. 22 , the engaging device  528  may comprise an inflatable device configured to dissipate a pressure applied to the substrate  520  by the distal portion  518  of the body  502 . In an alternate embodiment, shown in  FIG. 23 , the distal portion  518  may be positioned proximate to, but not in contact with, the substrate  520 . As such, the illumination sources  510  may be configured to project illuminating energy  512 A,  512 B to the substrate  520 . Optionally, one or more lenses may be in optical communication with the illumination source(s)  510  to aid in the projection of illumination energy  512 A,  512 B to the substrate  520 . 
         [0127]      FIG. 24  shows a block diagram of an embodiment of an imaging and analyzing system. As shown, the imaging system  600  includes an analyzing section  602 , a light transport section  604 , and a light delivery section  606  configured to deliver light to and receive information from a substrate  608 . As stated above, the analyzing section  602  may include any number of analyzing modules configured to process information received from the substrate  608 . In the illustrated embodiment, the analyzing section  602  includes an OPS imaging module  620 , a dark filed illumination module  622 , a reflectance spectrophotometry module  624 , an additional processor module  626 , a fluorescence module  628 , and/or a fluorescence lifetime module  630 . The additional processor module  626  may include one more processing module including, without limitation, Raman spectroscopy devices, fluorescence decay processors, PpIX analyzers, and/or OCT (Hb sat) analyzers, and/or CO2 analyzers. Referring to  FIGS. 18 and 21 , the analyzing section  602  may be configured to receive imaging information from the substrate  520  via the imaging passage  504  formed within the body  502 . Those skilled in the art will appreciate that the present system enables a user to selectively analyze a substrate using multiple imaging modalities, spectrophotometry modalities, and similar analyzing methods using a single device coupled to multiple analyzers. 
         [0128]    The light transport section  604  may comprise a body  634  configured to transport light to and from the substrate  608 . For example, the body  634  may include an image passage  504  and an optically isolated illumination passage  506  as shown in  FIG. 18 . Further, the light transport section  604  may include one or more internal illumination sources  636  positioned therein and configured to irradiate the substrate  608 . Optionally, one or more optical elements  634  may be positioned within the body  632 . Further, the body  632  may be configured to receive and transport light from an external light source  638  to the substrate  608 . 
         [0129]    Referring again to  FIG. 24 , the light delivery section  606  may comprise a direct illumination source  640  configured to be positioned proximate to the substrate  608  and providing direct illumination thereto. As such, the direct illumination source  640  is optically isolated from an image received from the substrate  608 . Exemplary direct illumination sources  640  include LLED&#39;s, LED&#39;s, and the like. Further, one or more white light illumination sources  642  may be used to illuminate the substrate  608 . In one embodiment, the light delivery section  606  may be configured to deliver materials to or receive materials  644  from the substrate  608 . For example, the light delivery section  606  may be configured to infuse therapeutic agents to the substrate  608 . Optionally, the light delivery section  606  may include one or more engaging devices  646  positioned thereon to assist in positioning the system during use. 
         [0130]    As stated above, the preceding imaging and analyzing systems disclosed herein may include one or more cap devices  464  which may be detachably coupled to the body  452 . (See  FIG. 17 ). Generally, the cap device  464  may comprise optically transparent materials configured to protect the body  452  during use. As such, the cap device  464  may be disposable.  FIG. 25  shows an alternate embodiment of a cap device  714 . As shown in  FIG. 25 , the imaging device  700  includes a body  702  having an imaging passage  704  and an illumination passage  706  optically isolated from the imaging passage  704  formed therein. The illumination passage  706  may include one or more conduits  708  coupled to one or more illumination sources  710  located therein. As shown in  FIG. 25 , one or more lenses  712  may be positioned within the imaging passage  704 . A cap device  714  may be coupled to the body  702 . The cap device  714  includes an illumination field  716  optically isolated from an imaging relief  718 . In the illustrated embodiment, the illumination field  716  is positioned proximate to the illumination sources  710  located within the body  702 . Similarly, the imaging relief  718  is positioned proximate to the imaging passage  704 . At least one isolation surface  720  optically isolates the illumination field  716  from the imaging relief  718 . For example, in the illustrated embodiment the isolation surface  720  include a reflective foil  722  thereon which is configured to prevent light from illumination sources  710  from directly entering the imaging passage  704  without first engaging a substrate under examination. Alternate isolation materials may be used on the isolation surface  722  including, without limitation, dyes, foils, impregnations, etc. Optionally, the cap device  714  may be disposable and may be configured to detachably couple to the body  702 . 
         [0131]      FIG. 26  shows yet another embodiment of a reflectance avoidance imaging system. As shown, the reflectance avoidance imaging system  810  includes an imaging device  812  having a spacer or tissue engaging tip  814  attached thereto. The imaging device  812  includes a body  816  having a distal portion  818  configured to receive and engage the spacer  814 . In one embodiment, the spacer  814  is detachably coupled to the body  816 . Optionally, the spacer  814  may be non-detachably coupled to the body  816 . 
         [0132]    Referring again to  FIG. 26 , the imaging body  816  includes one or more conduits formed therein. In the illustrated embodiment, the body  816  includes an imaging conduit  820  configured to project light from a light source (not shown) to a work surface. In addition, the imaging conduit  820  collects light reflected from the work surface and transports the reflected light to a sensor suite (not shown) in communication therewith. Exemplary sensor suites include, without limitation, a CCD or any other type of imaging or sensing device, spectral photometers, and the like. Optionally, a secondary imaging conduit  822  may be positioned within the body  816 . For example, the secondary imaging conduit  822  may be configured to measure CO 2  within tissue through the use of a CO 2  sensing dye. The CO 2  sensing dye enables the measurement of fluorescence decay and may utilize light received from and transmitted through the imaging conduit  820 . Optionally, one or more additional conduits  824  may be positioned within the body  816 . For example, any number of fluid conduits may be formed within the body  816 . 
         [0133]    The spacer  814  includes a spacer body  830  having a coupling portion  832  configured to engage and couple the distal portion  818  of the body  816 . The spacer body  830  further defines an orifice  834  which is in communication with the coupling portion  832 . In the illustrated embodiment, the spacer body  830  includes thread members  836  and attachment devices  838  formed or otherwise disposed thereon to enable the spacer body  830  to couple to the body  816 . Any number or type of thread members  836  and attachment devices  838  may be used to couple the spacer body  830  to the imaging device  812 . The distal portion of the spacer body  830  includes a flange  840  defining the orifice  834 . In the illustrated embodiment, the flange  840  includes one or more vacuum ports  842  portioned thereon, thereby permitting the flange  840  to engage or couple to the a work surface. 
         [0134]    In the illustrated embodiment, the spacer  814  includes one or more vacuum ports  842  which enable the spacer  184  to engage the work surface. Optionally, the spacer body  814  may be configured to avoid contacting the work surface. For example, the spacer body  814  may include an optical system comprised of one or more lenses to enable the imaging device  812  to project and receive light to and from a work surface from a distance without contacting the work surface. For example the optical system may include a zoom lens system. 
         [0135]    Further, the spacer  814  may be formed in any variety of shapes and size. For example, the spacer may include a doughnut-shaped spaces. Furthermore, the spacer  814  may include a bladder or cushion filled with any variety of fluids. Optionally, the fluid may be optically transparent. 
         [0136]      FIG. 27  shows a cross sectional view of an embodiment of a reflectance avoidance imaging system  810 . As shown, the body  816  includes the imaging conduit  820 , the secondary imaging conduit  822 , and the addition conduit  824  formed therein. In addition, vacuum conduits  856  and  858  are formed within the body  816  and a couple to a vacuum source. (not shown) The spacer  814  includes vacuum conduits  870  and  872  which are in communication with the vacuum conduits  856  and  858  of the body  816  and the vacuum ports  842  formed on the spacer  814 . Optionally, one or more attachment members  862  may be positioned on the body  816  to further enable coupling of the spacer  814  to the body  816 . 
         [0137]    In addition to the novel imaging devices described above, the present application describes a method of imaging and determining various biological parameters non-invasively and, if needed, treating an affected area. For example, when operating the above-described system in an OPS imaging mode, flow though the capillaries and related circulatory structures may be examined be viewing red blood flow therethrough. To operate the system in an OPS imaging mode, the user irradiates the examination substrate with white light. The white light is polarized by a polarizer prior to illuminating the examination substrate. Reflected light is captured by the light guide and transmitted to the polarizing section  42  of the OPS imaging module  30  (See  FIG. 2 ). Light reflected by the system optics and the patient&#39;s tissue surface undergoes a polarization shift as a function of scattering and, thus, is cancelled by the polarizing section  42 . As such, sub-surface reflected light fails to undergo a polarization shift and will be captured by the image capture device  50 , thereby enabling sub-surface imaging. Optionally, OPS imaging may be accomplished in combination with dark field illumination. 
         [0138]    Similarly, the imaging system described herein may be used to perform reflectance spectrophotometry using the reflectance spectrophotometry module. A spectrophotometer may be used with the present imaging system to examine the spectral reflectance of the tissue surface. Light from a light source illuminates an examination substrate. The light may comprise an internal light source  18 , external light source  20 , and/or an ancillary light source  22 . (See  FIG. 1 ). Light reflected by the examination substrate  16  is captured by a light transport section  14  and transmitted to a reflectance spectrophotometry module. The spectral characteristic of the reflected light may then be examined and used to determine the hemoglobin saturation, and/or hematocrit concentration within the surface of an organ under investigation. 
         [0139]    Lastly, the imaging system described herein may be used to determine the oxygenation and/or functional state of a tissue cell using the fluorescence imaging module. For example, an examination area may be illuminated with UV light thereby targeting the mitochondrial energy state therein. For example, light having a wavelength of about 360 nm may be used to illuminate the examination substrate. Thereafter, light reflected by the substrate may be captured by the light transport section  14  and transmitted to the analyzing section  12 . (See  FIG. 1 ) The captured light may undergo a lambda shift from 360 nm to about 460 nm. Thereafter, a fluorescence imaging module  34  may analyze the reflected light for to determine the presence of NADH in the cells, thereby showing availability of oxygen within the cells. 
         [0140]    The OPS imaging processor  52 , RFS processor  72 , and fluorescence imaging processor  92  may each contain any number of formulas, algorithms, models, databases, look-up tables, or related information to compute and display their respective reflectance measurements. For example, Beers-Lambert law may be used to determine the concentration of material in the examination substrate based on the absorbance of the light by the examination substrate. 
         [0141]    Also disclosed herein is a method of comprehensively monitoring the microcirculation of a patient. The method may include using any of the aforementioned imaging systems disclosed herein. In one embodiment, the method includes illuminating a tissue substrate, avoiding the reflection of light from the surface of the tissue substrate, receiving light from the tissue substrate, utilizing some of the received light to image microcirculatory flow in the tissue substrate, utilizing some of the received light to determine oxygen availability in the microcirculation, and utilizing some of the received light to determine the adequacy of oxygenation of the tissue cells. 
         [0142]    In one embodiment, the aforementioned method may include utilizing the microcirculatory flow information, the oxygen availability information, and the adequacy of oxygenation of tissue cells information, making an early and sensitive determination regarding states of shock, such as septic, hypovolemic, cardiogenic and obstructive septic shock, in patients, and guiding resuscitation therapies aimed at correcting this condition. 
         [0143]    In another embodiment the aforementioned method may also include utilizing the microcirculatory flow information, the oxygen availability information, and the adequacy of oxygenation of tissue cells information, and making an early and sensitive determination regarding cardiovascular disease and failure of the patient. 
         [0144]    In closing, it is understood that the embodiments of the invention disclosed herein are illustrative of the principals of the invention. Other modifications may be employed which are within the scope of the present invention. Accordingly, the present invention is not limited to that precisely as shown and described in the present disclosure.