Apparatus and method for fluorescence-based imaging and monitoring

Abstract

To provide a method for monitoring stem cells and cancer cells after transplantation, and for detecting contamination of other target surfaces, including non-living targets, in a minimally invasive or non-invasive manner. [Solution] An apparatus for imaging and monitoring a target based on fluorescence, comprising: a light source that emits light for illuminating the target, the light comprising at least one wavelength or wavelength band that causes at least one biomarker associated with the target to fluoresce; the apparatus further comprising a photodetector for detecting the fluorescence.

Description

[Technical Field]

[0001] Fluorescence-based imaging and monitoring devices and methods are disclosed. In particular, the devices and methods may be suitable for monitoring biochemicals and / or biological and non-biological materials for human and veterinary applications, such as in wound care. [Background technology]

[0002] Wound care is a major clinical challenge. Healing and chronic non-healing wounds are associated with numerous biological tissue changes, including inflammation, proliferation, connective tissue remodeling, and bacterial infection, which is a common and significant problem. Some wound infections are not clinically apparent and contribute to the growing economic burden associated with wound care, especially in the elderly population. Currently, gold-standard wound assessment involves direct visual inspection of the wound site under white light combined with the indiscriminate collection of bacterial swabs and tissue biopsies, which often results in delayed, costly, and insensitive bacteriological results. This may affect the timing and effectiveness of treatment. Qualitative and subjective visual assessments merely provide a gross visual observation of the wound site and do not provide information about the underlying biological and molecular changes occurring at the tissue and cellular levels. A relatively simple, complementary method utilizing "biological and molecular" information to improve early identification of such potential changes is desirable in clinical wound management. Early recognition of high-risk wounds will guide therapeutic intervention and, with response monitoring over time, will significantly reduce both morbidity and mortality, particularly from chronic wounds.

[0003] Wound care and management are major clinical issues that pose a significant burden and challenge to healthcare worldwide [Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3]. Wounds are generally classified into wounds without tissue loss (e.g., in surgery), wounds with tissue loss such as burn wounds, trauma, wounds caused as a result of abrasion, or as secondary events in chronic diseases (e.g., venous stasis, diabetic ulcers or pressure ulcers, and iatrogenic wounds such as skin graft donor sites and dermabrasion, pilonidal cysts, non-healing surgical wounds, and chronic cavity wounds). Wounds are also classified by the layers involved: superficial wounds involve only the epidermis; partial-thickness wounds involve only the epidermis and dermis; and full-thickness wounds involve subcutaneous fat or deeper tissues. While restoration of tissue continuity after injury is a natural phenomenon, infection, the quality and speed of healing, fluid loss, and other complications that increase healing time represent major clinical challenges. The majority of wounds heal without complications. However, chronic, non-healing wounds, which involve progressively more tissue loss, present a significant challenge to wound care practitioners and researchers. Unlike surgical incisions, where there is relatively little tissue loss and wounds generally heal without significant complications, chronic wounds also disrupt the normal healing process, which is often not sufficient by itself to achieve repair. Delayed healing generally occurs as a result of compromised wound physiology [non- [Patent Document 4], typically used for venous congestion, diabetic ulcers, or immunosuppressed elderly and These chronic conditions increase treatment costs and reduce patients' quality of life. As the numbers of these groups increase, so will the need for advanced wound care products.

[0004] Traditional clinical evaluation methods for acute and chronic wounds continue to be suboptimal. They are usually based on a qualitative and subjective clinical assessment of a complete patient history, under ambient white light and simple visual evaluation using the "naked eye," and may sometimes involve the use of color photography to capture the general appearance of the wound under white light illumination. 5 Furthermore, periodic reassessment of progress toward healing and appropriate modification of interventions is necessary. Wound assessment terminology is not uniform, many questions surrounding wound assessment remain unanswered, consensus has not yet been reached on the important wound parameters that should be measured in clinical practice, and there is variation in the accuracy and reliability of available wound assessment techniques. Visual assessment is frequently combined with swabs and tissue biopsies for bacteriological culture for diagnosis. Bacterial swabs are collected at the time of wound examination and have the distinct advantage of providing identification of specific bacterial / microbial species. [Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document However, often multiple swabs and / or biopsies are taken from the wound. Some swab collection techniques, which are randomly collected from the site, may actually spread microorganisms along with the wound during the collection process, thus affecting healing time and morbidity of the patient [Dow, 1999]. This is particularly true for swabbing and biopsy procedures that use current microbiopsy procedures. Suboptimal detection of bacterial presence (low diagnostic sensitivity) may be problematic in large, chronic (refractory) wounds, despite the large number of swabs collected. Therefore, current methods for obtaining swabs or tissue biopsies from wound sites for subsequent bacteriological culture are based on untargeted or "blind" swab or punch biopsy approaches, which are not optimized to minimize trauma to the wound or maximize the diagnostic yield of bacteriological testing. Additionally, obtaining swabs and biopsies for bacteriology can be laborious, invasive, painful, and expensive. More importantly, bacteriological culture results often take approximately 2-3 days to return from the laboratory and can be inconclusive [7; 8], thus delaying accurate diagnosis and treatment [3]. Thus, bacterial swabs do not provide real-time detection of the infectious status of wounds. The use of cotton swabs on wounds may seem straightforward, but if not performed correctly, it can lead to inadequate treatment, patient morbidity, and increased hospital stays [1; 2; 3; 6]. The lack of noninvasive imaging methods that objectively and rapidly assess wound repair at the in vivo level (which may be more detailed than based on appearance or morphology) and that support targeted collection of swabs and tissue biopsies for bacteriology is a major obstacle in clinical wound assessment and treatment. Alternative methods are highly desirable.

[0005] As a wound (chronic or acute) heals, many important biological changes occur at the wound site at the tissue and cellular levels [Non-Patent Document 2]. It involves a complex and dynamic interplay of biological processes that can be divided into four overlapping phases that affect the pathophysiology: hemostasis, inflammation, cell proliferation, and maturation and remodeling of connective tissue [9]. A common major complication that arises in the wound healing process, which can range from days to months, is infection caused by bacteria and other microorganisms [2; This can result in serious impairment of the healing process, leading to significant complications. All wounds contain bacteria at various levels, ranging from contamination, to colonization, through critical colonization, to infection, and the diagnosis of bacterial infection is based on clinical symptoms and signs (e.g., visual and olfactory cues).

[0006] The most commonly used terms for wound infection include wound contamination, wound colonization, wound infection, and more recently, critical colonization. Wound contamination refers to the presence of bacteria in the wound without a host response

[10] , wound colonization refers to the presence of bacteria in the wound that augments or initiates a host response

[10] , and critical colonization refers to bacterial proliferation that has not been previously reported but causes a delay in wound healing, usually accompanied by an increase in pain without an obvious host response

[10] . 1; Non-patent document 12]. Wound infection is a cellular infection in tissues with an associated host response. This refers to the accumulation and proliferation of bacteria

[10] . In fact, the term "critical colonization" can be used to describe wounds that are thought to be transitioning from colonization to localized infection. However, the challenge within the clinical setting is certainly to confidently and quickly recognize this situation and eliminate the bacteria as soon as possible, possibly through the use of topical antimicrobial agents. The goal is to reduce the bioburden of wounds. Potential wound pathogens can be classified into different groups, such as bacteria, fungi, spores, protozoa, and viruses, depending on their structure and metabolic capabilities

[13] . Viruses do not usually cause wound infections, but bacteria can infect skin lesions formed during certain viral illnesses. Such infections can occur in several environments, including medical settings (hospitals, clinics) and homes or chronic care facilities. The management of wound infections is increasingly complex; however, treatment is not always guided by microbiological diagnosis. Due to the high incidence of microbial diversity and polymicrobial flora in most chronic and acute wounds, it is believed that identifying one or more bacterial pathogens from wound cultures is valuable. Early recognition of the pathogen causing the wound infection can assist wound care professionals in taking appropriate measures. Furthermore, incomplete collagen formation occurs due to the increased bacterial load, resulting in the formation of excessively vascularized, friable, and loose granulation tissue, which usually leads to wound dehiscence

[14] .

[0007] Accurate and clinically relevant wound assessment is an important clinical tool. However, this process currently poses substantial challenges. Current visual assessment in clinical practice provides only a gross visual observation of the wound site (e.g., the presence of purulent material and eschar). Even best current clinical practice fails to fully utilize crucial objective information about key underlying biological changes occurring at the tissue and cellular level (e.g., contamination, colonization, infection, matrix remodeling, inflammation, bacterial / microbial infection, and necrosis). This is because such indicators i) are not readily available at the time of wound examination and ii) are not currently integrated into the traditional wound management process. Direct visual assessment of wound health using white light relies on detection of color and topographical / histological changes in and near the wound and therefore cannot detect subtle changes in tissue remodeling and may be unreliable. More importantly, direct visual assessment of wounds often fails to detect the presence of bacterial infection because bacteria are invisible to the naked eye under white light illumination. Clinical diagnosis of infection is made with microbiological tests used to identify organisms and their antibiotic susceptibility. Although physical signs of bacterial infection can be readily observed in most wounds using white light (e.g., purulent exudate, crusting, swelling, erythema), they are often significantly delayed, and the patient is already at increased risk of morbidity (and other complications associated with infection) and mortality. Thus, direct visualization using standard white light cannot detect the early presence of bacteria themselves or identify the type of bacteria within the wound. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Bowler et al., Clin Microbiol Rev. 2001, 14:244-269 [Non-patent document 2] Cutting et al., Journal of Wound Care. 1994, 3:198-201 [Non-licensed document 3] Dow et al., Ostomy / Wound Management. 1999, 45:23-40

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Non-licensed literature 9

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[0009] Stem cell transplantation has recently attracted interest, for example, for wound care and treatment. However, tracking the proliferation of stem cells after transplantation is currently a challenge. Tracking and identifying cancer cells is also a challenge. It would be desirable to be able to monitor such cells in a minimally invasive or non-invasive manner. Additionally, it would be useful to provide a method for detecting contamination of other target surfaces, including non-biological targets. [Means for solving the problem]

[0010] Fluorescence-based monitoring devices and methods are disclosed. In one aspect, the devices include optical (e.g., fluorescence and / or reflectance) devices for real-time, noninvasive imaging of biochemical and / or organic materials, e.g., wounds. The devices may be compact, portable, and / or handheld and may provide high-resolution and / or high-contrast images. Such devices may be easily integrated into current wound care practices. The imaging devices may quickly and conveniently provide clinicians / healthcare professionals with valuable wound biometric information, including imaging of connective tissue changes and early detection of bacterial contamination / infection. Furthermore, the devices may facilitate wound margin delineation, image-guided collection of bacterial swabs / biopsies, exogenous molecular biomarker targeting, and imaging of activated optical (e.g., absorption, scattering, fluorescence, reflectance) contrast agents, and may enable monitoring of therapeutic response over time for adaptive intervention in wound management. By utilizing wireless capabilities with dedicated image analysis and diagnostic algorithms, the device may be seamlessly integrated into telemedicine (e.g., e-health) infrastructures for remote access to wound care professionals. Such devices may have applications outside of wound care, including early detection of cancer, monitoring of emerging photodynamic therapy, stem cell detection and monitoring, and instrumentation in dermatology and cosmetic clinics, among other applications.

[0011] In one aspect, an apparatus for fluorescence-based target imaging and monitoring is provided, comprising: a light source that emits light to illuminate a target, the emission comprising at least one wavelength or band of wavelengths that causes at least one biomarker associated with the target to fluoresce; and a photodetector for detecting the fluorescence. In one example, the apparatus may further include a filter holder aligned with the photodetector, the filter holder having a plurality of optical filters selectively alignable with the photodetector, each optical filter for selecting a respective wavelength or wavelength band of light to be detected. In some examples, the target may be selected from at least one of a surgical site, a wound, a tumor, an organ, a skin target, a biological target, a non-biological target, a food product, a plant-based substance, an oral target, an ear, nose and throat target, an ocular target, a genital target, or an anal target. In some instances, all components of the device may be mounted on a portable frame or a stationary platform. In some examples, the device may further include a means or measuring component for measuring the distance from the device to the target. In some examples, the device may include at least two light sources spaced a distance apart for triangulating the distance from the device to the target. In some examples, the device may include an ultrasound source for measuring the distance from the device to the target. In some examples, the device may include a physical scale for measuring the distance from the device to the target. In some examples, the device may further include a data port for transmitting and receiving data. At least one biomarker may be selected from the group consisting of bacteria, fungi, yeast, spores, viruses, microorganisms, parasites, connective tissue, tissue components, exudates, pH, blood vessels, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), microorganisms, vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), epidermal growth factor, epithelial cell membrane antigen (ECMA), hypoxia-inducible factor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin, fibroblast growth factor, transforming growth factor (TGF), fibroblast activation protein (FAP), tissue inhibitor of metalloproteinases (TIMP), nitric oxide synthase (NOS), inducible endothelial NOS, cellular lysosomes, macrophages, neutrophils, and phospholipids. The protein may be selected from at least one of: lymphocytes, hepatocyte growth factor (HGF), anti-neuropeptide, neutral endopeptidase (NEP), granulocyte-macrophage colony-stimulating factor (GM-CSF), neutrophil elastase, cathepsin, arginase, fibroblasts, endothelial cells and keratinocytes, keratinocyte growth factor (KGF), macrophage inflammatory protein-2 (MIP-2), macrophage inflammatory protein-2 (MIP-2), and macrophage chemotactic protein-1 (MCP-1), polymorphonuclear neutrophils (PMN), macrophages, myofibroblasts, interleukin 1 (IL-1), tumor necrosis factor (TNF), nitric oxide (NO), c-myc, beta-catenin, endothelial progenitor cells (EPC), matrix metalloproteinases (MMPs), or MMP inhibitors. In some examples, the emitted light may include a wavelength or wavelength band selected from the range of about 400 nm to about 450 nm, the range of about 450 nm to about 500 nm, the range of about 500 nm to about 550 nm, the range of about 600 nm to about 650 nm, the range of about 650 nm to about 700 nm, the range of about 700 nm to about 750 nm, and combinations thereof. In some examples, the device may further include a memory for recording fluorescence data of the at least one biomarker. In some examples, the apparatus may further include a processor for comparing the fluorescence spectrum of the at least one biomarker to a look-up table of fluorescence spectra of predetermined biomarkers.

[0012] In one aspect, a kit for fluorescence-based target imaging and monitoring is provided, comprising the device described above; and a fluorescent imaging agent for labeling a target biomarker with a fluorescent wavelength or wavelength band detectable by the device. In one example, the biomarker may be bacteria and the imaging agent may be aminolevulinic acid (ALA) or PpIX. In certain examples, the imaging agent may be selected from at least one of a fluorescent dye, a chromogenic dye, a quantum dot (Qdot), a molecular beacon, a nanoparticle having a fluorescent agent, and a scattering or absorbing nanoparticle. In certain examples, the imaging agent may include at least one moiety for targeting a biomarker, for example, the at least one moiety may be selected from at least one of an antibody, an antibody fragment, a peptide, an aptamer, an siRNA, an oligomer, a receptor binding molecule, an enzyme inhibitor, or a toxin. In some examples, the biomarkers include bacteria, fungi, yeast, spores, viruses, microorganisms, parasites, connective tissue, tissue components, exudates, pH, blood vessels, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), microorganisms, vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), epidermal growth factor, epithelial cell membrane antigen (ECMA), hypoxia inducible factor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin, and fibroblast growth factor. Transforming growth factors (TGF), fibroblast activation proteins (FAP), tissue inhibitors of metalloproteinases (TIMP), nitric oxide synthase (NOS), inducible endothelial NOS, lysosomes of cells, macrophages, neutrophils, lymphocytes, hepatocyte growth factor (HGF), anti-neuropeptide, neutral endopeptidase (NEP), granulocyte-macrophage colony-stimulating factor (GM-CSF), neutrophil elastase, cathepsins, arginase, fibroblasts, endothelial cells, and keratinocytes , keratinocyte growth factor (KGF), macrophage inflammatory protein-2 (MIP-2), and macrophage chemotactic protein-1 (MCP-1), polymorphonuclear neutrophils (PMNs), macrophages, myofibroblasts, interleukin 1 (IL-1), tumor necrosis factor (TNF), nitric oxide (NO), c-myc, beta-catenin, endothelial progenitor cells (EPCs), matrix metalloproteinases (MMPs), or MMP inhibitors. In some instances, the kit may further include a calibration target for measuring or calibrating an imaging parameter.

[0013] In one aspect, a method for fluorescence-based target imaging and monitoring is provided, comprising illuminating the target with a light source that emits light at least one wavelength or wavelength band that causes at least one biomarker to fluoresce; and detecting the fluorescence of the at least one biomarker with an image detector. In some examples, the target may be selected from at least one of a surgical site, a wound, a tumor, an organ, a skin target, a biological target, a non-biological target, a food product, a plant-based substance, an oral target, an ear, nose and throat target, an ocular target, a genital target, or an anal target. In some examples, detecting fluorescence may include detecting a fluorescent band of a biomarker. In certain examples, the method may further include comparing the fluorescence band of the at least one biomarker to a look-up table of fluorescence spectra of predetermined biomarkers. In some instances, the at least one biomarker is selected from the group consisting of bacteria, fungi, yeast, spores, viruses, microorganisms, parasites, connective tissue, tissue components, exudates, pH, blood vessels, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), microorganisms, vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), epidermal growth factor, epithelial cell membrane antigen (ECMA), hypoxia inducible factor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin, fibroblast growth factor, transforming growth factor (TGF), fibroblast activation protein (FAP), tissue inhibitor of metalloproteinases (TIMP), nitric oxide synthase (NOS), inducible endothelial NOS, cellular lysosomes, macrophages, and neutrophils. , lymphocytes, hepatocyte growth factor (HGF), anti-neuropeptide, neutral endopeptidase (NEP), granulocyte macrophage colony-stimulating factor (GM-CSF), neutrophil elastase, cathepsin, arginase, fibroblasts, endothelial cells and keratinocytes, keratinocyte growth factor (KGF), macrophage inflammatory protein-2 (MIP-2), macrophage inflammatory protein-2 (MIP-2), and macrophage chemotactic protein-1 (MCP-1), polymorphonuclear neutrophils (PMN), macrophages, myofibroblasts, interleukin 1 (IL-1), tumor necrosis factor (TNF), nitric oxide (NO), c-myc, beta-catenin, endothelial progenitor cells (EPC), matrix metalloproteinases (MMPs), or MMP inhibitors. In certain instances, the method may further include labeling selected biomarkers in the target with at least one fluorescent-emitting imaging agent. In one example, the imaging agent may be aminolevulinic acid (ALA). In certain examples, the imaging agent may be selected from at least one of a fluorescent molecule, a chromogenic dye, a quantum dot (Qdot), a molecular beacon, a nanoparticle having a fluorescent agent, or a scattering or absorbing nanoparticle. In certain instances, the method includes labeling selected biomarkers in the target with a combination of two or more imaging agents, which may be specific for the selected biomarkers. In some instances, the method may further comprise providing the device or kit described above. In one example, the method includes imaging the illuminated target at separate time intervals to obtain multiple images of fluorescent signals from the target, and evaluating the fluorescent signals from each image. and determining a change in the fluorescent signal, which may be indicative of a change in the target. In some instances, the determined change may be compared to a known or expected change. In some instances, the target may be a biological target, and the target may be assessed to monitor the effectiveness of a therapeutic treatment over time. In some instances, the therapeutic treatment may be selected from at least one of drug treatment, treatment with a drug-containing biopolymer, wound debridement, photodynamic therapy, hyperbaric oxygen therapy (HOT), low-level phototherapy, treatment with an anti-matrix metalloproteinase, or treatment with a wound care product. In some instances, wound care products include hydrogels, Theramers™, gels containing silver, The wound dressing may be selected from at least one of artificial skin, ADD stem cells, a moisture-containing wound dressing, a hydrocolloid wound dressing, a transparent membrane wound dressing, an antibacterial agent, an anti-matrix metalloproteinase, an active wound dressing, or hyaluronic acid. In certain instances, the effect of a therapeutic treatment may be monitored at least one of the biological and physiological levels. In an example, detecting fluorescence may include detecting fluorescence from at least one of a surface of the target and a subsurface of the target. In some examples, the method may be used to provide image guidance in a medical or therapeutic procedure, for example, the medical or therapeutic procedure may be selected from at least one of swabbing, brushing, aspiration, biopsy, hyperbaric oxygen therapy, photodynamic therapy, or low-level light therapy. In some examples, the method may be used in combination with additional imaging techniques, such as those selected from at least one of thermal imaging, ultrasound, white light photography, or optical devices. In certain instances, the method may be used to monitor at least one of pharmacokinetics, biodistribution, and photobleaching in PDT. In some instances, the method may be used to detect the presence or location of a bacterial strain, such as at least one selected from at least one of Staphylococcus bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, Enterobacter sakazakii, Campylobacter species bacteria, coliform bacteria, Escherichia coli bacteria, Propionibacterium acnes, or Salmonella. In some instances, the method may be used to distinguish the presence or location of two or more different bacterial strains, for example, Staphylococcus aureus and Pseudomonas aeruginosa, and the different bacterial strains may be distinguished based on their autofluorescence characteristics. In some instances, this method may be used to evaluate a debridement or debridement procedure. In certain examples, the method may further include storing data regarding the detected fluorescence; and transmitting the data to a receiving device, which may be, for example, a component in a telemedicine system. In some instances, the transmission may occur wirelessly. In some instances, the target may be a human target or an animal target. In some instances, this method may be used for the detection of contamination. In some instances, the method may be used for the direct evaluation of swabs or swab cultures from wounds. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram of an apparatus for fluorescence-based monitoring. [Figure 1b] An example of a wound care clinical setting using a fluorescence-based monitoring device is shown. [Figure 2] 1 shows an image of a handheld embodiment of a device for fluorescence-based monitoring. [Figure 3] 1 shows images of live bacterial cultures captured using a fluorescence-based monitoring device. [Figure 3J] 1 shows an example of bacterial monitoring using a fluorescence-based monitoring device. [Figure 4] 1 shows images of a simulated animal wound model demonstrating non-invasive autofluorescence detection of bacteria using a fluorescence-based monitoring device. [Figure 5] 1 shows images of the skin surface of a pork sample demonstrating non-invasive autofluorescence detection of collagen and various bacterial species using a device for fluorescence-based monitoring. [Figure 6] 1 shows images of the muscle surface of a pork sample demonstrating the use of a fluorescence-based monitoring device for the detection of connective tissue and bacterial autofluorescence. [Figure 7] 1 shows images and spectral plots illustrating the use of a fluorescence-based monitoring device to detect fluorescence from bacteria growing in agar plates and on simulated wound surfaces on pork. [Figure 8] 1 shows images of bacterial cultures, illustrating the device for fluorescence-based monitoring, with and without contrast agents. [Figure 9]1 shows images illustrating the use of a fluorescence-based monitoring device for autofluorescence detection of connective tissue and various bacterial species on the skin surface of pork samples. [Figure 10] 1 shows images demonstrating the use of a fluorescence-based monitoring device for fluorescence contrast-enhanced detection of bacterial infection in pork samples. [Figure 10G] An example of the use of a fluorescence-based monitoring device for monitoring the effectiveness of photodynamic therapy is provided. [Figure 11] 1 shows images demonstrating the use of a fluorescence-based monitoring device for imaging of blood and microvasculature. [Figure 12] 1 shows images demonstrating the use of a fluorescence-based monitoring device for imaging of the oral cavity and skin surface. [Figure 12J] An example is given of the use of a fluorescence-based monitoring device for imaging of the skin surface. [Figure 13] 1 shows images demonstrating the use of a fluorescence-based monitoring device for the detection of exogenously derived fluorescent contrast agents in vivo. [Figure 14] 1 shows images illustrating the use of a fluorescence-based monitoring device for fluorescence image-guided surgery using an imaging contrast agent. [Figure 15] 10 shows images illustrating the use of a fluorescence-based monitoring device for recording fluorescence image-guided surgery. [Figure 16] 1 shows images demonstrating the use of a fluorescence-based monitoring device for autofluorescence image-guided surgical ablation of tissue in a mouse myocardial infarction model. [Figure 17] 1 shows images demonstrating the use of a fluorescence-based monitoring device for autofluorescence image-guided surgery of the mouse brain. [Figure 18] 1 shows images demonstrating the use of a fluorescence-based monitoring device in imaging cancer stem cells in mice. [Figure 19] 1 shows images demonstrating the use of a fluorescence-based monitoring device in imaging cancer stem cells in the liver and lung. [Figure 19H] An example of using a fluorescence-based monitoring device for tumor imaging is given. [Figure 19I] An example of using a fluorescence-based monitoring device for tumor imaging is given. [Figure 20] 1 shows images demonstrating the use of a fluorescence-based monitoring device in imaging a mouse model. [Figure 20B] An example of the use of a fluorescence-based monitoring device for imaging of small animal models is provided. [Figure 21] 1 shows the stages of wound healing over time. [Figure 22] 1 is a table showing examples of tissue, cellular and molecular biomarkers known to be associated with wound healing. [Figure 23] FIG. 1 compares healthy wounds with chronic wounds. [Figure 24] An example of chronic wound monitoring is shown. [Figure 24B] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24C] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24D] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24E] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24F] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24G] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24H] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24I] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24J] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24K] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24L] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24M] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24N] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24O] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24P] An example is given of the use of a fluorescence-based monitoring device to image wounds and conditions in clinical patients. [Figure 24Q] An example is given of the use of a fluorescence-based monitoring device for imaging bacterial response to photodynamic therapy. [Figure 24R] An example is given of the use of a fluorescence-based monitoring device for tissue imaging. [Figure 25] 1 is a flow chart showing the management of chronic wounds using a fluorescence-based monitoring device. [Figure 26] An example is given of the use of a fluorescence-based monitoring device for the detection of food contamination. [Figure 27] An example is given of the use of a fluorescence-based monitoring device for the detection of food contamination. [Figure 28] An example is given of the use of a fluorescence-based monitoring device to detect surface contamination. [Figure 28B] An example is given of the use of a fluorescence-based monitoring device to detect surface contamination. [Figure 28C] An example is given of the use of a fluorescence-based monitoring device to detect surface contamination. [Figure 29] An example of the use of a fluorescence-based monitoring device for forensic applications is given. [Figure 30] An example of the use of a fluorescence-based monitoring device for forensic applications is given. [Figure 31] An example of the use of a fluorescence-based monitoring device for forensic applications is given. [Figure 32] An example of the use of a fluorescence-based monitoring device to catalog animals is given. [Figure 33] 1 shows an example of a kit containing a device for fluorescence-based monitoring. [Figure 34] An example is given of the use of a fluorescence-based monitoring device for imaging of cosmetic or dermatological substances. DETAILED DESCRIPTION OF THE INVENTION

[0015] Wound progression is currently monitored manually. The National Pressure Ulcer Advisory Panel (NPUAP) has developed the Pressure Ulcer Scale for Healing (PUSH) tool, which summarizes the five-point scale for characterizing pressure ulcers. developed a tool that uses three parameters to determine a quantitative score that is used to monitor pressure ulcers over time. The quantitative parameters include wound size, tissue type, and amount of exudate or secretion, as well as thermal readings after wound dressing removal. Wounds can also be characterized by their odor and color. Such wound assessments currently do not include significant biometric and molecular information about the wound. Therefore, all wound descriptions are somewhat subjective and are manually written down by either the attending physician or nurse.

[0016] What is desired is a robust, cost-effective, non-invasive, and rapid imaging-based method or device for objectively assessing wound changes at the biological, biochemical, and cellular levels, and for rapidly, sensitively, and non-invasively detecting the earliest presence of bacteria / microorganisms within a wound. Such a method or device for detecting significant wound bioanalysis may serve as an adjunct to traditional clinical wound management procedures to guide important clinical pathology decisions in patient care. Such a device may be compact, portable, and capable of real-time, non-invasive and / or non-contact surveillance of wounds in a safe and convenient manner, seamlessly fitting into routine wound management practices and being easy to use for clinicians, nurses, and wound specialists. This may further include use of the device in home healthcare settings (including patient self-use) and military battlefield environments. Additionally, such an image-based device may provide the ability to monitor wound treatment response and healing in real time by incorporating valuable "biometric" image guidance into the clinical wound assessment process. This will ultimately lead to possible new diagnostics, treatment plans, therapeutic response monitoring, and therefore "tailored" interventions that will enable improved wound healing responses at the individual patient level. Accurate identification of the systemic, local, and molecular factors underlying wound healing problems in individual patients will allow for better tailored treatments.

[0017] Many imaging techniques are becoming available that may enable the fulfillment of the demand for improved clinical diagnosis and treatment of disease. Of these, fluorescence imaging appears promising for improving clinical wound assessment and management. When excited by short wavelength light (e.g., ultraviolet or short wavelength visible light), most endogenous biological components of tissues (e.g., connective tissue such as collagen, elastin, metabolic coenzymes, proteins, etc.) produce longer wavelength fluorescence in the ultraviolet, visible, near-infrared, and infrared wavelength ranges [DaCosta et al. al., Photochem Photobiol. 2003 Oct, 78(4):384-92]. Tissue autofluorescence imaging, the most clinically mature of the imaging techniques, has been used to improve endoscopic detection of early cancer and other diseases in a minimally invasive manner in the gastrointestinal tract [Dacosta (2002) J Gastroenterol Hepatol. Suppl:S85-104], oral cavity [Poh et al., Head Neck. 2007 Jan, 29(1):71-6], lungs [Hanibuchi et al., (2007) J Med Invest. 54:261-6], and bladder [D'Hallewin et al. (2002) Eur Urol. 42(5):417-25].

[0018] Tissue autofluorescence imaging offers a unique means of obtaining real-time biorelevant information of normal and diseased tissues, thus enabling differentiation between normal and diseased tissue conditions [DaCosta, 2003; DaCosta et al. J Clin Pathol. 2005, 58(7):766-74]. This section is based on the inherently different light-tissue interactions (e.g., light absorption and scattering), changes in tissue morphology, and changes in tissue blood volume that occur at the bulk tissue and cellular levels. In tissue, blood is the primary tissue component (i.e., chromophore) that absorbs light. This type of technology is suitable for imaging diseases in hollow organs (e.g., the gastrointestinal tract, oral cavity, lungs, and bladder) or exposed tissue surfaces (e.g., skin). Despite these implications, current endoscopic fluorescence imaging systems are bulky, involve complex diagnostic algorithms, and are expensive. To date, such equipment is found primarily in large clinical centers, with few commercially available systems. Currently, no such optical or fluorescence-based imaging devices exist for wound imaging. However, because wounds are easily accessible, autofluorescence imaging devices would be useful for rapid, noninvasive, noncontact, and real-time wound imaging, thereby detecting and utilizing a wealth of wound bioinformation to overcome current limitations and improve clinical care and management.

[0019] Methods and devices for fluorescence-based imaging and monitoring are disclosed. One embodiment of the device is a portable optical-digital imaging device. The device may utilize a combination of white light, tissue fluorescence, and reflectance imaging and may provide real-time wound imaging, assessment, recording / documentation, monitoring, and / or care management. The device may be handheld, compact, and / or lightweight. The device and method may be suitable for wound monitoring in humans and animals.

[0020] Other uses for the device may include: · Clinical and research-based imaging of small and large animals (e.g. livestock). Detection and monitoring of contamination (e.g. bacterial contamination) in food / animal product preparation in the meat, poultry, dairy, fish and agriculture industries. · Detection of "surface contamination" (e.g. bacterial or biological contamination) in public (e.g. healthcare) and private environments. Multispectral imaging and detection of cancer in human and / or veterinary patients. As a research tool for multispectral imaging and monitoring of cancer in experimental animal models of human disease (e.g. wounds and cancer). Forensic detection of latent fingerprints and body fluids on non-living surfaces. Imaging and monitoring of plaque, caries and cancer in the oral cavity. Imaging and monitoring devices in clinical microbiology laboratories. · Testing of antibacterial agents (e.g. antibiotics) and disinfectants.

[0021] The device generally includes i) one or more excitation / illumination light sources and ii) a detector device (e.g., a digital imaging detector device), which may include one or more optical emission filters or may be combined with a spectral filtering mechanism and may have a display / control screen (e.g., a touch-sensitive screen), image capture and zoom controls. The device may further include iii) a wired / wireless data transfer port / module, iv) a power supply and power / control and / or v) a housing that may be compact and / or lightweight and may have mechanisms for mounting the detector device and / or a handle. The excitation / illumination light source may be an LED array emitting at approximately 405 nm (e.g., ±5 nm) and may be combined with an additional bandpass filter centered at approximately 405 nm to remove / minimize side spectral bands of light from the LED array output so as not to cause light leakage into the imaging detector, which has its own optical filter. The digital imaging detector device may be, for example, a digital camera with at least ISO 800 sensitivity, more preferably ISO 3200 sensitivity, and may be combined with one or more optical emission filters or other equally effective (e.g., miniaturized) mechanized spectral filtering mechanisms (e.g., acousto-optical tunable filters or liquid crystal tunable filters). The digital imaging detector device may have a touch-sensitive display and / or control screen, image capture, and zoom controls. The housing may be an outer hard plastic or polymer shell that encloses the digital imaging detector device and has buttons so that all necessary device controls can be easily accessed and operated by the user. A small heat sink or small mechanical fan, or other heat dissipation device, may be embedded in the device to remove excess heat from the excitation light source, if necessary. The complete device, including its embedded accessories and attachments, may be powered using standard AC / DC power or by a rechargeable battery pack. Additionally, the complete device may be attached or mounted to an external mechanical device (e.g., a tripod or a mobile platform with a rotating arm) to allow mobility of the device for hands-free operation in the clinic. Alternatively, the device may be provided with a mobile frame to make it portable. The device may be cleaned with gauze moistened with water, while the handle may be cleaned with gauze moistened with alcohol.The device may include software that allows a user to control the device, including control of imaging parameters, image visualization, storage of image data and user information, transfer of images and / or associated data, and / or associated image analysis (e.g., diagnostic algorithms).

[0022] A schematic diagram of an example of the device is shown in FIG. 1. The device is shown positioned to image a target 10 or target surface. In the illustrated example, the device has a digital image capture device 1, such as a digital camera, video recorder, camcorder, cellular phone with an embedded digital camera, a "smart" phone with a digital camera, a personal digital assistant (PDA), a laptop / PC with a digital camera, or a webcam. The digital image capture device 1 has a lens 2, which may be aligned to point at the target 10 and detect optical signals emanating from the target 10 or surface. The device has an optical filter holder 3, which may house one or more optical filters 4. Each optical filter 4 may have a different, discrete spectral bandwidth or may be a bandpass filter. These optical filters 4 may be selected and moved from the digital camera lens to selectively detect specific optical signals based on the wavelength of the light. The apparatus may include a light source 5 that generates excitation light to illuminate the target 10 to induce an optical signal (e.g., fluorescence) to be imaged, for example, blue light (e.g., 400 nm to 450 nm) or any other combination of single wavelength or multiple wavelengths (e.g., wavelengths in the ultraviolet, visible, near-infrared, or infrared ranges). The light source 5 may include an LED array, laser diodes, and / or filtered light arranged in various configurations. The apparatus may include a method or apparatus 6 (e.g., a heat sink or cooling fan) to dissipate heat and cool the illumination source 5. The apparatus may include a method or apparatus 7 (e.g., an optical bandpass filter) to remove any undesired wavelengths of light from the light source 5 used to illuminate the target 10 being imaged. The apparatus may include a method or apparatus 8 to use optical means (e.g., the use of a compact, miniature laser diode emitting a collimated light beam) to measure and determine the distance between the imaging device and the target 10. For example, the device may be configured with two sensors as part of a triangulation system to maintain a constant distance between the device and the target 10. Two light sources, such as laser diodes, may be used. Other light sources are possible. The device may also use ultrasound or a physical measure, such as a ruler, to determine the constant distance to be maintained. The device may also include a method or device 9 (e.g., a pivot) to allow manipulation and orientation of the excitation light sources 5 and 8 to manipulate the excitation light sources 5 and 8 to change the illumination angle of the light impinging on the target 10 for different distances.

[0023] The target 10 may be marked with marks 11, allowing multiple images of the target to be obtained and then registered together for analysis. The marks 11 may, for example, involve the use of exogenous fluorescent dyes having different colors, which may produce multiple different light signals when illuminated by the light source 5 and be detectable in the image of the target 10, thereby allowing multiple images of the same area of ​​the subject (e.g., taken over time) to be oriented by registering the different colors and the distances between them together. The digital image acquisition device 1 may include one or more of an interface 12 for a head-mounted display device; an interface 13 for an external printer; an interface 14 for a tablet computer, laptop computer, desktop computer, or other computing device; an interface 15 for a device that allows for wired or wireless transfer of imaging data to a remote location or another device; an interface 16 for a global positioning system (GPS) device; an interface 17 for a device that allows for the use of additional memory; and an interface 18 for a microphone.

[0024] The device may include a power source 19, such as an AC / DC power supply, a compact battery bank, or a rechargeable battery pack. Alternatively, the device may be adapted for connection to an external power source. The device may have a housing 20 that houses all components in one entity. The housing 20 may include means for securing any digital imaging device therein. The housing 20 may be designed to be handheld, compact, and / or portable. The housing 20 may be one or more enclosures.

[0025] Further, referring to FIG. 1, (b) shows an example of the device in a typical wound care facility. (a) shows a typical clinical wound care setup, showing the examination chair and associated tables. (b-c) An example of the device is shown in its hard case container. The device may be integrated into routine wound care procedures to allow real-time imaging of patients. (d) To illustrate the size of the device, an example of the device (arrow) is shown placed on a "wound care cart." (e) The device may be used for imaging under white light illumination, while (f) shows the device being used to take fluorescent images of a wound under dim room light. (g) The device may be used, for example, in a telemedicine / telehealth infrastructure, e.g., to email fluorescent images of a patient's wound to a wound care professional at another hospital via a wireless communication device, such as a smartphone, using a wireless / WiFi internet connection. The device may be used to send high resolution fluorescent images as email attachments from remote wound treatment clinics to wound care professionals for immediate consultation with clinical experts, microbiologists, etc. at specialized clinical wound care and management centers. [Example]

[0026] Example An example of a device for fluorescence-based monitoring is described below. All examples are provided for illustrative purposes only and are not intended to be limiting. Parameters such as wavelengths, dimensions, and incubation times described in the examples may be approximate and are provided by way of example only.

[0027] In this example, the device uses two violet / blue (e.g., narrow emission spectrum, emitting at 405 nm ± 10 nm) LED arrays (Opto Diode Corporation, Newbury Park, California), each placed on either side of the imaging detector assembly as an excitation or illumination source. These arrays are 2.5 × 2.5 cm 2 The LED array may be used to illuminate the tissue surface from a distance of about 10 cm, resulting in a total optical power density on the skin surface of about 0.08 W / cm. 2 This means that at such low power, there is no known possible harm to the target wound or skin surface, or to the eyes, from the excitation light. However, it is best to avoid pointing the light directly at any individual's eyes during the imaging procedure. It should also be noted that 405 nm light does not pose a health hazard according to international standards formulated by the International Electrotechnical Commission (IEC), as shown in more detail on the website http: / / www.iec.ch / online_news / etech / arch_2006 / etech_0906 / focus.htm.

[0028] The one or more light sources may be articulated (e.g., manually) to change the illumination angle and spot size on the imaged surface, e.g., by using an internal pivot, and may be powered, e.g., by electrical connection to a wall outlet and / or a separate portable rechargeable battery pack. The excitation / illumination light beam may be generated by light sources including, but not limited to, individual or multiple light-emitting diodes (LEDs) in any configuration, including ring or array formats, wavelength-filtered incandescent bulbs, or lasers. Selected single and multiple excitation / illumination light sources with specific wavelength characteristics in the ultraviolet (UV), visible (VIS), far-infrared, near-infrared (NIR), and infrared (IR) ranges may be used and may consist of LED arrays configured in various shapes, organic LEDs, laser diodes, or filtered light. The excitation / illumination light sources may be "tuned" to adjust the light intensity emanating from the device during imaging. The light intensity may be variable. The LED arrays may be mounted on individual cooling fans or heat sinks to dissipate heat generated during their operation. The LED array may emit a narrow 405 nm light, which is filtered through a commercially available bandpass filter (Chroma Technology Corp, Rockingham, VT, By filtering the spectrum using a detector optical element (U.S.A.), the emitted light is This may reduce the possibility of "leakage" into the tissue. When the device is held over a tissue surface (e.g., a wound) to be imaged, the illumination source may illuminate the tissue / wound surface with narrow or broad bandwidth violet / blue wavelength or other wavelength or bandwidth light, thereby creating a flat and uniform field within the region of interest. The light may also illuminate or excite the tissue to a shallow depth. This excitation / illumination light may interact with normal and diseased tissue and induce optical signals (e.g., absorption, fluorescence, and / or reflection) within the tissue.

[0029] By changing excitation and emission wavelengths in this manner, the imaging device may interrogate tissue components (e.g., connective tissue and bacteria in a wound) at the surface and at certain depths in the tissue (e.g., a wound). For example, by changing from violet / blue (approximately 400 nm to 500 nm) wavelength light to green (approximately 500 nm to 540 nm) wavelength light, excitation of deeper tissue / bacterial fluorescent sources may be achieved, e.g., in a wound. Similarly, by detecting longer wavelengths, fluorescent emissions from tissue and / or bacterial sources deeper within the tissue may be detected at the tissue surface. For wound assessment, the ability to interrogate surface and / or subsurface fluorescence may be useful, for example, in the detection and possible identification of bacterial contamination, colonization, critical colonization, and / or infection, which may occur at the surface and often deep within the wound (e.g., a chronic, non-healing wound). In one example related to FIG. 6, c) shows detection of bacteria below the skin surface (i.e., deep) after wound debridement. This use of the device to detect bacteria on the surface and in the depths of wounds and surrounding tissues is beneficial for wound care centers. The clinical signs and symptoms may also be evaluated in relation to other clinical signs and symptoms conventionally used in clinical practice.

[0030] An exemplary embodiment of the device is shown in Figure 2. The device may use any standard compact digital imaging device (e.g., a charge-coupled device (CCD) or a complementary metal-oxide semiconductor (CMOS) sensor) as the image acquisition device. The exemplary device shown in a) has a commercially available digital camera securely mounted in a lightweight metal frame with an external power source, two LED arrays to illuminate the object / surface to be imaged, and a convenient handle for imaging. A multi-band filter is held in front of the digital camera, allowing for wavelength filtering of the detected light signal emitted from the object / surface being imaged. The camera's video / USB output cable transfers the imaging data to a computer for storage and later analysis. This example uses a commercially available 8.1-megapixel Sony digital camera (Sony Cybershot DSC-T200 Digital Camera, Sony Corporation, North America). This camera would be preferred because: i) it would be easily integrated into the housing frame; Its slim vertical design, ii) a large 3.5-inch widescreen touch panel LCD for easy control, iii) a Carl Zeiss 5x optical zoom lens, and iv) low light The device may have a built-in flash unit that allows for standard white light imaging (e.g., high-resolution still images or high-resolution video with audio recording output). A camera interface port allows for wired (e.g., USB) or wireless (e.g., Bluetooth, WiFi, and similar) connectivity to a variety of external devices such as head-mounted displays, external printers, tablet computers, laptop computers, personal desktop computers, wireless devices, etc. The digital camera may support (digital) data transfer or third-party add-on modules to allow imaging data to be transferred to remote locations / other devices, Global Positioning System (GPS) devices, devices that allow for additional storage, and microphones. The digital camera may be powered by a rechargeable battery or an AC / DC power source. Digital imaging devices may include, but are not limited to, digital cameras, webcams, digital SLR cameras, camcorders / video recorders, cellular phones with embedded digital cameras, smartphones™, personal digital assistants (PDAs), and laptop / tablet PCs or personal desktop computers, all of which include or are connected to digital imaging detectors / sensors.

[0031] The light signals generated by the excitation / illumination light source are filtered by the imaging device using optical filters (e.g., those available from Chroma Technology Corp, Rockingham, VT, USA) that reject the excitation light but detect selected wavelengths of emission from the tissue, forming an image on a display. As shown in Figure 2 b) and c), an optical filter holder, which may house one or more optical filters with different discrete spectral bandwidths, is attached to the housing frame from the digital camera lens. b) shows the device with a single emission filter in place and an LED array that emits bright purple / blue light when turned on. c) shows the device using a multiple optical filter holder that is used to select the appropriate filter for desired wavelength-specific imaging. d) shows the device being held in one hand during imaging of the skin surface of the foot.

[0032] By selecting and aligning these bandpass filters in front of the digital camera lens, specific optical signals from the tissue / wound surface may be selectively detected based on the desired wavelength of light. Spectral filtering of the detected optical signals (e.g., absorption, fluorescence, reflection) may be achieved using, for example, a liquid crystal tunable filter (LCTF) or an acousto-optic tunable filter (AOTF), which is an electronically tunable solid-state spectral bandpass filter. Spectral filtering may involve the use of continuously variable filters and / or manual bandpass optical filters. These devices may be placed in front of the imaging detector to selectively detect specific optical signals (e.g., absorption, fluorescence, reflection) from the tissue / wound surface based on the desired wavelength of light. , multispectral imaging, hyperspectral imaging, and / or wavelength-selective imaging of tissue may be performed.

[0033] The device may be modified using optically or variably oriented polarizing filters (e.g., linear or circular in combination with the use of optical waveplates) appropriately attached to the excitation / illumination light source and imaging detector device. In this manner, the device may be used to image tissue surfaces using white light reflectance and / or fluorescence imaging with polarized illumination and non-polarized detection, or vice versa, or polarized illumination and polarized detection. This may enable imaging of wounds with minimized specular reflection (e.g., glare from white light imaging) and imaging of fluorescence polarization and / or anisotropy-dependent changes in connective tissue (e.g., collagen and elastin) within the wound and surrounding normal tissue. This may provide useful information about the spatial orientation and organization of connective tissue fibers associated with wound remodeling during healing [Yasui et al., (2004) Appl. Opt. 43: 2861-2867].

[0034] All components of the imaging device may be integrated into a single structure, such as an ergonomically designed, closed structure with a handle, allowing it to be comfortably held in one or both hands. The device may even be provided without a handle. The device may be lightweight and portable and may enable real-time digital imaging (e.g., still images and / or video) of any target surface (e.g., accessible skin and / or oral cavity) using white light, fluorescence, and / or reflectance imaging modes. For imaging, the device may be scanned across the body surface by holding it at a variable distance from the body surface, or may be used in a lit environment / room to image white light reflectance / fluorescence. The device may be used in a dim or dark environment / room to optimize tissue fluorescence signals and minimize background signals from room lighting. The device may be used for direct (e.g., with the naked eye) or indirect (e.g., via a display screen on a digital imaging device) visualization of wounds and surrounding normal tissue.

[0035] The device further need not be implemented to be hand-held or portable, but may be implemented to be attached to a mounting mechanism (e.g., a tripod or stand) for use as a relatively stationary optical imaging device, for example, for white light, fluorescence, and reflectance imaging of objects, materials, and surfaces (e.g., the body). This allows the device to be used on a desk or table, or for "assembly line" imaging of objects, materials, and surfaces. In some embodiments, the mounting mechanism may be mobile.

[0036] Other features of this device include digital image and possibly video recording with audio, It may also include documentation (e.g., with image storage and analysis software), as well as wired or wireless data transmission capabilities for telemedicine / e-health needs. For example, Figures 2e and 2f show an embodiment of the device where the image acquisition device is a mobile communication device such as a cellular phone. The cellular phone used in this example is a Samsung Model A-900, which is equipped with a 1.3 megapixel digital camera. The story is fitted into a holding frame for convenient imaging. e) The "Wound" is marked with fluorescent ink. f) shows the use of the device to image a piece of paper bearing the words "P. acnes." f) shows the imaging of a finger stained with fluorescent ink and the detection of the common skin bacterium P. acnes. Images from a cellular phone may be wirelessly transmitted to another cellular phone or wirelessly (e.g., via Bluetooth® connectivity capabilities) to a personal computer for image storage and analysis. This is in addition to the device's real-time handheld fluorescence imaging and remote / off-site imaging as part of a telemedicine / e-health wound care infrastructure. Indicates the ability to transmit wirelessly to a person.

[0037] To demonstrate the capabilities of the imaging device in wound care and other related applications, a number of feasibility experiments were performed using the specific examples described. During all fluorescence imaging experiments, a Sony camera (Sony Cybershot DSC-T200 Digital Camera, Sony Corporation, North America) was set up with no flash and in "macro" imaging mode. Images were captured at 8 megapixels. A flash was used to capture white light reflectance images. All images were stored on an xD memory card for later transfer to a PC for long-term storage and image analysis.

[0038] All white light reflectance and fluorescence images / videos captured by the instrument were imported into Adobe Photoshop for image analysis. However, image analysis software was also used, such as MatLab. TM (Mathworks) and various image-based spectral algorithms (e.g., red-to-green fluorescence ratio) were used to generate directly relevant results for quantitative detection / diagnostic value. Image post-processing also included mathematical manipulation of the images.

[0039] Imaging of bacteriological samples The imaging device may be useful for imaging and / or monitoring in clinical microbiology laboratories. The device may be used for quantitative imaging of bacterial colonies and quantification of colony growth in routine microbiological assays. Fluorescence imaging of bacterial colonies may be used to measure growth kinetics. Software may be used to automatically count bacterial colonies.

[0040] To demonstrate the utility of this device in a bacteriology / culture laboratory, live bacterial cultures were grown on sheep blood agar plates. The bacterial species were Streptococcus pyogenes, Serratia marcescens, and Staphylococcus aureus. Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa Pseudomonas aeruginosa (American Type Culture Collection (ATCC)) was included. These were grown and maintained at 37°C under standard incubation conditions and used for experiments during the "exponential growth phase." Once colonies were detected on the agar plates (~24 h after inoculation), the agar plates containing individual bacterial species were imaged in the dark using the instrument. Using violet / blue (~405 nm) excitation light, the instrument imaged both combined green and red autofluorescence (emissions between ~490 nm and ~550 nm and ~610 nm and ~640 nm) and red-only autofluorescence (~635 ± 10 nm, the peak emission wavelength for fluorescent endogenous porphyrins) of each agar plate. Fluorescence images of each bacterial species were captured over time for comparison and to monitor colony growth.

[0041] Referring now to Figure 3, a) shows the device being used to image live bacterial cultures growing on sheep's blood agar plates to detect bacterial autofluorescence. b) shows an image of autofluorescence emitted by Pseudomonas aeruginosa. Additionally, the device was used to detect, quantitate, and / or monitor bacterial colony growth over time using fluorescence, as shown in c) using fluorescence imaging of autofluorescent Staphylococcus aureus growth on an agar plate 24 hours after inoculation. Note the presence of clearly distinguishable single bacterial colonies in the bottom image. Using purple / blue (e.g., 405 nm) excitation light, the device was used to detect, quantify, and / or monitor bacterial colony growth over time using fluorescence, as shown in c) using fluorescence imaging of autofluorescent Staphylococcus aureus growth on an agar plate 24 hours after inoculation. Note the presence of clearly distinguishable single bacterial colonies in the bottom image. Using purple / blue (e.g., 405 nm) excitation light, the device was used to detect Streptococcus pyogenes, shown in d), Serratia marcescens, shown in e), Staphylococcus aureus, shown in f), Staphylococcus epidermidis, shown in g), and Escherichia coli, shown in h). We have detected both combined green and red (e.g., 490 nm to 550 nm + 610 nm to 640 nm) and red-only (e.g., 635 ± 10 nm, the peak emission wavelength for fluorescent endogenous porphyrins) emission autofluorescence from several live bacterial species, including E. coli, and Pseudomonas aeruginosa shown in i). Note that the autofluorescence images of bacterial colonies obtained with this device may provide useful image contrast for simple, time-lapse, quantitative measurements of bacterial colonization and growth kinetics, as well as a possible means of monitoring response to therapeutic interventions, such as antibiotics, photodynamic therapy (PDT), low-level light therapy, hyperbaric oxygen therapy (HOT), or advanced wound care products.

[0042] The combination of the high spatial resolution of the camera detector and the significant bacterial autofluorescence signal-to-noise imaging of this device enabled the detection of very small colonies (e.g., less than 1 mm in diameter). This provided a portable and sensitive means of imaging individual bacterial colonies growing on standard agar plates. This provided a means to quantify and monitor the kinetics of bacterial colony growth, as shown in c), and potentially monitor response to therapeutic interventions, such as antibiotics or photodynamic therapy (PDT), over time using fluorescence. Thus, this device will serve as a useful tool in microbiology laboratories.

[0043] Figure 3J shows a) an example of the use of the imaging device in standard bacteriological laboratory work. b) Here, fluorescence imaging of a Petri dish containing Staphylococcus aureus, combined with custom, proprietary image analysis software, allows for rapid enumeration of bacterial colonies. Here, the fluorescence image of the culture dish shows ~182 (±3) colonies (bright bluish-green dots) growing at 37 °C on agar (excitation ~405 nm, emission ~500 nm to 550 nm (green), emission >~600 nm (red)).

[0044] In addition to detecting bacterial strains, it is also possible to detect different bacterial strains (e.g., the presence and The device may be used to distinguish between different species and / or locations. This may be based on the distinct autofluorescence emission characteristics of different bacterial strains, including those within the emission wavelength bands of 490 nm to 550 nm and 610 nm to 640 nm, when excited by violet / blue light, such as light near 405 nm. Other wavelength combinations may be used to distinguish other species on the image. This information may be used to select appropriate treatment, such as antibiotic selection. Such imaging of bacteriological samples may be applicable to wound care monitoring.

[0045] Use in monitoring wound healing The device may be scanned over any wound (e.g., on the body surface) by irradiating the wound area with excitation light. The wound may then be examined using the device by an operator viewing the wound in real time, for example, via a viewer on the imaging device or an external display device (e.g., a head-up display, television display, computer monitor, LCD projector, or head-mounted display). Images obtained from the device may be displayed in real time ( (e.g., via wireless communication) to a remote viewing location, e.g., for telemedicine purposes. Alternatively, the image may be sent directly to a printer or computer memory storage. Imaging may be performed during the routine clinical evaluation of a patient with a wound.

[0046] Prior to imaging, fiducial markers (e.g., using indelible fluorescent ink pens) may be placed on the skin surface near the edge or periphery of the wound. For example, four dots, each of a different fluorescent ink color selected from separate indelible fluorescent ink pens that may be provided to the clinician in a kit, may be placed on the normal skin surface near the edge or border of the wound. These colors may be selected using multispectral band filters that match the excitation light and emission wavelengths of the four ink dots. The wound may be imaged by the device. Image analysis may then be performed by co-registering the fiducial markers for image-to-image alignment. Thus, the user may not need to reposition the imaging device between different imaging sessions. This technique facilitates imaging of wounds over time, and thus the clinical operator may be able to image wounds over time without having to reposition the imaging device during every image acquisition.

[0047] Additionally, to aid in intensity calibration of the fluorescence image, a disposable, simple, fluorescent, standard "strip" may be placed in the field of view during wound imaging (e.g., using a mild adhesive to temporarily attach the strip to the skin). The strip may be impregnated with one or more different fluorescent dyes at various concentrations, which can produce a predetermined, calibrated fluorescence intensity when illuminated by an excitation light source. The fluorescence may have a single (e.g., 405 nm) or multiple fluorescence emission wavelengths or wavelength bands for image intensity calibration. The disposable strip may also have the four dots (e.g., each with a different diameter or size, each with a different fluorescent ink color, and each with a unique black dot next to it) selected from separate indelible fluorescent ink pens. With the strip placed on the normal skin surface near the wound edge or border, the device may be used to capture white-light and fluorescence images. The strip may provide a convenient way to take multiple images of a given wound over time and then align the images using image analysis. Additionally, the fluorescent "intensity calibration" strip may include an additional linear measurement device, such as a ruler of a fixed length, to assist in spatial distance measurement of the wound. Such a strip may be an example of a calibration target that may be used with the device to assist in calibrating or measuring image parameters (e.g., wound size, fluorescence intensity, etc.), although other similar calibration targets may also be used.

[0048] Because tissue fluorescence intensity may fluctuate slightly if the distance between the device and the wound surface varies during multiple imaging sessions, it may be desirable to increase the consistency of imaging results and reproduce the distance between the device and the wound surface. Thus, in some embodiments, the device may have two light sources, such as low-power laser beams, that can be used to triangulate the individual beams to the skin surface to determine a constant or variable distance between the device and the wound surface. This may be done using a simple geometric arrangement between the laser light sources, allowing the clinician to easily visualize the laser target point on the skin surface and adjust the distance between the device and the wound surface during multiple imaging sessions. Other methods for maintaining a constant distance may include the use of ultrasound or a physical ruler.

[0049] Use in white light imaging The device may be used to capture white-light images of the entire wound, along with surrounding normal tissue, using a measuring device (e.g., a ruler) placed within the imaging field of view. This allows for visual assessment of the wound and the calculation / determination of quantitative parameters such as wound area, circumference, diameter, and topographical profile. Wound healing may be assessed by planimetric measurements of the wound area at multiple time points (e.g., at clinic visits) until wound healing. The time course of wound healing may be compared to predicted healing time calculated from multiple measurements of wound radius reduction using the equation R = √A / π (R: radius, A: planimetric wound area, π: constant 3.14). This quantitative wound information may be used to track and monitor changes in wound appearance over time to assess and determine the extent of wound healing by natural means or with any therapeutic intervention. This data may be electronically stored in the patient's health record for future reference. White-light imaging may be performed during the operator's initial clinical evaluation of the patient.

[0050] Use in autofluorescence imaging The device may be designed to detect all or most of the tissue autofluorescence (AF). For example, using a multispectral band filter, the device may image, under excitation at, for example, 405 nm, the following tissue biomolecules: collagen (types I, II, III, IV, V, etc.), which appears green; elastin, which appears greenish-yellow-orange; reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), which emit a blue-green autofluorescence signal; and tissue autofluorescence emitted by bacteria / microorganisms, which appear to have mostly broad (e.g., green and red) autofluorescence emissions; and light absorption associated with blood.

[0051] Image analysis may include calculating the ratio of red to green AF in the image. Intensity calculations may be obtained from regions of interest within the wound image. The pseudocolored image may be mapped to a white light image of the wound.

[0052] Examples in wound healing Referring now to Figure 4, the device was tested in a bacterially contaminated wound model. For this test, pork was purchased from a butcher with the skin attached. To simulate a wound, a scalpel incision was made in the skin, measuring 1.5 cm. 2 4cm from 2 The imaging ranged in size from 100 to 1200 mm, and was performed deep enough to visualize the muscle layer. Several meat samples were imaged with the device without adding bacteria to the mock wound. To do so, the meat samples were left at room temperature for 24 hours to allow bacteria to grow on the meat, and then imaged with the device using both white light reflectance and autofluorescence for comparison.

[0053] To test the device's ability to detect several common bacteria present in connective tissue and typical wounds, six small 1.5cm spots were placed on the skin surface. 2Pork samples with mock wounds were prepared by applying six bacterial species: Streptococcus pyogenes, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa to each wound incision site. An additional small incision was made in the skin of the meat to serve as a control, without adding any bacteria. However, it was anticipated that this site would likely become contaminated over time with bacteria from the other six incisions. The device was used to image the bacteria-rich meat samples. Imaging was performed using white light reflectance and violet / blue light-induced tissue autofluorescence with both a dual-emission band (450 nm to 505 nm and 590 nm to 650 nm) and a single-band (635±10 nm) emission filter on the left side, as well as a single-band filter, at 24-hour intervals over a 3-day period, during which the meat samples were maintained at 37° C. Imaging was also performed on the Styrofoam containers in which the meat samples were stored for 3 days.

[0054] Figure 4 shows the results of using this device for noninvasive autofluorescence detection of bacteria in an animal mock wound model. Under standard white-light imaging, bacteria were not visible to the naked eye within the wound site, as shown in a) and magnified in b). However, under violet / blue excitation light, the device was able to identify the presence of bacteria within the wound site based on the dramatic increase in red fluorescence from bacterial porphyrins against the bright green fluorescent background from connective tissue (e.g., collagen and elastin), as seen in c) and magnified in d). Comparing b) and d) shows the dramatic increase in red fluorescence from bacterial porphyrins against the bright green fluorescent background from connective tissue (e.g., collagen and elastin). It was notable that bacterial colonies were also detected on the skin surface based on their green fluorescence emission, which revealed individual colonies as discrete green dots on the skin with autofluorescence. These were not visible under white-light examination. Fluorescent imaging of connective tissue aids in determining the wound edges as seen in e) and f) and in some areas of the skin (in c) marked with "* ") appeared more red fluorescent than other areas, indicating a possible subcutaneous infection with porphyrin-producing bacteria. e) and f) further demonstrate that the device detected red fluorescent bacteria within the surgical wound, which were invisible to the naked eye under white light imaging.

[0055] The device maps the biodistribution of bacteria within the wound site and on the surrounding skin, thus assisting in targeting specific tissue areas requiring swabbing or biopsy for microbiological testing. Furthermore, the imaging device may be used to monitor the response of bacterially infected tissue to various medical therapies, including the use of antibiotics and other therapies such as photodynamic therapy (PDT), hyperbaric oxygen therapy (HOT), low-level light therapy, or anti-matrix metalloproteinase (MMP). The device may be useful for visualizing the biodistribution of bacteria on the surface of the wound and within tissue deep within the wound, as well as in surrounding normal tissue. The device may therefore be useful for showing the spatial distribution of infection.

[0056] Use of the device with contrast agents in wound monitoring The device may be used with an exogenous imaging agent, such as a low dose of the prodrug aminolevulinic acid (ALA), which may be administered topically to the wound and imaging performed 1-3 hours later to enhance the red fluorescence of wound bacteria.

[0057] The prodrug aminolevulinic acid (ALA) induces porphyrin formation in almost all living cells. Exposure to ALA can induce protoporphyrin IX (PpIX) fluorescence in many bacterial species. The use of ultra-low doses of ALA may induce PpIX formation in bacteria, resulting in enhanced red fluorescence emission and thereby improving the red-to-green fluorescence contrast of bacteria imaged with this device. While ALA itself is non-fluorescent, PpIX fluoresces near 630 nm, 680 nm, and 710 nm, with the strongest emission at 630 nm. The imaging device may then be used to image the green and red fluorescence from the wound and surrounding tissue. After ALA (~20 μg / mL) is applied to the wound, the time required to obtain a significant / measurable increase in red (e.g., peak at 630 nm) fluorescence using the imaging device ranges from 10 to 30 minutes, but this time can be optimized and depends on the ALA dose, which can also be optimized.

[0058] Therefore, clinical personnel can premix ALA. ALA is typically available commercially in a lyophilized form with physiological saline or in other types of commercially available creams, ointments, hydrogels, wound dressings, etc., in a given dose, and clinical personnel can administer the agent locally by spraying, pouring, or carefully applying it to the wound area before imaging. After approximately 10 to 30 minutes (this time may vary), fluorescent imaging can be performed in a dimly lit or darkened room. Under white light, bacteria invisible to the naked eye and possibly poorly autofluorescent will appear as bright red fluorescent areas in and around the wound. Fluorescent imaging can be used to direct targeted swabbing, biopsy, and / or fine needle aspiration of the wound for bacterial culture based on the unique bacterial fluorescent signal, which may be performed at various depths for superficial and deep wounds.

[0059] Furthermore, the device may be used in combination with exogenous "prodrug" agents, including but not limited to 5-ALA, which is FDA approved for clinical therapeutic applications, to increase the endogenous production of porphyrins in bacteria / microorganisms, thereby increasing the intensity of the unique "porphyrin" fluorescent signal emitted by these bacteria and improving the detection sensitivity and specificity of the device. Thus, the device may be used for subsequent image-guided targeted swab / biopsy, or for other treatments such as photodynamic therapy (PDT) or hyperbaric oxygen therapy. For treatment with HOT, photosensitizer-induced fluorescence (e.g., PpIX) in bacteria growing in culture or in a patient's wound may be conveniently imaged. The device has the ability to increase signal-to-background for sensitive detection of bacteria in and near wounds, for example, when used with commercially available consumable fluorescent contrast agents. Note that ALA is commercially available.

[0060] In one example, the device was used to image live bacterial cultures (S. aureus grown on agar plates for 24 hours prior to imaging) using violet / blue excitation light. After incubation of S. aureus with ~20 μg / mL ALA for 30 minutes at 37°C, a significant increase in red fluorescence from the bacteria was detected compared to colonies that did not receive any ALA. Thus, the device may be used to increase signal-to-background for sensitive bacterial detection, for example, in wounds, utilizing a contrast agent approach. The time required for ALA to increase bacterial PpIX fluorescence to significant levels in culture was approximately 0.5 hours, suggesting that this method may be clinically practical. Testing of simulated bacterially contaminated meat samples yielded results similar to those obtained from bacterial cultures. Topical application of 0.2 μg / mL ALA by spraying onto wounds on pig skin resulted in a dramatic increase in the red fluorescence contrast of bacterial porphyrins approximately 2 hours after ALA administration, indicating that bacterial contamination within the wound site and other parts of the skin surface, previously invisible to the naked eye by white-light imaging, may be detectable by fluorescence imaging with this device.

[0061] Uses involving exogenously derived molecularly targeted and activated imaging agents - Patent Application 20070122997 The availability of commercially available fluorescent molecular bacteriological detection and viability kits may provide additional uses for the device in wound care. Such kits may be used to rapidly and quantitatively distinguish between live and dead bacteria, even in mixed populations containing a range of bacterial species. Traditional direct enumeration assays for bacterial viability are typically based on metabolic signatures or membrane integrity. However, methods that rely on metabolic signatures often only work for a limited subset of bacterial populations, and methods that assess bacterial membrane integrity commonly have high levels of background fluorescence. Both of these types of measurements also suffer from the disadvantage of being highly sensitive to growth and staining conditions.

[0062] using commercially available fluorescent labeling kits, such as Alexa Fluor active esters and kits (e.g., Zenon Antibody Labeling Kits and / or EnzChek Protease Assay Kits, Invitrogen) for labeling proteins, monoclonal antibodies, nucleic acids, and oligonucleotides (Invitrogen); Suitable exogenous optical molecular targeting probes may be prepared, for example, fluorescent dye bioconjugates that are luminescent in the following wavelength ranges: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 540, Alexa Fluor 550, Alexa Fluor 560, Alexa Fluor 570, Alexa Fluor 580, Alexa Fluor 590, Alexa Fluor 600, Alexa Fluor 610, Alexa Fluor 620, Alexa Fluor 630, Alexa Fluor 640, Alexa Fluor 650, Alexa Fluor 660, Alexa Fluor 670, Alexa Fluor 680, Alexa Fluor 690, Alexa Fluor 700, Alexa This covers the following dyes: Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. The numbers shown here are , indicates the excitation wavelength of the dye. These kits may provide well-differentiated fluorescence emission spectra, allowing for multiple options for multicolor fluorescence detection and fluorescence resonance energy transfer based on appropriate selection of fluorescence emission filters associated with the imaging device. These fluorescent dyes provide high absorbance at the wavelengths of the maximum output of common excitation sources, and they are bright and exceptionally photostable fluorescent of their bioconjugates, facilitating conjugation in clinical laboratories and providing sufficient water solubility to prevent conjugate precipitation and aggregation. The fluorescence spectra of the dyes are insensitive to a wide range of pH. This makes them particularly useful for wound imaging, as wound pH can vary. In addition, There are other commercially available and non-commercially available fluorescent agents that are suitable for in vivo imaging of wounds and that can be combined with the above-described devices, including, for example, fluorescent stagnation agents from VisEn Medical (Boston, Mass., USA) and various wound enzyme or protease-activated probes.

[0063] These targeted fluorescent bioconjugates may be prepared using such labeling kits and stored in a light-proof container to avoid photobleaching prior to clinical examination of the wound using the imaging device in fluorescence mode. Such fluorescent bioconjugates may be prepared as solutions of a known appropriate concentration prior to fluorescence imaging of the wound using the device and then administered / applied directly to the wound and surrounding normal tissues locally (e.g., using aerosol / spray, irrigation techniques, etc.), or systemically via oral administration in a drink, or via intravenous infusion.Such dyes may be targeted to specific biological components by targeting moieties, such as bacteria, fungi, yeast, spores, viruses, microorganisms, parasites, exudates, pH, blood vessels, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), microorganisms, certain types of connective tissue (e.g., collagen, elastin), tissue components, vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), epidermal growth factor, epithelial cell membrane antigen (ECMA), hypoxia inducible factor (HIF-1), carbonic anhydrase IX (CAIX), laminin, fibrin, fibronectin, fibroblast growth factor, transforming growth factor (TGF), fibroblast activation protein (FAP), enzymes (e.g., caspases, matrix metalloproteinases (MMPs), etc.), metalloproteins, etc. Tissue inhibitors of enzymes (e.g., TIMPs), nitric oxide synthase (NOS), inducible endothelial NOS, lysosomes of cells, macrophages, neutrophils, lymphocytes, hepatocyte growth factor (HGF), anti-neuropeptides, neutral endopeptidase (NEP), granulocyte-macrophage colony-stimulating factor (GM-CSF), neutrophil elastase, cathepsins, arginase, fibroblasts, endothelial cells, and keratinocytes, keratinocyte growth factor (KGF), macrophage inflammatory protein-2 (MIP-2), and macrophage chemotactic protein-1 (MCP-1), polymorphonuclear neutrophils (PMNs) and macrophages, myofibroblasts, interleukin-1 (IL-1), and tumor necrosis factor (TNF), nitric oxide (NO) (Calbiochem, Model DAF-2 DA kit), and bone marrow-derived c-myc and beta-catenin, circulating endothelial progenitor cells (EPCs). Exogenous optical agents may include, but are not limited to, activated (e.g., targeted) molecular beacons, nanoparticles with fluorescent agents (e.g., labeled on their surface and / or containing or carrying fluorescent agents), and scattering or absorbing nanoparticles (e.g., gold, silver).

[0064] The LIVE / DEAD BacLight™ Bacterial Viability Kit (Invitrogen, Molecular Probes) assay uses SYTO® 9 green fluorescent nucleic acid stain and red fluorescent nucleic acid stain. This study utilizes a mixture of acid stains, propidium iodide, but these fluorescent dyes may be replaced with other existing or emerging fluorescent dyes. These stains differ both in their spectral characteristics and their ability to penetrate healthy cells. When used alone, SYTO9 stain labeled both bacteria with intact and damaged membranes. In contrast, propidium iodide penetrated only bacteria with damaged membranes, comparable to SYTO9 stain for nucleic acid binding sites when both dyes were present. When mixed in the recommended ratio, SYTO9 stain and propidium iodide produced green fluorescent staining of bacteria with intact membranes and red fluorescent staining of bacteria with damaged membranes. Thus, live bacteria with intact membranes fluoresce green, while dead bacteria with damaged membranes fluoresce red. The background remains virtually non-fluorescent. As a result, the ratio of green to red fluorescent intensity provides a quantitative indicator of bacterial viability.

[0065] Observation of live and dead bacteria was performed using imaging equipment with suitable optical filter sets. These fluorescent agents may be performed separately or simultaneously, depending on the device. Similarly, similar fluorescent assay kits are available for Gram stain (i.e., positive / negative) identification of bacteria; such identification is a useful parameter in wound treatment planning and may be used in conjunction with an imaging device. Such fluorescent agents are general and applicable to most bacterial species, and may be used to determine bacterial viability and / or Gram stain directly on / in the wound, or in culture samples from ex vivo swabs or tissue biopsies obtained from the wound site (e.g., superficially or deeply) for real-time quantitative evaluation using an imaging device. Such fluorescent agents may be prepared as solutions of a known appropriate concentration prior to fluorescent imaging of the wound using the device, and then administered / applied directly to the wound and surrounding normal tissues, either topically (e.g., using aerosol / spray, irrigation techniques, etc.), or systemically, perhaps via intravenous infusion. After a defined time for the fluorescent agent to react with the target, imaging may then be performed accordingly. Prior to imaging with the device, it may be necessary to wash away unlabeled fluorescent agent. Physiological saline solution may be used for this purpose. Target-bound fluorescent agent may remain within the wound and surrounding tissue for fluorescent imaging.

[0066] Thus, when used with a fluorescent reporter system, the imaging device may provide a relatively rapid means of assessing bacterial viability after contact with an antimicrobial agent. The ability to measure the same patient or animal repeatedly reduces variability within treatment experiments, resulting in equal or greater confidence in determining treatment efficacy. This non-invasive, portable imaging technique reduces the number of animals used during such studies and is applicable to the evaluation of test compounds in drug discovery.

[0067] Many commercially available organic fluorophores have properties that depend on hydrogen ion concentration, making them useful as probes for measuring pH; they typically have pH-sensitive UV / visible light absorption properties. Most commercially available pH-sensitive fluorescent dyes used in intracellular studies have low fluorescent signals in acidic media, or alternatively, the pKa of the dye is outside the critical intracellular pH window between 5 and 8 pH units. However, other pH-sensitive fluorescent agents respond by increasing their fluorescence intensity. For example, Invitrogen / Molecular Probes is a collection of various fluorescent pH indicators, their conjugates, and methods for measuring pH in biological systems. Other reagents for the determination of pH are provided. Among these are several probes with unique optical responses and specialized localization properties: for example, visible-light-excitable SNARF pH indicators allow researchers to determine intracellular pH in the physiological range using dual-emission or dual-excitation ratiometric techniques, thus providing a useful tool for confocal laser scanning microscopy and flow cytometry. LysoSensor probes and indicators based on the Oregon Green fluorophore may be used to estimate the pH of acidic organelles in cells. Fluorescent pH indicators conjugated to dextran may also be used. After application to cells, the indicator dextran is well retained, may not bind to cellular proteins, and may have little tendency to compartmentalize. Again, such fluorescent agents may be prepared as solutions of a known appropriate concentration prior to fluorescent imaging of the wound using the device, and then administered / applied directly to the wound and surrounding normal tissue either locally (e.g., using aerosol / spray, lavage techniques, etc.) or systemically, for example, by intravenous injection or orally.

[0068] Example Referring now to Figure 24, as an example, imaging devices may be used clinically to determine the healing status of chronic wounds and the success of wound excision. For example, a typical foot ulcer from a diabetic patient is shown in the figure with (i) a non-healing margin (callus) containing ulcer-initiating cells with molecular markers indicative of impaired healing, and (ii) phenotypically normal, but physiologically damaged, cells that can be stimulated to heal. Despite the appearance of the wound after wound excision, It may not be healing and may require evaluation for the presence of specific inhibitory molecular markers and / or hyperkeratotic tissue (e.g., c-myc and β-catenin). Using an imaging device in combination with exogenously derived fluorescently labeled molecular probes for such molecular targets, clinicians can determine the in situ expression of molecular biomarkers. Once the wound is debrided using this device, fluorescent imaging and image analysis of the wound area may allow targeted biopsy for subsequent immunohistochemistry, which may determine whether the wound was adequately debrided. If the wound was not adequately debrided, as shown in the lower left image, cells positive for c-myc (appearing green) and nuclear β-catenin (appearing purple) can be identified based on their fluorescence, indicating the presence of ulcerogenic cells that prevent the wound from healing properly and the need for further wound debriding. Non-healing may also be indicated by an increased epidermal thickness, an increased keratinized layer, and the presence of nuclei within the keratinized layer. When wound excision is successful, as shown in the lower right panel, no staining for c-myc or β-catenin is observed, indicating the absence of ulcerogenic cells and successful wound excision. While these inhibitory markers may be useful, the goal is actual healing, as defined by the appearance of new epidermis, reduction in wound area, and absence of drainage / purulent discharge. This information may be collected using a fluorescent imaging device and stored electronically in the patient's medical record, providing an objective analysis linked to pathology and microbiology reports. By comparing predicted healing time with actual healing (i.e., healing progression) time using an imaging device, adaptive treatment strategies may be implemented on a patient-by-patient basis.

[0069] Figure 24B shows an example of the use of the device to image wound healing in a pressure ulcer. a) Shows a white-light image obtained with the device of the right foot of a diabetic patient with a pressure ulcer. b) The corresponding fluorescence image shows bright red fluorescence of bacteria not visible with standard white-light examination (bacteriological results confirmed the presence of aggressive growth of Staphylococcus aureus) (yellow arrow). Note the aggressive growth of Staphylococcus aureus near the margins of the non-healing wound (long yellow arrow). c-d) Shows spectrally separated (unmixed) red-green-blue images of the raw fluorescence image in b), which are used to form a spectrally coded image map of green (e.g., collagen) and red (e.g., bacteria) fluorescence intensities calculated using a mathematical algorithm and displayed in pseudocolor using a color scale. f-g) An example of an image processing method is shown in which the red / green fluorescence intensity ratio is calculated to increase the contrast of the internally derived bacterial autofluorescence signal, revealing the presence and biodistribution of bacteria (red-orange-yellow) in and near an open wound. These data demonstrate the ability to use custom or commercially available image analysis software to perform mathematical analysis of the fluorescence images acquired by this device and display them in a manner meaningful for clinical use, even in real time (scale 1 cm).

[0070] Figure 24C shows an example of the use of this device to image a chronic, non-healing wound. a) A white-light image obtained with this device of the left breast of a female patient with pyoderma gangrenosum shows the chronic, non-healing wound (blue arrow) and the healed wound (red arrow). Bacteria are typically not visualized by standard white-light visualization used in conventional clinical wound examinations. b) A corresponding fluorescence image of the same wound (in this example, using 405 nm excitation, 500-550 nm emission (green), and >600 nm emission (red)) is shown. The non-healing wound appears dark under fluorescence (primarily due to blood absorption of excitation and fluorescence emission), while bacteria appear as intermittent bright red dots in the healed wound (red arrow). Under fluorescence, normal surrounding skin appears cyan-green due to endogenous collagen fluorescence (405 nm excitation). In contrast, the non-healing wound (blue arrow) appears to have a very bright band of red fluorescence near the wound border, and swab cultures (bacteriology) showed intense growth of Staphylococcus aureus (few gram-positive bacilli and sparse Microscopy confirmed the wound contained numerous gram-positive cocci. c) White-light images of the healed wounds in a and b), and d) the corresponding fluorescent image showing bright red fluorescence from bacteria not visible to the naked eye under white light (pink arrows). e) White-light image of a non-healing chest wound and f) its corresponding fluorescent image. Bacteria (Staphylococcus aureus) appear to be primarily localized near the wound edge / border (yellow arrows), while fewer bacteria are present within the wound (X) as judged by bacterial biodistribution directly visualized using fluorescent imaging but not visible under white light (black arrows, e) (scale scale in cm).

[0071] Figure 24D further illustrates imaging of a chronic, non-healing wound using an exemplary imaging device. a) White-light image of the left breast of a female patient with pyoderma gangrenosum, obtained with the device, shows the chronic, non-healing wound (blue arrow) and the healed wound (blue arrow). Bacteria cannot be visualized by standard white-light visualization used in conventional clinical wound examination. b) Corresponding fluorescence image of the same wound (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)). While the nipple appears normal under white light with no obvious bacterial contamination, fluorescence imaging shows the presence of bacteria emanating from the milk duct. A nipple swab sample showed the bacteria to be Staphylococcus epidermidis (occasional growth was observed on culture) (scale in cm).

[0072] Figure 24E shows the central region and border of a chronic, non-healing wound imaged using the imaging device. a) White-light image of the left breast of a female patient with pyoderma gangrenosum, obtained with the device, showing the central region and border of the chronic, non-healing wound. a) White-light image and b) corresponding fluorescence image of the non-healing chest wound are shown (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)). Bacteria (Staphylococcus aureus; indicated by bacterial swabs) appear to be primarily localized near the wound edge / border, while fewer bacteria are located within the wound (X) as judged by bacterial biodistribution directly visualized using fluorescence imaging but not visible under white light (scale in cm).

[0073] Figure 24F shows additional images of a chronic, non-healing wound obtained using the imaging device. a) A white-light image of the left breast of a female patient with pyoderma gangrenosum, obtained using the device, shows a chronic, non-healing wound. Bacteria cannot be visualized by standard white-light visualization used in clinical wound examinations. b) A corresponding fluorescence image of the same wound (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)) is shown. Fluorescence imaging shows the presence of bacteria near the wound edge and border before (b) and after (c) cleaning. In this example, cleaning involved wiping the wound surface (inside and outside) with standard gauze and phosphate-buffered saline for 5 minutes. After cleaning, the red fluorescence of the bacteria is significantly reduced, indicating that some red-fluorescent bacteria may reside beneath the tissue surface near the wound edge. A small amount of bacteria (red fluorescence) remained within the wound after cleaning. This demonstrates real-time monitoring of the effectiveness of wound cleansing using an imaging device. As a further example, d) shows a white-light image of a chronic, non-healing wound on the left calf of the same patient. e) shows the corresponding fluorescent images before (e) and after (f) cleansing. Swab sampling of the central region of the wound revealed occasional growth of Staphylococcus aureus, along with aggressive growth of Staphylococcus aureus at the edges (yellow arrows). Cleansing resulted in a reduction of fluorescent bacteria (S. aureus) on the wound surface, as determined using a handheld optical imaging device. Using the imaging device, bacteria invisible to the naked eye under white light were detected in real time. This allowed for a change in patient treatment strategy, either to completely re-clean the wound and surrounding area (bacterially contaminated) after fluorescent imaging, or to clean it for the first time for de novo detection of bacteria. Furthermore, disposable adhesion measurement-calibration devices were used to assist imaging-focusing. Note the use of a "strip." This "strip" may be attached to any portion of the body surface (e.g., near the wound) to allow spatial measurement of the wound. This calibration strip may also be differentially fluorescent and used to add patient-specific information to the image, including using multiple exogenous fluorescent dyes for "barcoding" purposes, which can be directly integrated into the fluorescent image of the wound (scale in cm).

[0074] Figure 24G shows the use of an imaging device to monitor wound healing over time. The imaging device was used to track changes in the healing status and bacterial biodistribution (e.g., contamination) of a non-healing chronic wound from the left breast of a female patient with pyoderma gangrenosum. White-light images (a-m) and corresponding fluorescence images of (b-n) healed wounds and (c-o) chronic non-healing wounds are shown over the course of 6 weeks, taken using the imaging device in both white-light and fluorescence modes (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)). In (b-n), the presence of small, bright red fluorescent bacterial colonies was detected (yellow arrows), and their localization changed over time within the healed wound. Bacterial swabs confirmed that no bacteria were detected under a microscope and no bacterial growth was observed in culture. In contrast, non-healing wounds (c-o) have a very bright red fluorescent band near the wound border, and swab cultures (bacteriology) are confirmed to contain robust growth of Staphylococcus aureus (confirmed by microscopy with few gram-positive bacilli and sparse gram-positive cocci), which changes in biodistribution over time (i.e., c-o). These data demonstrate that imaging devices can provide real-time biological and molecular information and can be used to monitor morphological and molecular changes in wounds over time.

[0075] Figure 24H shows another example of the use of the device to monitor wound status over time. The imaging device is used to track changes in healing and bacterial biodistribution (e.g., contamination) of a wound on the left calf of a 21-year-old female patient with pyoderma gangrenosum. White-light images (a-i) and corresponding fluorescence images (b-j) of a wound being treated with hyperbaric oxygen therapy (HOT) are shown over the course of 6 weeks (fluorescence parameters: 405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)). a-i) The white-light images reveal distinct changes in the wound under the microscope as it heals, indicated by a reduction in size (e.g., cessation) over time from week 1 (~2 cm major diameter) to week 6 (~0.75 cm major diameter). In b-j), real-time fluorescence imaging of endogenous bacterial fluorescence (autofluorescence) within and near the wound can be tracked over time and correlated with white-light images and wound closure measurements (a-i). b) shows a distinct green band of fluorescence at the exact wound border (yellow arrow; indicating contamination with aggressive Staphylococcus aureus growth), which changes over time as the wound heals. Red-fluorescent bacteria are also seen further away from the wound (orange arrow), and their biodistribution changes over time (b-j). The wound-wound edge-normal tissue interface is clearly visible in fluorescence in image j). Connective tissue (in this example, collagen) in normal skin appears as light green fluorescence (j). Connective tissue remodeling during wound healing can be monitored over time during various wound treatments, including hyperbaric oxygen therapy for chronic wounds in this case.

[0076] Figure 24I shows the use of an imaging device to target bacterial swabs during routine wound assessment in a clinic. Under fluorescent imaging, the swab can be directed or targeted to specific bacterially contaminated / infected areas using real-time fluorescent image guidance. This reduces the potential for contamination of non-infected tissue by reducing bacterial dispersion during the routine swabbing procedure, which can be problematic with traditional wound swab sampling methods. The swab result for this sample was determined to be Staphylococcus aureus (confirmed microscopically with few gram-positive bacilli and sparse gram-positive cocci).

[0077] Figure 24J shows an example of co-registration of a) white-light images and b) corresponding fluorescence images generated using an imaging device in a patient with a non-healing foot ulcer associated with diabetes. Using a non-contact temperature measurement probe with cross-laser aiming (inset in a), direct temperature measurements were taken on normal skin (yellow "3 and 4") and within the foot ulcer (yellow "1 and 2") (confirmed by bacteriological culture to be infected with Pseudomonas aeruginosa). This demonstrates the ability to add temperature-based information to wound assessment during clinical examination. Infected wounds exhibit elevated temperatures, as shown by a mean of 34.45°C compared with 30.75°C on normal skin surfaces. These data demonstrate the potential for multimodality measurements, including white-light, fluorescence, and thermal information, to assess wound health / infection in real time. Note that both non-healing wounds on this patient's right leg contained (in addition to gram-positive cocci and gram-negative bacilli) aggressive growth of Pseudomonas aeruginosa, which in this example appears as bright green fluorescent areas within the wound (b).

[0078] Figure 24K shows an example of the use of an imaging device for monitoring pressure ulcers. a) A white-light image of the right foot of a Caucasian diabetic patient with a pressure ulcer taken with the imaging device is shown. b) The corresponding fluorescence image shows bright red fluorescence of bacteria not visible with standard white-light examination (yellow arrow). (Bacteriological results confirmed the presence of aggressive Staphylococcus aureus growth.) Necrotic skin appears as a white / pale light-green color (white arrow). Note the aggressive growth of Staphylococcus aureus near the margins of a non-healing open wound (yellow arrow). c) Fluorescence imaging of a topically applied silver antimicrobial wound dressing is shown. The imaging device may also be used to detect endogenous fluorescent signals from advanced wound care products (e.g., hydrogels, wound dressings, etc.) or from such products formulated with fluorescent dyes with emission wavelengths detectable by the device's imaging detector. The device may be used for image-guided delivery / application of advanced wound care treatment products and then monitoring their distribution and clearance over time.

[0079] Figure 24L shows an example of the use of the device for monitoring pressure ulcers. a) A white-light image of the right foot of a Caucasian diabetic patient with a pressure ulcer taken with the device is shown. b) The corresponding fluorescence image shows bright red fluorescent areas of bacteria at the wound edge (bacteriological results confirmed the presence of aggressive growth of Staphylococcus aureus, SA), and bright green fluorescent bacteria (bacteriological results confirmed the presence of aggressive growth of Pseudomonas aeruginosa, PA), both of which are invisible to standard white-light examination. c) Fluorescence spectroscopy of the wound revealed unique spectral differences between these two bacterial species: SA has a characteristic red (approximately 630 nm) autofluorescence emission peak, while PA lacks red fluorescence but has a strong green autofluorescence peak near 480 nm.

[0080] Figure 24M shows an example of the use of this device to monitor chronic, non-healing wounds. a) White-light images of a chronic, non-healing wound from a 44-year-old Black male patient with type II diabetes obtained with the imaging device are shown. Bacteria cannot be visualized by standard white-light visualization (a–g) used in traditional clinical wound examinations. b–h) Corresponding fluorescent images of the same wound (405 nm excitation, 500–550 nm emission (green), >600 nm emission (red)) are shown. This patient presented with multiple open, non-healing wounds. Swab cultures obtained from each wound area using fluorescent image guidance revealed intense growth of Pseudomonas aeruginosa (yellow arrows), which appears fluorescent green, and Serratia marcescens (circled), which appears fluorescent red (scale scale in cm).

[0081] FIG. 24N is a schematic diagram illustrating the use of a "calibration" target, which may be custom designed, multi-purpose, and / or disposable, for use during wound imaging with an imaging device. The strip, in this example, is adhesive and may include one or more combinations of spatial measurement tools (e.g., length scales), information barcodes for integrating patient-specific medical information, and concentration gradients of impregnated fluorescent dyes for real-time fluorescence image calibration during imaging. For the latter, multiple concentrations of various exogenous fluorescent dyes or other fluorescent agents (e.g., quantum dots) may be used for multiplexed fluorescence intensity calibration, for example, when two or more exogenous fluorescently labeled probes are used for tissue / cell / molecule-targeted molecular imaging of wounds in vivo.

[0082] Figure 24O shows an example of the use of an embodiment of the imaging device for monitoring bacteria, for example to monitor treatment response. a) Fluorescence spectroscopic image of a live / dead bacterial stain (i.e., BacLight product) sold by Invitrogen Corp. b) Invitrogen Fluorescence spectroscopic image of Gram stain bacterial marker stain sold by Biotech Corp. An imaging device (c) may be used with such a product to distinguish live (green) and dead (red) bacteria (e) in real time ex vivo (e.g., on a swab or tissue biopsy) following bacterial swabbing of a wound or other body surface, as in d), for example, in the use of a buccal swab in the oral cavity. Assessment based on this real-time bacterial Gram stain or live / dead image would be useful for real-time or relatively rapid bacteriological results that may be used to improve treatments, such as antibiotic or other antiseptic treatments, or to monitor therapeutic response.

[0083] Figure 24P shows an example of the device being used to image a toenail infection. A) White light and b) corresponding autofluorescence of a subject's right toe demonstrates that fluorescence imaging shows improved infection contrast compared to white light visualization (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).

[0084] Figure 24Q shows an example of imaging using the device to monitor the response of bacterially infected meat to disinfectants, such as hydrogen peroxide (Virox5™). a) Ex vivo porcine tissue samples were prepared in Petri dishes and b) contaminated with Staphylococcus aureus prior to topical administration of Virox5™ and fluorescence imaging (using a handheld device). c) Tissue degradation, triggered by the disinfectant, began to occur rapidly, while over time, particularly after gentle agitation of the sample, changes in the bacterial fluorescence characteristics became apparent (e.g., red fluorescence begins to change to orange fluorescence, as seen in d) after approximately 5 minutes of incubation in Virox5™ solution. These data suggest the use of the device for monitoring bacterial disinfection in clinical and non-clinical settings, for example (405 nm excitation; 490 nm to 550 nm and >600 nm emission).

[0085] In addition to fluorescence-enhancing prodrugs, advances in medicine have enabled the widespread use of fluorescent biomarkers to diagnose disease at the molecular level. Accurate measurement of fluorescent biomarker signals in living tissues may be a key parameter toward obtaining biomolecular information about disease progression and treatment response, but has historically posed significant challenges. To date, this type of advanced molecular imaging has not been reported in the context of wound care.

[0086] The devices described herein may further be used in combination with exogenous fluorescent imaging agents that are fluorescent, light scattering, or light absorbing, and that can be used passively and / or directed to unique and specific molecular targets within the wound to improve the detection and diagnosis of wound infection. These molecular targets may be any biological and / or molecular components in the wound or normal surrounding tissue (e.g., normal tissue and wound biomarkers) that have known detection and / or diagnostic value. All exogenous materials may be delivered to the wound locally and / or systemically, including, but not limited to, appropriate wavelength-selected fluorescent / scattering agents. The device may include any exogenous substance / drug (e.g., encapsulated liposomes, beads, or other biocompatible carrier materials) that can be coupled / conjugated with a disrupting moiety (e.g., organic fluorescent dyes, quantum dots and other fluorescent semiconductor nanoparticles, colloidal metals (e.g., gold, silver, etc.)). Fluorescent agents / probes and / or light scattering agents / probes, and / or chromogenic (i.e., absorbing) agents / dyes may be prepared using standard bioconjugation techniques to include moieties for targeting specific biomarkers. Such moieties may include monoclonal antibodies (e.g., whole and / or fragments) and other tissue-specific moieties (including, but not limited to, peptides, oligomers, aptamers, receptor-binding molecules, enzyme inhibitors, toxins, etc.). Furthermore, the device may be used to image the in situ, activatable, promoter-controlled expression of light-generating proteins in preclinical wound models. Furthermore, the imaging device may be used to detect wound infections and treat them using photothermal therapy, such as light-absorbing gold nanoparticles conjugated with specific antibodies that specifically target bacteria.

[0087] Figure 24R shows an example of the use of an imaging device for imaging fluorescent dyes / probes / agents on biological tissue. a) White-light imaging of a piece of flesh (ex vivo) shows no fluorescent dye, while in b) the device allows for accurate fluorescent detection and monitoring of the biodistribution of fluorescent dyes. While not shown for ex vivo tissue, these capabilities may be transferred to in vivo applications, including, but not limited to, imaging the biodistribution of fluorescent photosensitizers within tissue for photodynamic therapy (PDT) of wounds, cancer, infection, or other diseases. White-light imaging may provide anatomical context for fluorescence imaging. Furthermore, these capabilities may be used to monitor photobleaching of fluorescent agents (including photosensitizers) and for image-guided delivery of multiple PDT treatments (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)). The device may be used to monitor pharmacokinetics, biodistribution, and / or photobleaching during PDT. Similarly, the device may be useful for monitoring low level light therapy.

[0088] The device may also be used with other molecular sensors, such as "molecular beacons" or "smart probes," that fluoresce only in the presence of their own specific biological targets (e.g., enzymes associated with wound health). Such probes may be useful, for example, for identifying specific bacterial species or Gram stains. For example, skin wound healing is a highly complex process involving five overlapping stages (inflammation, granulation tissue formation, epithelialization, matrix production, and remodeling) associated with numerous migratory and remodeling events that are thought to require the action of matrix metalloproteinases (MMPs) and their inhibitors, TIMPs. In vivo analyses of human acute and chronic wounds and a variety of different wound healing models have implicated functional roles for MMPs and TIMPs during normal wound repair, while deregulation of their activity is thought to contribute to impaired wound healing. Extracellular matrix degradation is required to remove damaged tissue and provisional matrix, allowing for angiogenesis and re-epithelialization. In contrast, in chronic and non-healing wounds, overexpression of inactive proteinases is thought to contribute to the underlying pathology and inhibit normal tissue repair processes. Molecular beacons are activatable fluorescent reporters that control fluorescence emission in response to specific biological stimuli by using the fluorescence resonance energy transfer (FRET) principle. They typically contain a disease-specific linker that brings a quencher into proximity with the fluorophore, quenching its fluorescence. Upon specific linker-target interactions (e.g., nucleic acid hybridization, protease-specific peptide cleavage, phospholipase-specific phospholipid cleavage, etc.), the quencher is removed from the vicinity of the fluorophore, restoring its fluorescence. These smart probes may offer several orders of magnitude greater sensitivity than targeted probes due to their built-in high degree of signal amplification, ranging from non-fluorescent to highly fluorescent. Depending on their specific linker-target interactions, they can further influence protein or gene expression levels. In addition, smart probes may be able to detect specific molecular abnormalities in tissues. Because of these advantages, smart probes have recently been described as a "quantum leap" over conventional probes for early cancer detection. Such exogenous materials may be used, for example, for relatively rapid, non-invasive, sensitive, and specific optical wound infection detection, to identify specific bacterial / microbial species present and in situ microbial diagnostics, monitor wound hygiene, and report on the effectiveness of treatment and care in real time.

[0089] Additionally, when used in combination with exogenous optical agents, the device may be used to identify patients who are minimally responsive to various established and experimental therapies, allowing for rapid, non-invasive or non-contact visual quantitative assessment of treatment response, allowing timely changes in therapy to be made to optimize treatment outcomes.

[0090] Furthermore, imaging devices may be used to monitor antibacterial effects in real time in vitro and in animal model test systems, improving fundamental understanding of antibiotic action and facilitating original studies of disease in vivo.

[0091] Example Figure 5 shows an example in which the device was used for noninvasive autofluorescence detection of collagen and various bacterial species on the skin surface of a pork sample. In contrast to white-light imaging, autofluorescence imaging was able to detect the presence of several bacterial species (i.e., Streptococcus pyogenes, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa) 24 hours after topical application of these species to a small incision made in the skin. a) shows a white-light image of the pork used for testing. Several bacterial species were administered on day 0 through a small incision made in the skin and labeled as follows: 1) Streptococcus pyogenes, 2) Serratia marcescens, 3) Staphylococcus aureus, 4) Staphylococcus epidermidis, 5) Escherichia coli, and 6) Pseudomonas aeruginosa. Using an imaging system, collagen and bacterial autofluorescence were detected over time. Connective tissue fluorescence was also strong and easily detected. Some bacterial species (e.g., Pseudomonas aeruginosa) produced significant green fluorescence (450 nm to 505 nm) that saturated the system's camera. (b) shows the autofluorescence image on day 0, and (c) shows a magnified version.

[0092] The device was also able to detect the spread of bacteria across the surface of the meat over time. d) shows an image from the first day, and f) shows an image from the third day, with the meat sample maintained at 37°C. Red fluorescence is visible in some of the wound sites (5, 6) in c). As shown in d) and magnified in e), after 24 hours, the device detected a dramatic increase in bacterial autofluorescence from wound sites 5) Escherichia coli and 6) Pseudomonas aeruginosa, with the latter producing significant green and red autofluorescence. c) and e) show the device detecting wound surface fluorescence using a two-band filter (450 nm to 505 nm green and 590 nm to 650 nm) on the left and a single-band filter (635 ± 10 nm) on the right. As shown in f), by day 3, the device detects a significant increase in bacterial autofluorescence (in green and red) from other wound sites, as well as bacterial contamination on the Styrofoam container holding the meat sample (indicated by the arrow in f). The device was also able to detect bacterial spread across the surface of the meat. This demonstrates the device's ability to provide detection of bacterial species on mock wounds, their growth over time, and monitoring of bacterial growth in the wound over time. The device can provide important information regarding bacterial biodistribution on the wound surface, which may be useful for targeting bacterial swabs and tissue biopsies. d) and Note in a) and f) the strong green fluorescent signal from endogenous collagen at the edge of the pork sample.

[0093] This example demonstrates the use of the device to detect connective tissue biotransformation and bacterial growth in real time based solely on autofluorescence, suggesting the practical ability of the device to provide temporal monitoring of bacterial growth in wounds.

[0094] Reference is now made to Figure 6, which shows an example of the device being used to detect connective tissue (e.g., collagen, elastin) and bacterial autofluorescence on the muscle surface of a pork sample. In a), a white-light image of the pork used for testing shows no obvious signs of bacterial / microbial contamination or spoilage. However, as can be seen in b), imaging the same area with the device under blue / violet light excitation reveals bright red fluorescent areas of the muscle, indicating possible bacterial contamination compared to the adjacent side of the muscle. Extremely bright green autofluorescence of collagen is also seen at the skin edge. In c), the device was used to further surgically explore suspicious red fluorescence, providing targeted biopsies for subsequent pathology or bacteriology. Note also the device's ability to detect contamination of surgical instruments (e.g., forceps) (arrow) during surgery by fluorescence. In d), the device was used to target a collection of fluorescent spectra at a suspected bacterially infected area using a fiber-optic probe (the inset shows the device being used to advance the spectroscopic probe at the same area of ​​red-fluorescent muscle in b and c). In e), the device was used to detect contamination with various thin layers of bacteria on the surface of a Styrofoam container in which a meat sample was stored. Bacterial autofluorescence appears under violet / blue excitation light as stripes of green and red fluorescence from the various bacterial species previously applied to the meat. Thus, the device can detect bacteria on non-biological surfaces, even when they are invisible to the naked eye under standard white-light observation (as in a).

[0095] In addition to detecting bacteria in wounds and on the skin surface, the device can also identify areas of suspicious muscle tissue, which may then be further explored by surgical or targeted biopsy for pathological verification, or by other optical means such as fluorescence spectroscopy using a fiber optic probe. Furthermore, it detected contamination with various bacteria on the surface of a Styrofoam container in which meat samples were held. Bacterial autofluorescence appears under violet / blue excitation light as stripes of green and red fluorescence from the various bacterial species previously applied to the meat.

[0096] To determine the autofluorescence characteristics of bacteria growing in culture and simulated skin wounds, we quantitatively measured the fluorescence intensity spectra from the bacteria under violet / blue excitation light using hyperspectral / multispectral fluorescence imaging. See Figure 7. In Figure 7, we used this device to detect fluorescence from bacteria growing on agar plates and on the surface of simulated wounds in pork, as described above for Figures 4 and 5. Using this device, bacterial autofluorescence was detected in the green and red wavelength ranges in the culture (a) and meat sample (d). Hyperspectral / multispectral imaging was used to image bacteria (E. coli) in culture (b) and quantitatively measure the fluorescence intensity spectra from the bacteria (red line - porphyrin, green - cytoplasm, blue - agar background) (c). The red arrow indicates the 635 nm peak of porphyrin fluorescence detected in the bacteria. Hyperspectral / multispectral imaging also confirmed the strong green fluorescence from Pseudomonas aeruginosa (*, right rectangle in d) (with slight porphyrin fluorescence, yellow line in f) compared to E. coli (left rectangle in d), where significant porphyrin red fluorescence was detected. e) and g) are color-coded hyperspectral images corresponding to Pseudomonas aeruginosa and E. coli, respectively, from the surface of meat after 2 days of growth (incubated at 37°C). Figure 1 shows a multispectral image; Figures 2a and 2b show the corresponding color-coded fluorescence spectra. In Figure 1c, excitation-emission matrices (EEMs) were further measured for various bacterial species in solution, demonstrating the ability to select optimal excitation and emission wavelength bandwidths for use with optical filters in the imaging device. The EEM for Escherichia coli shows strong green fluorescence and significant red fluorescence from bacterial porphyrins derived from the body (arrows).

[0097] This example demonstrates that bacteria emit both green and red autofluorescence, with some species (e.g., Pseudomonas aeruginosa) producing more of the former. Escherichia coli produced significant red autofluorescence from endogenous porphyrins. Such inherent spectral differences between bacterial species are significant because they may provide a means to distinguish between different bacterial species using autofluorescence alone. Excitation-emission matrices (EEMs) were also measured for each of the bacterial species used in these pilot studies, confirming that under violet / blue light excitation, all species produced significant green and / or red fluorescence, the latter caused by porphyrins. Spectral information derived from the excitation-emission matrices may aid in optimizing the selection of excitation and emission wavelength bandwidths for use with optical filters in imaging devices, enabling differentiation between bacterial species ex vivo and in vivo. In this manner, the device may be used to detect subtle changes in the presence and quantity of endogenous connective tissue (e.g., collagen and elastin) and other microorganisms, such as bacteria and / or yeast, fungi and mold, within wounds and surrounding normal tissues based on the unique autofluorescence properties of these biological components.

[0098] In addition to fluorescence-enhancing prodrugs, advances in medicine have enabled the widespread use of fluorescent biomarkers to diagnose disease at the molecular level. Accurate measurement of fluorescent biomarker signals in biological tissues may be an important parameter toward obtaining biomolecular information about disease progression and treatment response, but has historically posed significant challenges. To date, this type of advanced molecular imaging has not been reported in the context of wound care. The device described herein will enable imaging and monitoring of such biomarkers for diagnostic purposes.

[0099] Imaging of wound models using exogenously derived contrast agents When used in wound assessment, tissue autofluorescence imaging may detect relative changes in connective tissue remodeling during wound healing, as well as the early presence of bacteria contaminating, colonizing, and / or infecting the wound (including, but not limited to, bacterially induced wound exudate and inflammation). When most wounds are illuminated with violet / blue light, endogenous tissues within the connective tissue matrix (e.g., collagen and elastin) emit a characteristic strong green fluorescent signal, while endogenous bacteria emit a unique red fluorescent signal due to the production of endogenous porphyrins. These bacteria include, but are not limited to, common species typically found at wound sites (e.g., Staphylococcus, Streptococcus, Escherichia coli, and Pseudomonas species). Autofluorescence may be used to obtain important wound information in real time, providing a means of early detection of key biological determinants of wound health, thereby assisting in patient stratification for wound treatment and management optimization.

[0100] The prodrug aminolevulinic acid (ALA) induces porphyrin formation in almost all living cells. Many bacterial species exposed to ALA can induce protoporphyrin IX (PpIX) fluorescence [Dietel et al., (2007). Journal of Photochemistry and Photobiology B: Biology. 86: 77-86]. To improve the red-to-green fluorescence contrast of bacteria using an imaging device, we investigated the use of ultra-low doses of ALA, which induce PpIX formation in bacteria and thus increase red fluorescence emission. Using this device, we obtained the following results: Violet / blue excitation light was used to image live bacterial cultures (Staphylococcus aureus grown on agar plates for 24 hours prior to imaging) as shown in Figure 8. Figure 8 shows the device in use in a bacteriology / culture laboratory.

[0101] In a), the device was used to image a live bacterial culture (S. aureus grown on an agar plate for 24 hours prior to imaging) under white light (circled). In b), violet / blue excitation light reveals red bacterial autofluorescence, which is distinguishable from the weak background green autofluorescence from the agar growth medium. In c), to increase the red-to-green fluorescence contrast of S. aureus against the background agar, an ultra-low dose (~20 μg / mL) of the photosensitizer aminolevulinic acid (ALA, in phosphate-buffered saline), commonly used in photodynamic therapy (PDT), was added locally to some of the colonies on the agar plate (marked "ALA+" in the circle), while the remainder of the agar plate was ALA-negative. After 30 minutes of incubation at 37°C, the agar plate was again imaged using the device under violet / blue light excitation, revealing a significant increase in red fluorescence (from ALA-induced protoporphyrin IX, PpIX) from Staphylococcus aureus bacteria compared to colonies that did not receive any ALA (boxed). Comparing b) to c) indicates that the addition of ALA may be beneficial in increasing bacterial fluorescence. d) shows the RBG image from c) in which the green fluorescence from the agar plate has been removed, thus revealing increased red bacterial fluorescence in the ALA-treated Staphylococcus aureus colonies. This demonstrates the device's ability to utilize contrast agent techniques to increase signal-to-background for sensitive bacterial detection, for example, in wounds. The time required for ALA to increase PpIX fluorescence to detectable levels was 30 minutes, indicating that this technique may be clinically practical. Furthermore, this indicates that the device may be used conveniently to image photosensitizer fluorescence (e.g., PpIX) in bacteria growing in culture or in a patient's wound for subsequent treatment with PDT.

[0102] After 30 minutes of incubation of Staphylococcus aureus with ~20 μg / mL ALA at 37°C, a significant increase in red fluorescence from the bacteria was detected compared to colonies (boxed) that did not receive any ALA. This demonstrates the device's ability to utilize contrast agent techniques to increase signal-to-background for sensitive bacterial detection, for example, in wounds. The time required for ALA to increase bacterial PpIX fluorescence in culture to detectable levels was approximately 0.5 hours, indicating that this technique may be clinically practical. Tests on simulated bacterially contaminated meat samples showed results similar to those obtained from bacterial cultures. Topical application of 0.2 μg / mL ALA by spraying onto wounds on pig skin resulted in a dramatic increase in the red fluorescence contrast of bacterial porphyrins approximately 2 hours after ALA administration. This will allow for the detection of bacterial contamination with fluorescence imaging within the wound site and elsewhere on the skin surface that was previously invisible to the naked eye with white light imaging, as shown with reference to Figures 9 and 10.

[0103] Figure 9 shows an example of the use of this device for detecting connective tissue and autofluorescence of various bacterial species on the skin surface of a pork sample. To determine whether the intensity of bacterial fluorescence could be enhanced for imaging with this device, the nontoxic prodrug aminolevulinic acid (ALA) (~0.2 mg / mL PBS) was applied topically to the skin surface by spraying with a standard spray bottle. The pork sample was then placed in a light-tight incubator at 37 °C for approximately 3-4 hours before white light and fluorescence imaging with this device.

[0104] Please refer to Figure 9. a) shows a white light image of the pork used for the test. b) Several bacterial species were applied to small incisions made in the skin [(1) Streptococcus pyogenes, (2) Serratia marcescens, (3) Staphylococcus aureus, (4) Staphylococcus epidermidis, (5) Escherichia coli, and (6) Pseudomonas aeruginosa]. Under violet / blue excitation light, the device shows bacterial autofluorescence (green and red fluorescence at the wound site). The presence of endogenous porphyrin red fluorescence can also be seen in other areas of the skin surface (red arrow). Bright collagen fluorescence can also be seen at the edge of the sample (blue arrow). Bacteria on the surface of a Styrofoam container holding a pork sample were also detected by autofluorescence using the device, but were invisible to the naked eye under white light (left panel). This demonstrates that the device can be used to detect and image the presence of bacteria or microorganisms and other pathogens on a variety of surfaces, materials, and instruments (such as surgical instruments) in hospitals, long-term care facilities, elderly care facilities, and other healthcare settings where contamination may be a major source of infection. The device may be used in conjunction with standard detection, identification, and enumeration of indicator organisms and pathogen response strategies.

[0105] In c), the nontoxic prodrug aminolevulinic acid (ALA) (0.2 μg / mL) was applied topically to the skin surface to determine whether bacterial fluorescence would increase. Approximately 1 hour after ALA administration, bacterial porphyrin fluorescence (bright red fluorescence) dramatically increased in both the skin tissue and wound site, as well as on the surface of the Styrofoam container holding the pork sample (arrows). This indicates the potential for using this device, for example, for fluorescence image-guided biopsy targeting and for detecting infected areas and subsequent treatment with PDT.

[0106] Figure 10 shows an example of the use of this device for detecting bacterial infection in pork samples with enhanced fluorescence contrast. (a) White-light images of pork are shown. Several bacterial species were applied to a small incision made in the skin (arrows). (b) The nontoxic prodrug aminolevulinic acid (ALA) (~0.2 mg / mL) was applied topically to the skin surface by spraying with a standard spray bottle, and the resulting red fluorescence of ALA-induced protoporphyrin IX (PpIX) was imaged using the imaging device. Imaging of the skin surface (~2 h after ALA administration) using violet / blue light (405 nm) resulted in a dramatic increase in the red fluorescence contrast of bacterial porphyrins, demonstrating the presence of bacterial contamination detected by fluorescence imaging within the mock surgical wound incision (arrow) and elsewhere on the skin surface (circled in a and b), which was previously invisible to the naked eye in white-light imaging. Note that areas of the skin surface that were not exposed to oxygen because the sample was placed "skin-side down" in the container do not emit bright red fluorescence, presumably due to the presumed oxygen dependence of bacterial PpIX production. Some bacteria produce a bright green fluorescent signal, which is also detected by the device. In c), in another pork sample, bacteria invisible to the naked eye in white-light imaging (circled) are easily detected by autofluorescence imaging alone (inset). However, as shown in d), topical application of low-dose ALA causes a dramatic increase in bacterial fluorescence after 2 hours, demonstrating the use of exogenously derived prodrugs as fluorescence imaging contrast enhancers to improve the detection of bacterial infections. Note the bright green autofluorescence of endogenous collagen and elastin in the connective tissue of the sample. In e) and f), ALA-induced fluorescence enabled the detection of bacteria invisible to the naked eye on the skin surface (circled), potentially enabling the use of the device for, for example, image-guided biopsy targeting and the detection of infected areas and subsequent treatment with PDT.

[0107] Furthermore, the device may be used in combination with exogenous "prodrug" agents, including but not limited to 5-ALA, which is approved by the FDA for clinical therapeutic indications, to increase the endogenous production of porphyrins in bacteria / microorganisms, thereby increasing the intensity of the unique "porphyrin" fluorescent signal emitted by these bacteria, which can be detected by the device. Sensitivity and specificity may be improved. Thus, the device may be used to conveniently image photosensitizer-induced fluorescence (e.g., PpIX) in bacteria growing in culture or in a patient's wound for subsequent treatment with image-guided targeted swab / biopsy or photodynamic therapy (PDT) [Jori et al. Lasers Surg Med. 2006 Jun; 38(5):468-81; Dougherty et al. (1998) J. Natl. Cancer Inst. 90, 889-905; Carruth (1998) Int. J. Clin. Pract. 52, 39-42; Bissonnette et al. (1997) Dermatol. Clin. 15, 507-519]. When antibiotics are no longer effective (e.g., drug-resistant strains), PDT may supplement or replace current antibiotic treatment. Available evidence suggests that multiply antibiotic-resistant strains are as readily killed by PDT as naive strains, and that bacteria may not readily develop resistance to PDT. This may be essential for wound care in patients undergoing cancer treatment, HIV patients with antibiotic resistance, and elderly people with persistent oral infections [Hamblin et al. (2004) Photochem Photobiol Sci. 3:436-50].

[0108] The device may be used to detect bacteria and microorganisms in wounds and surrounding normal tissues using low-power excitation / illumination blue / violet light, which may then be immediately destroyed using, for example, PDT or other therapies. High-power red excitation / illumination light may be used to destroy endogenous porphyrins in bacteria and microorganisms within the wound site using PDT. Thus, the device may have the potential to serve as an all-in-one, non-invasive or non-contact "detect and treat" instrument for clinical wound care. Furthermore, once bacteria or microorganisms are detected, the device may be used to treat and / or disinfect the wound site with PDT, and the wound site may be immediately re-imaged to determine the effectiveness of the PDT treatment. In some embodiments, the device may be used solely for detection / diagnostic purposes without any therapeutic treatment itself. The device may be used continuously until the entire wound and surrounding normal tissue is disinfected, after which the wound may be monitored over time as part of standard clinical follow-up. Fluorescence images from this device may be used to determine the biodistribution of PDT photosensitizers or photoproducts [Gudgin et al. (1995) J. Photochem. Photobiol. B: Biol. 29, 91-93; Konig et al. (1993) J. Photochem. Photobiol. B: Biol. 18, 287-290]. Because most of these are inherently fluorescent, the device may therefore serve as a means to target PDT treatment light. Thus, the device may guide, via imaging, the completion of the PDT treatment. Similarly, the device may be used to guide other therapies.

[0109] Some photosensitizers are known to photobleach [Jongen et al. (1997) Phys. Med. Biol. 42, 1701-1716; Georgakoudi et al. (1997) Photochem. Photobiol. 65, 135-144; Rhodes et al. (1997) J. Investig. Dermatol. 108, 87-91; Grossweiner (1986) Lasers Surg. Med. 6, 462-466; Robinson et al. (1998) Photochem. Photobiol. 67, 140-149; Rotomskis et al. (1996) J. Photochem. Photobiol. B: Biol. 33, 61-67], the fluorescence imaging capabilities of the device may be used to measure the extent or rate of photobleaching of photosensitizers. This information will be useful in optimizing PDT dosimetry to ensure adequate disease treatment while simultaneously minimizing damage to surrounding normal tissue [Grossweiner (1997) J. Photochem. Photobiol. B: Biol. 38, 258-268]. In certain embodiments, the device may be used with an excitation light source, which may be selected for a specific excitation wavelength and intensity, to deliver light for PDT in combination with any commercially available and / or experimental PDT photosensitizer. It may thus have utility in existing clinical applications of PDT (e.g., on the skin surface or hollow organs) and / or in commercial / academic research and development of future PDT photosensitizers, both preclinical and clinical.

[0110] Figure 10G shows an example of the use of the device to monitor bacterial response to photodynamic therapy (PDT). Ex vivo porcine tissue was prepared in a Petri dish and bioluminescently labeled (BL) After 24 hours of contamination with Staphylococcus aureus, BL and fluorescence imaging of the samples were performed using this device. Bioluminescence and corresponding fluorescence imaging were performed on a, d) uncontaminated muscle tissue, and b, e) SA-contaminated muscle tissue before and after PDT. Note that Staphylococcus aureus produced a red fluorescence color (white arrow in e). PDT was performed on the bacterially contaminated meat samples (marked with yellow circles) as follows: The samples were incubated with a common photosensitizer called methylene blue (MB) for approximately 30 minutes, followed by removal of excess MB (and rinsing with PBS), and then illuminated with a light source (here, an LED array) at approximately 670 nm for approximately 10 minutes at ~10 J / cm. 2 Photodynamic treatment was performed by irradiating with light. Comparison of the BL intensity scales in (e) and (f) showed a significant decrease in BL intensity in the treated meat samples after PDT (e.g., PDT killed a measurable proportion of bioluminescent bacteria, thus reducing BL signal intensity), and changes in the fluorescence properties (e.g., intensity and biodistribution) of Staphylococcus aureus bacteria (red) could be seen using a handheld imaging device after PDT. Note that the strong green fluorescence on the meat samples (pink arrow in (e)) was caused by unintentional cross-contamination of the meat samples with non-BL Pseudomonas aeruginosa during the experiment (bacteriologically confirmed), which was detected by the device. These data suggest the use of the device to monitor the use of PDT for the treatment of bacterial contamination in living (and non-living) samples. (Excitation at 405 nm; emission at 490 nm to 550 nm and >600 nm).

[0111] The device may be used as an imaging and / or monitoring device in a clinical microbiology laboratory. For example, the device may be used for quantitative imaging of bacterial colonies and for quantifying colony growth in common microbiology assays. Fluorescence imaging of bacterial colonies may be used to measure growth kinetics.

[0112] Imaging blood in wounds Angiogenesis, or the growth of new blood vessels, is a critical natural process required for wound healing and for restoring blood flow to tissues after injury or damage. Angiogenic therapies, designed to "turn on" new capillary growth, are revolutionizing medicine by providing a unified approach to the treatment of harmful and life-threatening conditions. Angiogenesis is a physiological process required for wound healing. Immediately after injury, angiogenesis is initiated by multiple molecular signals, including hemostatic factors, inflammation, cytokine growth factors, and cell-matrix interactions. New capillaries proliferate through a cascade of biological events, forming granulation tissue in the wound bed. This process may continue until the end-stage of the wound, when angiogenesis is halted by declining levels of growth factors, resolution of inflammation, stabilized tissue matrix, and endogenous angiogenesis inhibitors. Defects in the angiogenic pathway impair granulation and delay healing, which are evident in chronic wounds [Tonnesen et al. (2000) J Investig Dermatol Symp Proc. 5(1):40-6]. By illuminating the tissue surface with light of a selected narrow bandwidth (e.g., blue, green, and red components) or detecting the reflection of white light within some narrow bandwidth of the visible spectrum (e.g., wavelengths of peak absorption selected from the blood absorption spectrum of white light), the device may be used to image the presence of blood and microvascular networks within the wound and nearby, including surrounding normal tissue, thus revealing areas of erythema and inflammation.

[0113] Referring now to Figure 11, the device may demonstrate the possibility of imaging blood and microvasculature in a wound using individual optical filters (e.g., 405 nm, 546 nm, 600 nm, each ±25 nm). A white light image of the wound may be collected with the device, and then the device with a triple bandpass filter (e.g., 405 nm, 546 nm, 600 nm, each ±25 nm) may be placed in front of the imaging detector to image the reflected light components from the wound in separate narrow bandwidths of blue (B), green (G), and red (R). These wavelength bands are: Within the visible wavelength range, the wavelength may be selected based on the peak absorption wavelength of blood, including both oxygenated and reduced hemoglobin. The resulting image may provide the relative absorption and reflection of visible light by blood in the field of view. The resulting "blood absorption" image provides a high-contrast image of the presence of blood and / or microvascular networks in the wound and surrounding normal tissue. The clinician may select an appropriate optical filter set for use with the device to obtain an image of the blood and / or microvascular distribution within the wound and combine this information with either or both fluorescence imaging and imaging using an exogenous contrast agent. This may provide a comprehensive set of information about the wound and surrounding normal tissue at morphological, topographical, anatomical, physiological, biological, and molecular levels that may not currently be possible in conventional wound care practice.

[0114] Figure 11 shows an example of the device being used to image blood and microvasculature in a wound. The device was used to image a blood-stained piece of filter paper (a) and a mouse ear during surgery (b). White-light images of each specimen were collected using the device in nonfluorescence mode. Then, by equipping the device with a triple bandpass filter (e.g., 405 nm, 546 nm, 600 nm, ±25 nm each) placed in front of the imaging detector, the reflected light components from the specimen in separate narrow bandwidths of blue (B), green (G), and red (R). These wavelength bands were selected based on the peak absorption wavelength of blood within the visible wavelength range (inset in a). The absorption spectra characteristic of oxygenated and reduced hemoglobin in blood are shown. This demonstrates that it is possible to combine the three B, G, and R images into a single "white-light equivalent" image using a simple multiband pass filter to measure the relative absorption of light by blood in a field of view. The resulting "blood absorption" image provides a high contrast image showing the presence of blood containing both oxygenated and reduced hemoglobin. The device may also be used with narrower bandwidth filters to provide even higher contrast images of blood absorption, for example, in a wound.

[0115] The regulation of angiogenesis over time during wound repair in vivo has been largely unexplored due to the difficulty in observing events within blood vessels. Although initial testing of the imaging device was exploratory, simple modifications to existing prototypes may enable the time-lapse imaging of dynamic changes in the blood supply and microvascular network during the wound healing process in vivo.

[0116] Skin and oral cavity imaging The device may be suitable for imaging the skin, mouth, and oral cavity, and may enable detection of connective tissue changes due to minor skin injuries (e.g., cuts, abrasions, etc.) and endogenous bacteria (e.g., Propionibacterium acnes or P. acnes) commonly found in normal skin. .

[0117] The device may also be suitable for multispectral imaging and / or monitoring of dental calculus, caries, and / or cancer in the oral cavity. The device may be used to detect the presence of dental calculus, periodontal disease, caries, and cancer, as well as localized oral infections, based on the presence of unique autofluorescence characteristics in abnormal or cancerous tissue. The device may provide real-time detection and diagnosis of periodontal disease, calculus, caries, and cancer in the oral cavity using white-light imaging, fluorescence imaging with or without autofluorescence or exogenous fluorescent agents, and reflectance imaging. The device may record images for medical record cataloging. Unlike direct (i.e., naked-eye) observation approaches using existing products, such as the VELscope System by Vancouver-based LED Medical Diagnostics Inc. (LED-MD), the device may provide digital imaging and recording of white-light, fluorescence, and reflectance information from tissue.

[0118] In dermatology, the device may be used to detect bacteria on normal skin. For example, Figure 12 shows high-resolution autofluorescence imaging of normal skin on a patient's face, revealing distinct red fluorescence from the common bacterium Propionibacterium acnes.

[0119] Figure 12 shows an example of the use of this device for noninvasive, high-resolution digital still or video imaging of a patient's oral cavity and skin surface. As shown in a), this device can be used to image the mouth and oral cavity. Violet / blue light excitation excites autofluorescence from teeth, which appears as strong green fluorescence compared to blood-rich gums. Periodontal disease and caries can be easily detected using this device based on the autofluorescence of teeth and gum tissue. At the edge of the lip, red fluorescence from Propionibacterium acnes (P. acnes), which is commonly found within pores, is detected. This red fluorescence is caused by endogenous bacterial porphyrins. Note the detection of P. acnes in individual pores (red arrows) on the lip. Similarly, in b), red fluorescence from endogenous porphyrins in the normal tongue flora is easily detected as a bright red fluorescent "mask" on the tongue surface. Additionally, the device may be used to detect early cancers in the oral cavity based on differences in optical properties (e.g., absorption, scattering, autofluorescence, etc.) between normal tissue and pre-neoplastic and neoplastic tissue. The device may also be used to "scan" the oral cavity for mucosal cancers or to assess the effectiveness of anti-cancer treatments such as PDT or other therapies. Furthermore, the device may be used to image the skin surface. In c)-e), the device images the skin on a patient's face by detecting autofluorescence caused by violet / blue light excitation on the skin surface. Red fluorescence from P. acnes may be readily detected in the facial area (e). The device may also be used to image and / or monitor the potential effects of dermatological interventions (e.g., topical creams, medications, and other antibiotics) on a patient's skin. In f) and g), the device may also be used to image minor cuts (arrow h), scratches and abrasions, and psoriasis on a finger (arrow i) on a patient's skin. Under violet / blue light, the device detected tissue autofluorescence from connective tissue components (e.g., collagen and elastin) from the wound site and surrounding normal skin, providing high-resolution images of faint skin lesions. P. acnes, the causative agent of acne vulgaris (i.e., acne), normally resides in the pilosebaceous glands of human skin and is invisible to the naked eye under white-light visualization.These autofluorescence images were obtained without the need for exogenous substances / drugs, demonstrating the device's ability to detect bacterial porphyrin fluorescence even in single pores.

[0120] Figure 12J shows an example of the use of this device for real-time fluorescent detection of common bacterial flora on the skin. a) Red fluorescence from Propionibacterium acnes (P. acnes), commonly found in pores, is detected on and near the nose. b) Fluorescence imaging may be used to simultaneously detect two or more bacterial species on the skin; for example, Propionibacterium acnes appears as red fluorescence (red arrow), while P. aeruginosa appears as bright green (green arrow). These data suggest the use of this device to distinguish the relative concentrations / levels of various bacterial species, determine their biodistribution on the body surface, and monitor responses to antimicrobial treatments in dermatological and cosmetic applications. c) shows an example of a fluorescent image of a culture grown on agar from a swab sample obtained from normal skin on the nose of a healthy volunteer. Bacteriological results indicated the presence of P. aeruginosa.

[0121] Such an ability to image and document the presence and biodistribution of bacteria on the skin surface makes the device potentially useful in the dermatology and cosmetic fields, for example, for dermatological treatment and / or pharmaceutical / chemical treatment of normal and abnormal skin conditions, including but not limited to scarring, hyperpigmentation, acne, psoriasis, eczema, rashes, etc. Fluorescence imaging may be performed before, during, and after application of cosmetic formulations (e.g., topical creams, drugs and other antibiotics, skin disinfectants, acne treatments, etc.). Fluorescence / reflectance image-guided tattoo removal (e.g., using surgery or available laser treatments) may also be an option with this device. Using this device, minor cuts, scratches, and abrasions on a patient's skin were imaged, and tissue autofluorescence from connective tissue components (e.g., collagen and elastin) at the wound site and in the surrounding normal skin under violet / blue light assisted in detecting changes in connective tissue during healing of minor skin wounds that are invisible to the naked eye under white light (as shown in Figures 12h and 12i). Additionally, this device may further serve as a practical, cost-effective, and sensitive imaging-based tool for the noninvasive early detection of invisible skin cancers and noncancerous (i.e., benign) lesions [Chwirot et al. (1998) Eur J Cancer. 34(11):1730-4]. Next, the device It may be used to provide image guidance for surgical excision of lesions or for PDT. In the latter case, PDT response may be monitored by fluorescence imaging, and multiple image scans of the affected area over time may determine whether treatment is complete over time. The device may be used in real time to determine PDT photosensitizer localization and biodistribution and photobleaching, which may be mapped onto white-light images of the area to be treated for anatomical comparison. Changes in optical properties between normal tissue and diseased or burned tissue may be detected using both the white-light and fluorescence imaging capabilities of the device. The device may also be used to image, assess, and monitor the healing process in burns over time, or to determine the response of skin grafts or temporary skin replacements in the treatment of burn patients [Bishop (2004) Crit Care Nurs Clin North Am. 200416(1):145-77]. The device may be used to detect and monitor radiation-induced skin damage following treatment of a patient with ionizing radiation [Charles (2007) J Radiol Prot. 27(3):253-74].

[0122] Additionally, the device may be used to image the mouth and oral cavity, particularly in embodiments in which the device is small and compact. Pilot imaging studies have shown that the device may detect endogenous bacteria within the oral cavity (e.g., on the tongue surface and between the teeth above the gum line), suggesting use in the clinical detection of dental caries and periodontal disease [Pretty (2006) J Dent. 34(10):727-39]. Additionally, tissue autofluorescence has been shown to be useful in the detection of oral cancer. It has been shown [Kois et al. (2006) Dent Today. 25(10):94, 96-7]. The device may be used to detect early cancers in the oral cavity based on differences in optical properties (e.g., absorption, scattering, autofluorescence, etc.) between normal, pre-neoplastic, and neoplastic oral tissues. In addition, the device may be used to "scan" the oral cavity for mucosal cancers and monitor response to treatment.

[0123] In general, the device may be used to image and / or monitor targets such as skin targets, oral targets, ear, nose and throat targets, ocular targets, genital targets, anal targets, and any other suitable targets of a subject.

[0124] Use in malignant wounds Malignant wounds are also known as tumor necrosis, fungating wounds, ulcerated cancerous wounds, or malignant skin wounds. Malignant wounds can be an emotional and physical challenge for patients, families, and even experienced clinicians. Fungating and ulcerated wounds can be unsightly, foul-smelling, and painful. These wounds represent an advanced stage of disease and can become infected, leading to delayed / impaired healing and associated morbidity and a reduced quality of life for patients.

[0125] Many cancer patients live with the knowledge that their disease is progressive and incurable. For a very small minority of these people, this reality may manifest in the form of foul-smelling, exudative, necrotic skin lesions, which can be a constant physical reminder of the disease's progression (Mortimer PS. In: Doyle et al. editors. Oxford Textbook of Palliative Medicine, 1999). (2nd ed. Oxford: Oxford University Press, 1998, 617-27; Englund F. RCN Contact 1993; Winter: 2-3). These lesions are commonly known as "fungiotic wounds." The term "mycosis" refers to the malignant process of both ulcerative and proliferative growth (Grocott P. J Wound Care 1995; 4(5): 240-2). Lesions that ulcerate may develop into nodular "fungal" or "cauliflower" lesions, while ulcerating lesions result in wounds with a crater-like appearance (Grocott P. J Wound Care 1999, 8(5): 232-4; ​​Collier M. Nurs Times 1997; 93(44): suppl 1-4). Such lesions may also have a mixed appearance, with both proliferating and ulcerating areas (Young T. Community Nurse 1997; 3(9): 41-4).

[0126] A malignant wound may develop in one of the following ways: As a result of a primary skin tumor such as squamous cell carcinoma or melanoma. · Through direct invasion of skin structures by an underlying tumor, for example, breast cancer, or by a hematologic malignancy such as cutaneous T-cell lymphoma (mycosis fungoides). From the metastatic spread of distant tumors. Metastasis can occur along cell surfaces, capillaries, or lymphatic vessels.

[0127] Malignant wounds are often difficult to manage due to their location, odor, excessive exudate, and tendency to bleed. Each malignant wound may be unique in its appearance and the symptoms it presents. Common symptoms associated with malignant wounds include foul odor, excessive exudate, infection, bleeding, periwound skin separation and abrasion, pruritus, pain, and reduced aesthetic and cosmetic results due to wound dressings. Currently, approaches to care are holistic and primarily palliative, with the goals of managing symptoms at the wound site and reducing the impact of the wound on the patient's daily life by identifying bacterial / microbial infections and monitoring for signs of healing. If the condition is not controlled, these wounds are not expected to heal.

[0128] The above-described device may be useful for performing clinical evaluations of such wounds (e.g., physical examinations and biopsies). The device may provide thorough image-based wound assessments (i.e., longitudinal monitoring) at baseline and at regular intervals throughout treatment; wound assessment of wound location, size, and color, type, and amount of any exudate or drainage; and serial white-light (e.g., for color changes) and fluorescent (e.g., for histoarchitectural, cellular, biological, and molecular) images of chronic malignant wounds. The device may also provide real-time assessment of any signs and symptoms of infection that would affect treatment planning and efficacy. The device may be integrated into current clinical practice for the evaluation and treatment of such malignant wounds.

[0129] Imaging of exogenously derived fluorescent contrast agents The development of highly efficient analytical methods capable of probing biological systems at the systems level is a key challenge needed to meet the requirements of the emerging field of systems biology. Optical molecular imaging is an extremely powerful tool for studying the temporal and spatial dynamics of specific biomolecules and their interactions in real time in vivo. Several advances have been made in optical molecular imaging in recent years, including the development of molecular probes (e.g., FPs and semiconductor nanocrystals, also known as quantum dots) that make imaging brighter, more stable, and more biologically informative; the development of imaging strategies that provide higher resolution and greater tissue penetration; and applications for measuring biological events from the molecular to organism level. These advances may be applied to disease diagnosis (e.g., wound care) and drug screening. However, current fluorescence imaging devices involve large, complex, and expensive optical components and highly sensitive camera detectors, which make such systems prohibitively expensive. The device developed here is suitable for preclinical or research studies and the potential translation of such methods to clinical settings, where these cost-constrained systems are required. Provide an alternative to the

[0130] Referring now to Figure 13, the present device was used to image animals for whole-body observation under fluorescence and measure the degree of fluorescence from a BPD photosensitizer throughout the skin surface. Figure 13 demonstrates the applicability of the present device for real-time imaging and highly sensitive detection of exogenous fluorescent contrast agents in vivo (e.g., quantum dots, Qdots). In a), the present device was used to image an exogenous fluorescent contrast agent in a sacrificed rat bearing human breast tumor cells metastasized to the bones of its hind leg. The rat had previously been injected with a fluorescent photosensitizer called a benzoporphyrin derivative (BPD) for an unrelated photodynamic therapy experiment. The rat received subcutaneous injections of two separate fluorescent semiconductor nanoparticle solutions (here, Qdots) that fluoresce at 540 (±15) nm and 600 (±15) nm, respectively, into the left hind leg. The injections were performed approximately 1 cm apart. The rat's entire body was then imaged using the present device with violet / blue excitation light. The rat's skin appeared red. This was likely due to a combination of fluorescence from the benzoporphorin derivative (BPD) photosensitizer administered to the rats prior to the experiment for subsequent PDT, and dust and food contamination from the cages in which the rats were housed.

[0131] Still referring to Figure 13, in b), fluorescence from green and red Qdots (inset) was easily detected beneath the skin at the injection site. The red Qdots emitted a brighter signal, due to the greater tissue penetration of red light. c) A magnified image of the hind leg shown in b) is shown. The device was able to simultaneously detect multiple fluorescent contrast agents along with background tissue autofluorescence with sufficient signal-to-noise (green and red arrows), enabling preclinical and potential clinical in vivo fluorescence imaging of multiplexed molecularly targeted fluorescent contrast agents. Note that the green fluorescence is weaker than the red fluorescence. This is because both the violet / blue excitation light and the subsequent green Qdot fluorescence are preferentially absorbed by blood, while the red Qdot fluorescence light has a greater tissue penetration depth. In d), the device was used to image the animal for whole-body observation under fluorescence to measure the degree of BPD photosensitizer fluorescence throughout the skin surface. The device may be useful for guiding intravenous injections using a needle by detecting superficial blood vessels beneath the skin. Thus, the device may be used to detect fluorescent tumors, such as tumors transfected with fluorescent proteins and grown subcutaneously in xenograft or orthotopic models, and thus may be used for visualization of multiple wound healing and / or infectious biomarkers using multiplexed exogenous fluorescent molecular targeting agents (e.g., for in situ image-based bacteriology).

[0132] To improve the use of fluorescent contrast agents in preclinical research and ultimately for the translation of optical molecular imaging techniques into clinical practice, it is desirable to be able to relatively quickly distinguish and identify different fluorescent agents. In e) and f), the device was used as a means to relatively quickly identify which fluorescent contrast agent was in the syringe prior to injection, something that was not possible under standard white light. This demonstrates the utility of the device as a cost-effective fluorescent imaging-guided technique to rapidly provide useful information during fluorescent image-guided surgery and / or PDT procedures, where fluorescent compounds are commonly used, perhaps even in emerging wound care technologies.

[0133] Fluorescence image-guided surgery One emerging field is the use of fluorescence imaging for diagnostic screening and image-guided surgery. Overcoming the limitations of standard white-light surgery, fluorescence imaging may be used to assist in surgical resection of tumors in vivo based on fluorescence (e.g., autofluorescence or fluorescence from exogenous targeted / non-targeted contrast agents) and checking the completeness of tumor removal (e.g., clear margins). Fluorescence image-guided surgery has shown improved survival rates preclinically and clinically [Bogaards et al. (2004) Lasers Surg Med. 35:18 For example, in experimental surgery on rats, the device was used to measure the pressure at the surgical site. Standard white light imaging may be provided.

[0134] Referring now to Figure 14, several tests were performed to demonstrate the applicability of this device for fluorescence image-guided surgery in small animals. Experimental surgery was performed on a euthanized female rat using the imaging device. Figure 14 shows an example of the use of this device for fluorescence image-guided surgery using an imaging contrast agent. During the experimental surgery, the device provided standard white-light imaging of the surgical site, here the abdomen of a female rat (a). The surgeon used the device's display screen to guide the procedure and easily and quickly switched between white-light mode and fluorescence mode. In b), using violet / blue excitation light, the device provided additional contrast between different types of tissue that was not possible during white-light imaging. For example, connective tissue in the abdomen appeared bright green fluorescent (green arrow), while the skin surface (with a red-fluorescent photosensitizer (BPD)) appeared red (red arrow), and Qdots previously injected into the hind leg appeared bright red (blue arrow). Fluorescence imaging was used to detect contamination of surgical instruments and supplies (e.g., gauze, tape, blankets, etc.) during the surgical procedure. In c), the device further demonstrated utility by detecting soiled / contaminated surgical gauze during the surgical procedure. Compared to standard white light, where all gauze appeared clean, gauze used to clean the skin and surgical site during surgery appeared fluorescent red (left side) compared to clean gauze (right side).

[0135] The device was also used for real-time detection of exogenously derived fluorescent contrast agents in animal models (e.g., for labeled cell tracking and outcome in in vivo experiments and for cell engineering research in regenerative medicine). To this end, the device was used in fluorescence mode to image the presence of red fluorescent QDots injected into the myocardium and lungs of a rat during surgery (d). Under violet / blue excitation light, the red QDots could be easily detected in the heart (e) and lungs (f), appearing dark due to the high blood concentration in these organs. This demonstrates the device's utility for guiding and targeting biopsies or microsurgical procedures, particularly those aimed at cancer detection and removal (e.g., using autofluorescence or fluorescence contrast enhancement). Note the bright red fluorescence detected by the device from ingested food material in the colon. In g), the device demonstrated utility in imaging fluorescent tumor phantoms commonly used in small-animal imaging studies. Solid spherical polymer tumor phantoms of various sizes dosed with fluorescent dye were prepared and placed within the surgical site to demonstrate the device's ability to provide rapid "high-contrast" fluorescent imaging in small animal cancer models.

[0136] These results indicated that the device would be useful in detecting submillimeter-sized lesions under fluorescence guidance. It would be useful for targeting biopsies or microsurgical procedures, particularly those aimed at cancer detection and removal (e.g., using autofluorescence or fluorescence contrast enhancement). The device may also have utility in imaging fluorescent tumor phantoms commonly used in small animal imaging studies.

[0137] Figure 15 shows an example of the device used for video recording of the high-resolution fluorescence image-guided surgery of the rat in Figure 9. The device may be capable of providing both still digital images and video footage taken under standard white light (WL) (a) and fluorescence (FL) (b), which may be easily switched between. Here, the device was used to capture digital video of a surgical procedure in a rat using both white-light and fluorescence imaging. The surgeon used the device's digital display screen to guide the complete surgical procedure, using fluorescence when the white-light image did not provide sufficient information. In c)-e), for example, under violet / blue light excitation, fluorescence imaging provided the surgeon with significant image contrast between different types of tissue. Blood vessels were clearly visible under fluorescence, and connective tissue could be distinguished from the gastrointestinal tract. Digestive The device may also distinguish between ingested foods. The device may provide real-time imaging for image-guided surgical intervention or biopsy, allowing surgeons to make critical decisions during the surgical procedure. Capturing digital still and / or video images of the procedure allows for later analysis of the procedure for the patient's medical record and for future technical training of medical personnel. Additionally, the device may be used to record audio during the surgical procedure, thereby allowing a complete record of each procedure to be collected. The utility of the device has also been demonstrated as an extremely useful tool for image-guided minimally invasive microsurgery in animal and potentially human procedures.

[0138] Figure 16 shows an example of the device used for autofluorescence imaging-guided surgical resection of tissue in a mouse myocardial infarction model (a). During the experimental surgery, the device provided standard white-light (WL) imaging of the open surgical site, here the mouse's abdomen (b). The surgeon used the device's display screen to guide the procedure and easily and quickly switched between white-light and fluorescence modes. Using violet / blue excitation light, the device provided high contrast between different tissue types that was not possible during white-light imaging (c). For example, high-resolution autofluorescence imaging was used to visualize various internal organs. In d), intact animals can be imaged using fluorescence before and during surgery (e).

[0139] Figure 17 shows an example of the device being used for non-invasive, real-time autofluorescence image-guided surgery of a mouse brain. During the experimental surgery, the device provides standard white light (WL) imaging of the open surgical site (a), with the mouse skull visible. The surgeon can view the device's display screen (b). The procedure was guided using a microscope, allowing for easy and rapid switching between WL and fluorescence (FL) modes. (b) shows a view of the surgical site (here, an intact skull) provided by the imaging device under tissue autofluorescence. Note that the surgical area is dark, primarily due to the absorption of violet / blue excitation light and the resulting autofluorescence by blood. The nose and eyes appear bright red fluorescent compared to the bright green fluorescence from the fur. (c) shows the surgical site with the calvaria removed under WL, while (d) shows an autofluorescence image of the brain surface with the imaging device using violet / blue excitation light. Direct injection of an exogenous contrast agent (here, red fluorescent quantum dots) into the right hemisphere of the brain results in bright red fluorescence (arrow) (e). This demonstrates the utility of this device for imaging fluorescent contrast agents, particularly for high-resolution fluorescence image-guided surgery.

[0140] Use in clinical care While current wound management practices aim to reduce wound morbidity and mortality in patients, one limitation is the availability of medical resources. The possibility of incorporating telemedicine technology into wound care needs is currently being explored. Wound care represents care for chronic, debilitating conditions that require long-term, specialized nursing care. The significant effects of improved survival conditions and advances in medical care have led to people living longer worldwide. Thus, the proportion of elderly people and people worldwide with chronic medical conditions that will require medical attention is increasing. With rising medical costs and an industry pushing toward outpatient care, this is part of a medical crisis that requires immediate attention.

[0141] The device may provide biometric information about the wound and may utilize the emerging telemedicine (e.g., e-health) infrastructure to provide a solution for mobile wound care technology, which will have a significant impact on wound medical treatment. Wound care accounts for a large percentage of home visits performed by nurses and medical professionals. Despite the best techniques, some wounds do not heal as expected and require the attention of clinical professionals. The device described herein may enable access to specialized clinical resources to assist in the treatment of wounds from the convenience of the patient's home or long-term care facility, which would reduce client travel time, increase utilization of clinical wound professionals, and reduce costs to the healthcare system. .

[0142] Various uses of imaging devices have been discussed for wound assessment, monitoring, and nursing management. The devices can be used to detect and monitor changes in connective tissue (e.g., collagen, elastin) and blood / vascular supply during the wound healing process, to monitor tissue necrosis and exudate in wounds based on fluorescence, to detect and diagnose wound infection, potentially including providing important "clinically significant" classifications of bacterial and microbial presence at the wound surface and in deeper areas (e.g., to detect contamination, colonization, critical colonization, and infection) [Kingsley, Ostomy Wound Manage. 2003 Jul; 49(7A Suppl):1-7], and to monitor wound healing. It may provide topographical information about the wound and identify the wound edges and surrounding normal tissue. Tissue fluorescence and reflectance imaging data may be "mapped" onto a white-light image of the wound, thereby enabling visualization of essential wound biochemistry and photobiological (e.g., fluorescence) information within the wound and surrounding normal tissue, previously unavailable. Real-time imaging of the wound over time may monitor changes in wound healing and potentially monitor treatment effectiveness by providing useful information about underlying biological changes occurring at the tissue / cellular level (e.g., matrix remodeling, inflammation, infection, and necrosis). This may provide quantitative and objective wound information for detection, diagnosis, and treatment monitoring in patients. In particular, the device may be used to monitor and / or track the effectiveness of therapeutic treatments at the organismal level (i.e., at the bacterial level), thereby providing more information than monitoring microscopic / morphological appearance alone using white light.

[0143] The device may provide real-time, noninvasive, image-guided biopsy targeting, clinical procedure guidance, and tissue characterization, and may enable image-guided treatment using traditional and emerging modalities (e.g., PDT). Additionally, the imaging device may be used to correlate important biological and molecular wound information obtained through fluorescence (e.g., internally derived tissue autofluorescence and / or externally derived molecular biomarker-targeted fluorescent contrast agents) with existing and emerging clinical wound care assessment and treatment guides, such as NERDS and STONES proposed by Sibbald et al. (Sibbald et al. Increased Bacterial Burden and Infection: The Story of NERDS and STONES. ADV SKIN WOUND CARE 2006;19:447-61). Fluorescence imaging data obtained using the device may be used to spatially and spectrally characterize bacterial balance and burden at superficial and deep levels of the wound. The device may provide real-time, non-invasive, image-guided biopsy targeting, clinical procedure guidance, tissue characterization, and enable image-guided treatment using traditional and emerging modalities (e.g., photodynamic therapy, PDT). The device may be used in clinical settings and integrated with traditional clinical wound care procedures, and may have a distinct role in the field of infectious diseases. It is also noted that the device may be used for real-time analysis, monitoring, and treatment of chronic and acute wounds in animals and companion animals via traditional veterinary medicine.

[0144] The device may enable real-time wound healing assessment for a large, concurrent patient population base. Elderly, diabetic, immunosuppressed, and restrained individuals, in particular, are at increased risk of chronic wounds and other skin afflictions, such as bed sores, stasis ulcers, and diabetic ulcers, due to poor circulation and restraint. These chronic conditions significantly increase healthcare costs and reduce patients' quality of life. These populations are growing in number, creating a greater need for advanced wound care products. The device will impact patient care by enabling a cost-effective means of monitoring chronic and acute wounds in critical areas in hospitals, urgent care clinics, long-term care facilities, home health care, emergency rooms, and other healthcare facilities. Furthermore, such a "handheld" and portable imaging device would be convenient for nursing staff and ambulance crews. The device may be easily carried and used by crew members. Early identification of scarring and bacterial infections associated with connective tissue generation and remodeling in wounds, currently difficult, can be detected and appropriately treated. In addition, recent developments in advanced wound care products, including multiple types of wound dressings (e.g., films, hydrocolloids, foams, antimicrobials, alginates, and permeable), hydrogels, wound cleansers, debridements, tissue-engineered products (e.g., skin substitutes, substitutes, and tissue-engineered products, such as synthetic, polymer-based living tissue and growth factors), wound irrigation, pharmaceuticals, and physical therapy, may also benefit from the device developed herein, as it would enable image-based monitoring of the effectiveness of such treatments over time. Physical therapy may include hydrotherapy, electrical stimulation, electromagnetic stimulation devices, ultraviolet light therapy, hyperbaric oxygen therapy, ultrasound devices, laser / light-emitting diode (LED) devices, and wound imaging / documentation.

[0145] Wound tissue analysis is typically required for the evaluation of cutaneous wound healing. The proportion of granulation tissue, fibrin, and necrosis in a wound, as well as their changes during treatment, may provide useful information that may guide wound treatment. Image analysis may involve advanced statistical pattern recognition and classification algorithms to identify individual pixels within fluorescent wound images collected using the device based on optical information from the wound and surrounding normal tissue. In this way, image analysis may enable mapping of wound images to various components of the wound, including total wound area, epithelialization, granulation tissue, wound cover, necrotic, overgrown granulation tissue, infection, erosion, and surrounding tissue margins. This has the added benefit of providing a relatively rapid measurement of wound healing rate, informing patient management decision-making.

[0146] Figure 25 shows a planned management workflow for an imaging device in a clinical wound care setting. The device may be easily integrated into routine wound assessment, diagnosis, treatment, and response monitoring over time, providing important real-time biological and molecular information of the wound for rapid decision making during adaptive intervention.

[0147] The device may be easily integrated into existing medical computer infrastructure (such as desktop and pocket PCs used by a growing number of physicians and other medical professionals) for longitudinal image cataloging for patient wound management within traditional clinical settings. The device's wireless data reception and transmission capabilities may enable wound care monitoring and remote healing via existing and future wireless telemedicine infrastructure. The device may also be used to transmit vital medical data (e.g., wound health status) to remote locations via the Internet or wireless services such as cellular phone, PDA, or smartphone services, thereby enabling remote medical intervention and furthering its utility in military medical applications for battlefield wound management. The device may enable real-time surface imaging of the wound site and may be easily carried by medical personnel in close proximity to the patient in a clinical setting. Cost-effective, highly sensitive, commercially available digital imaging devices such as digital cameras, cellular phones, PDAs, laptop computers, tablet PCs, webcams, and smartphones may be used as image capture or recording components to provide image-based documentation of wound healing and treatment efficacy tracking. Furthermore, this technology may be adapted to function in a "wireless" mode, perhaps adapting it for use with high-resolution digital cameras embedded in commercially available cellular phones to enable remote medical intervention.

[0148] By utilizing a web-based telemedicine and telemedicine monitoring infrastructure, imaging devices may be integrated into a "store and forward" concept of wound assessment systems. In addition to providing digital images, such systems may present a comprehensive set of clinical data that meets the recommendations of clinical practice guidelines. The presently disclosed devices may be used to visualize and visualize wounds (e.g., images). The device may be incorporated into a computer-based wound assessment system (with image analysis software) and used in healthcare facilities to improve existing clinical databases and support the implementation of evidence-based practice guidelines. Such an integrated telehealth infrastructure may be used to monitor patients at home or in long-term care facilities who could benefit from routine monitoring by a qualified clinician but currently do not have access to such care. The device may be further developed into a portable, handheld, near-patient system, which would represent a major advance in detecting, monitoring, treating, and preventing the spread of infectious diseases in developed and developing countries. Such knowledge would greatly improve the diagnostic tools available to practitioners treating chronic wounds in settings where quantitative cultures are difficult to obtain.

[0149] The device may enable digital imaging using optical and digital zoom capabilities (e.g., those embedded in commonly available digital imaging devices). Still or video quality may be in "high definition" format to achieve spatially high resolution imaging of tissue surfaces. Images may be recorded as still / time-lapse frames and / or in video / moving format and may be printed using standard imaging printing protocols that require a PC (e.g., connected via USB) or do not require a PC (e.g., PictBridge). Image / video data may be transferred to a PC for data archiving and / or image display and / or analysis / manipulation. Additionally, the device may support wired or wireless capabilities (e.g., Bluetooth®). ) may be used to transfer the data to a printer or personal computer. Visualization may be performed on the screen of a handheld device and / or in addition to simultaneous display on a video screen / monitor (e.g., head-mounted display, eyeglasses, etc.) using a standard output video cable. The device may also enable quantitative measurement of distance over time (e.g., monitoring tissue morphology / topology changes) by displaying, combined or separately, optical wavelength and fluorescence / reflectance intensity information along with the spatial dimensions of the imaged scene. Furthermore, the device may enable digital image / video storage / cataloging of images and associated patient medical data, e.g., using dedicated software with imaging analysis capabilities and / or diagnostic algorithms.

[0150] Image analysis Image analysis may be used with the device to quantitatively measure the fluorescence intensity and relative changes in multiple fluorescence spectra (e.g., multiplexed imaging) of exogenously derived optical molecular targeting probes in wounds and surrounding normal tissues. The biodistribution of fluorescent probes may be determined based on collected fluorescent images, which may be monitored over time between individual clinical wound imaging sessions to see if there are any changes. By quantitatively measuring the presence and relative changes in abundance of each and every spectrally unique fluorescent probe using the device, clinical personnel may determine the health and / or healing status of a given wound and its response to treatment over time in real time or near real time, for example, by using a look-up table in which specific tissue, cellular, and molecular signals are displayed in correlation with the wound's health, healing, and response status, an example of which is shown in FIG. 21 (adapted from Bauer et al., Vasc & Endovasc Surg 2005, 39:4). This allows clinicians to perform tasks not otherwise possible using existing technology. Based on wax, biological and molecular information, it may be possible to determine whether a wound is healing. Furthermore, the presence and abundance of bacteria / microorganisms and their response to treatment may provide a means to adapt treatment in real time, instead of causing delays in response assessment that accompany traditional bacteriological testing of wound cultures.

[0151] Using image analysis technology, a portable device placed within the field of view during imaging with this device is A suitable fluorescent standard may be used to calibrate the initial or first image of the wound. Image analysis may enable a pseudo-color display on the monitor to differentiate between different biological (e.g., tissue, cellular, and molecular) components of the wound and surrounding normal tissue, including biomarkers identified by autofluorescence and those identified by the use of exogenous, targeted, or untargeted fluorescent / absorbent contrast agents.

[0152] Examples of such biomarkers are listed in Figure 22 (adapted from Brem et al. Journal of Clinical Investigation, 117:5, 2007) and shown in Figure 23. Figure 23 illustrates the mechanisms of wound healing in healthy individuals versus those with diabetic wounds. In healthy individuals (left), the acute wound healing process is induced and maintained through the integration of multiple molecular signals (e.g., in the form of cytokines and chemokines) released by keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. During wound-induced hypoxia, vascular endothelial growth factor (VEGF) released by macrophages, fibroblasts, and endothelial cells induces the phosphorylation and activation of eNOS in the bone marrow, resulting in increased NO levels, which triggers the mobilization of bone marrow EPCs into the circulation. For example, the chemokine SDF-1α promotes the homing of these EPCs to the injury site, where they participate in neovascularization. A diabetic rat model (right) In diabetic wounds, eNOS phosphorylation in the bone marrow is impaired, which directly limits EPC mobilization from the bone marrow into the circulation. SDF-1α expression is reduced in epithelial cells and myofibroblasts in diabetic wounds, which prevents EPC homing to the wound and therefore limits wound healing. Establishing a hyperoxic state in wound tissue (e.g., via HBO therapy) has been shown to activate multiple NOS isoforms, increase NO levels, and improve EPC mobilization to the circulation. However, local administration of SDF-1α was required to trigger the homing of these cells to the wound site. These results suggest that the combination of HBO therapy and SDF-1α administration may be a potential therapeutic option to accelerate diabetic wound healing, either alone or in combination with existing clinical protocols.

[0153] Pre-assigned color maps may be used to simultaneously display biological components of the wound and surrounding normal tissue, including connective tissue, blood, microvascularity, bacteria, microorganisms, etc., as well as fluorescently labeled drugs / agents, which may allow real-time or near real-time (e.g., less than one minute) visualization of the health, healing, and infection status of the wound area.

[0154] The image analysis algorithm may provide one or more of the following features: Patient digital image management Integration of various image acquisition devices Record all imaging parameters, including all exogenous fluorescent contrast agents Multiple scales and calibration environments Built-in spectral image unmixing and computational algorithms for quantitative measurement of tissue / bacterial autofluorescence and exogenous material fluorescence signals - Convenient annotation tools Digital archiving ·Web publication Basic Image Processing and Analysis Complete suite of image processing and quantitative analysis functions An image stitching algorithm allows a series of panoramic or overlapping images of a wound to be stitched into a single image in either an automated or manual mode. Easy to use measuring tools -Intuitive setting of processing parameters - Convenient manual editor Report Generation Powerful imaging report generator with professional templates that may be integrated into existing clinical reporting infrastructure or telemedicine / eHealth patient health data infrastructure. Reports may be exported to e.g. PDF, Word, Excel, etc.

[0155] A large library of automated solutions Customized automated solutions for wound assessment in various fields, including quantitative image analysis.

[0156] While image analysis algorithms, techniques or software have been described, this description also extends to computing devices, systems and methods for performing this image analysis.

[0157] Stem Cell Therapy and Cancer Monitoring The device may be used for imaging and detection of cancer in humans and / or animals. The device may be used to detect cancer based on the inherent differences in fluorescence properties between cancer and surrounding normal tissue in a patient. Additionally, the device may be used for image-based cancer detection in pets, for example, in a veterinary environment.

[0158] Additionally, the device may be used as a research tool for multispectral imaging and monitoring of cancer in experimental animal models of human disease (e.g., wounds or cancer). The device may be used to detect and / or image the presence of cancer and track tumor growth in animal models of cancer, particularly using fluorescent (e.g., in the visible and NIR wavelength ranges) protein-transfected tumor cell lines.

[0159] The device may be used in conjunction with both existing and emerging cell therapies useful for reconditioning chronic wounds and accelerating their healing. To this end, fluorescently labeled stem cells may be administered to the wound site prior to imaging with the device. Pluripotent stem cells (PSCs), the precursors to all more differentiated stem cells, are all involved in healing. Bone marrow can differentiate into a variety of cell types, including fibroblasts, endothelial cells, and keratinocytes, which are important cellular components of the body. A recent report of an uncontrolled clinical trial suggests that direct application of autologous bone marrow and its cultured cells may accelerate the healing of refractory chronic wounds (Badiavas et al., Arch Dermatol. 2003; 139(4): 510-16). Given the pathophysiological abnormalities present in chronic wounds, it is possible that stem cells may reconstitute the dermal, vascular, and other components required for optimal healing. The device may be used to visualize and track labeled stem cells over time at the wound site to determine their biodistribution and therapeutic efficacy. For example, the use of the exogenously derived fluorescent molecular targeted agents described above may confirm stem cell differentiation in vivo and further assist in determining the wound's response to this treatment.

[0160] For example, the device may be used to identify, track, and / or monitor cancer tumor stem cells and stem cells in general (e.g., in preclinical small animal experimental cancer models and other clinical models). An example is shown in the figure. Additionally, the device may be useful for imaging clinical cell therapy, including stem cell-based disease treatment.

[0161] We now refer to Figure 18. In a) we show the mouse model using white light. In b) we use a fluorescent imager to clearly see the individual organs of the mouse. In c) we show the mouse liver imaged using the device, no fluorescence is visible. In d) we show the mouse liver in white light. e) shows the lung of a mouse imaged using this device, with cancer tumor stem cells clearly visible as bright fluorescent dots.

[0162] Reference is now made to Figure 19. In a), the liver of the mouse model of Figure 18 is not visible in fluorescent imaging. b), d), and f) show different images showing the mouse lungs in white light. c), e), and g) show corresponding images showing the mouse lungs imaged using the device, clearly showing the cancer tumor stem cells as bright fluorescent dots.

[0163] Figure 19H shows an example of the use of this device for the detection of human ovarian tumor-bearing nude mice. a) White-light images of virus-treated and untreated control mice show the open abdominal cavity. b) Corresponding fluorescence images of treated and control mice show orange-red fluorescence from optically labeled virus in tumor nodules in the mesentery compared to controls (yellow arrows). c) A close-up of the mesentery is shown, and d) a comparison with control mice demonstrates the biodistribution of the viral optical probe within tumor nodules and the ability to detect submillimeter tumor nodules (blue arrows). Note that the probe fluorescence may be distinguished from background intestinal tissue autofluorescence. These data demonstrate the potential use of this device for imaging therapeutic responses, including but not limited to viral and cell therapy, and for image-guided surgical resection of fluorescent tumor specimens (c; inset) (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).

[0164] Figure 19I shows an example of the use of this device for detection / visualization in a nude mouse bearing a murine colon tumor that was administered a fluorescent cocktail of separate exogenously derived green and red tumor cell-targeting probes postoperatively. a) White-light image of the open abdominal cavity, and b) the corresponding multispectral fluorescence image, showing simultaneous detection of both green (green arrow) and red (red arrow) molecular probes, may be analyzed using spectral unmixing software. The device may also be modified to enable endoscopic imaging. In this example, c) a rigid endoscopic probe was attached to a handheld imaging device, and d) white-light and e) fluorescence images of surgically resected tissue from the mouse in images a and b) were obtained. These data suggest the use of this device with an endoscopic probe accessory for portable in vivo endoscopic real-time fluorescence imaging in human and veterinary patients for a variety of detection, diagnostic, or treatment monitoring applications (clinical and research-based). f) The device (e.g., with endoscopic capabilities) may be capable of fluorescent imaging of multiple spectrally unique "probes" that may be used in vivo (405 nm excitation; 490 nm to 550 nm and >600 nm emission channels).

[0165] The device may be used for multispectral imaging and detection of cancer in humans and animals. Additionally, the device may be used to detect cancer based on the inherent differences in fluorescence properties between such cancer and surrounding normal tissue in a patient. Furthermore, the device may be used for image-based cancer detection in animals, such as pets or livestock, within a veterinary environment, for example.

[0166] Additionally, the device may be suitable as a research tool for multispectral imaging and monitoring of cancer in experimental animal models of human disease (e.g., wounds and cancer). The device may be used to detect and / or image the presence of cancer and track tumor growth in animal models of cancer, particularly using fluorescent (e.g., in the visible and NIR wavelength ranges) protein-transfected tumor cell lines.

[0167] Image guidance The device further eliminates the use of dyes or markers, for example, in surgical procedures. may also be useful for providing fluorescence image guidance. Certain tissues and / or organs may have different fluorescence spectra (e.g., autofluorescence) when viewed with an imaging device or under certain excitation light conditions, for example.

[0168] Figure 20 shows the usefulness of this device for surgery assisted by fluorescence imaging. With the aid of fluorescence imaging using this device, various organs of the mouse model can be more clearly distinguished than under white light. b, c, and g show the mouse model under white light. a, d-f, and h-j show the mouse model imaged with this device.

[0169] Figure 20B shows an example of the use of this device to image a small animal model. Here, the dorsal skinfold window chamber of a mouse is imaged under white light (a, c) and fluorescence (b, d). Note the high-resolution white light and fluorescence images obtained with this device. The paws and face appear bright red fluorescent due to autofluorescence from the cage bedding and food powder materials (405 nm excitation; 490 nm to 550 nm and >600 nm emission channels).

[0170] bioengineered skin Several bioengineered skin products or skin equivalents are available on the market for the treatment of acute and chronic wounds and burns. These have been developed and tested in human wounds. Skin equivalents may contain living cells such as fibroblasts or keratinocytes or both, while others may consist of acellular material or extracts of living cells (Phillips. J Dermatol Surg Oncol 1993; 19(8): 794-800). These structures The clinical efficacy of these constructs is 15-20% better than conventional "control" therapies, although there is debate as to what constitutes adequate control. Bioengineered skin may work by delivering living cells, known as "smart materials," because they have the ability to adapt to their environment. There is evidence that some of these living constructs are able to release growth factors and cytokines (Falanga et al. J Invest Dermatol 2002; 119(3): 653-60). The association of exogenously derived fluorescent molecular substances with such skin substitutes has They may be used in the determination of graft integrity and the biological response of the wound to treatment. Healing of full-thickness skin defects may require extensive synthesis and reconstruction of dermal and epidermal components. Fibroblasts play a key role in this process and are being incorporated into the latest generation of artificial dermal substitutes.

[0171] The imaging device described herein may be used to determine the outcome of fibroblasts seeded into a skin substitute and the effect of the seeded fibroblasts on cell migration, and to determine dermal substitute degradation after implantation into a wound site. A wound may be treated with a dermal substitute seeded with autologous fibroblasts or an acellular substitute. The seeded fibroblasts, labeled with fluorescent cell markers, may then be detected in the wound with a fluorescence imaging device and then quantitatively evaluated using image analysis, for example, as described above.

[0172] Polymer-Based Therapeutics There are many commercially available medical polymer products designed for wound care. For example, Rimon Therapeutics manufactures Theramers™ (www.rimontherapeutics.com), medical polymers that are bioactive in and of themselves, without the use of drugs. Rimon Therapeutics offers wound care products that are manufactured to emit unique fluorescence when excited with 405 nm excitation light, including: Angiogenic Theramers™, which induce the development of new blood vessels (i.e., angiogenesis) in wounds or other ischemic tissue; MI Theramer™, which inhibits the activity of matrix metalloproteinases (MMPs), a ubiquitous group of enzymes implicated in numerous conditions in which mammalian cells are damaged or disrupted; AM Theramer™, a thermoplastic that kills Gram-positive and Gram-negative bacteria without harming cells; and AM Theramer™, a thermoplastic that reversibly transforms from a liquid to a strong gel at around body temperature. These are manufactured by the company with wavelengths of, for example, 405 nm and 405 nm respectively. The fluorescent dyes can be made to fluoresce with light of a longer wavelength by the addition of fluorescent dyes or fluorescent nanoparticles selected to be excited by longer wavelength fluorescent emissions.

[0173] Using an imaging device, the application of such fluorescent polymer agents may be guided in real time by fluorescence imaging. This allows for precise (e.g., topical) delivery / application of the Theramer agent to the wound site. After application of the agent to the wound, a fluorescence imaging device may then be used to determine the therapeutic effect of the Theramer on the wound and to noninvasively track its biodistribution in these wounds over time in vivo. Furthermore, molecular beacons, perhaps with different fluorescence emission wavelengths, that can fluoresce in the presence of wound enzymes (e.g., MMPs) may be added to the MI Theramer™, thereby indicating the wound's response to the MI Theramer™ in real time. It may be possible to use one fluorescence emission for image-guided application of the Theramer to the wound site, another different fluorescence emission for therapeutic response monitoring, and other fluorescence emissions for other measurements. The relative effectiveness of MMP inhibition and antimicrobial treatment may be determined simultaneously over time. Image analysis may allow for real-time comparison of the fluorescence changes of these signals in the wound. This adds a quantitative dimension to the device, increasing its clinical usefulness.

[0174] It should be noted that other custom, biosafe fluorescent agents may be added to the following materials currently used for wound care: The fluorescent materials may then be imaged and monitored using the present device.

[0175] Moist wound dressings: These provide a moist, promoting environment for better healing rates compared to conventional wound dressings. The primary consumer base targeted by manufacturers for these wound dressings is people aged 65 and above who suffer from chronic wounds such as pressure ulcers and stasis ulcers. People suffering from diabetes and the resulting advanced ulcers form part of the target population.

[0176] Hydrogels: These hydrate dry wounds, creating a favorable environment for faster healing. Their additional feature is that they may also be used in infected wounds. They are designed for dry to mildly exudative wounds.

[0177] Hydrocolloid wound dressings: Hydrocolloids seal the wound bed and prevent moisture loss. They form a gel when they absorb exudate, providing a moist healing environment. They are used for mild to moderately exudative wounds that are not infected.

[0178] Alginate wound dressings: These absorb wound exudate and form a gel that provides a moist environment for healing. They are primarily used for severely exuding wounds.

[0179] Foam wound dressings: These absorb wound exudates and maintain a moist wound surface, allowing for an environment conducive to wound healing. They are used for moderately exudative wounds.

[0180] Transparent film wound dressings: These are non-absorbent but allow water vapor to pass through, thus ensuring a moist wound surface. They are intended for dry to slightly exudative wounds. Examples include alginate foam transparent film wound dressings.

[0181] Antibacterial: These provide antibacterial action to disinfect wounds. Of particular importance is the use of nanocrystalline silver wound dressings. The extended release of silver significantly reduces the accumulation of bioburden, especially proteases and toxins released by bacteria that interfere with healing and cause pain and exudation.

[0182] Active wound dressings: These include highly advanced tissue-engineered products. Biomaterials and skin substitutes fall into this category; they consist entirely of biopolymers such as hyaluronic acid and collagen, or biopolymers combined with synthetic polymers such as nylon. These wound dressings actively promote wound healing by interacting directly or indirectly with the wound tissue. Skin substitutes are bioengineered devices that mimic the structure and function of skin.

[0183] Hyaluronic acid: It is a natural component of the extracellular matrix and plays an important role in the formation, re-epithelialization and remodeling of granulation tissue. Hyaluronic acid provides hydration to the skin and acts as an absorbent.

[0184] Other wound care products that may be imaged using the disclosed device include Theramer, silver-containing gels (e.g., hydrogels), artificial skin, ADD stem cells, anti-matrix metalloproteinase, and hyaluronic acid. Fluorescent agents may be added to other products to enable imaging with the device. In some cases, these products may already be luminescent and may not require the addition of fluorescent agents. The device may also be used to monitor the effectiveness of such treatment over time.

[0185] Application to food products The imaging device may be useful for monitoring contamination of food products (e.g., meat products). This may be useful, for example, in the preparation of food / animal products in the butcher, poultry, dairy, fishing, and agricultural industries. The device may be used as part of an integrated, multidisciplinary approach to analytical laboratory operations within this sector, thereby providing capabilities including image-based contamination detection and guidance to obtain specimens for testing. The device may be used for real-time detection, identification, and monitoring of bacterial and other microbial meat contamination / contamination levels in food products. It may also be used for bacterial contamination tracking in food processing plant environments, thus providing an image-based method for determining food safety and quality. In embodiments where the device is handheld, compact, and portable, the imaging device may be useful in food preparation areas to determine the safety of food products against bacterial / microbial contamination. The device may be used for relatively rapid detection and analysis of bacteria / microorganisms in collected or sampled meat specimens (and cooking surfaces) during processing and in finished food products, for example, as part of a food safety and quality control inspection process. The device may be used in conducting food safety testing / detection procedures to meet food safety and quality requirements in the butcher, horticulture, and aquaculture industries. The device may be used to detect food contaminants, such as those found in the butcher, poultry, and fishing industries. This technology may also be useful as a fecal contamination detection system, because fecal bacteria produce porphyrins that will be easily detected by the device.

[0186] The detection and accurate identification of foodborne pathogens, such as Listeria monocytogenes (LM), in food samples and processing lines can be important both to ensure food quality assurance and to track outbreaks of bacterial pathogens in the food supply. Current detection methods used in food manufacturing and processing facilities typically involve multiple random surface sampling (e.g., swabbing) of equipment followed by subsequent Current methods rely on molecular-based diagnostic assays (e.g., real-time polymerase chain reaction, RT-PCR), which typically provide quantitative confirmation of the presence of LM within 24-72 hours. However, due to time and cost constraints, typically only randomly selected zones of a given food production facility are tested for pathogen contamination at one time, and the significant potential for under-sampling during "first-pass" surface swabbing of equipment can result in undetected pathogens, potentially causing catastrophic health and economic problems. Additionally, there are limitations: i) the inability to rapidly sample all surface areas during "first-pass" swabbing to identify areas with a high probability of infection; ii) the inability to visually document this initial screening process (e.g., to date, no imaging methods are available); iii) delays in obtaining laboratory results; iv) the high costs associated with current methods; and v) More importantly, the potential for missing deadly pathogen infections has prompted efforts to improve early and accurate detection of foodborne pathogens in a cost-effective manner.

[0187] The device may be useful in providing a relatively rapid and accurate method for detecting such pathogens. Using highly specific gene probe technology, the device may be used in conjunction with assays for multicolor fluorescent probe "cocktails" (e.g., combinations of two or more imaging agents) that may unambiguously identify (and visualize) only viable L. monocytogenes from other Listeria species. This may allow for specific detection of live LM in real time, possibly minimizing the need for time-consuming standard enrichment. Furthermore, this method may be useful for detecting Enterobacter sakazakii, Campylobacter species (C. coli), and other pathogens. , Campylobacter jejuni (C. jejuni) and Campylobacter lari (C. lari) The imaging method may be expanded to include detection of other pathogens of interest, including coliform bacteria and bacteria of the Escherichia coli species (including lactose-negative and indol-negative Escherichia coli strains), Salmonella (genus), all bacteria belonging to the Staphylococcus aureus species, and, separately, all bacteria belonging to the Staphylococcus genus, and Pseudomonas aeruginosa. Other bacteria may be detectable by selecting a suitable probe or combination of suitable probes. For example, a combination of two or more imaging agents may be designed to be specific for a certain bacterium, resulting in a unique detectable fluorescence signature when imaged with an imaging device.

[0188] The imaging device (e.g., when combined with an applied exogenous bacteria-specific imaging agent, including a multi-target probe or probe combination) may be used for relatively rapid "first-pass" screening of food preparations and handling of surfaces for targeted swab sampling and microbial testing. The device may enable relatively rapid, image-based surveys of any surface of equipment and food products and may capture the fluorescent signatures of foodborne bacteria / pathogens in real time. The device may be used in conjunction with assays of multicolor fluorescent probe "cocktails" (and combinations thereof) that may unambiguously identify (and visualize) viable Listeria monocytogenes from other Listeria species, for example, using highly specific gene probe technology, as described above. Such probe "cocktails" may be designed to specifically target certain pathogens based on certain probe combinations known to be sensitive to such pathogens and known to give characteristic fluorescent responses. In addition to detecting such pathogens, the device may be able to distinguish the presence and / or location of different strains based on their distinct signature fluorescent responses.

[0189] Figure 26 shows an example of the use of imaging devices for real-time inspection of meat products in the food supply, where a) white light imaging and b) corresponding autofluorescence imaging of a piece of pork shows the differences between various tissues, including bone and tendon (white arrow), fat and muscle. c) white light imaging and b) corresponding autofluorescence imaging of a bone "edge notch", where Under fluorescence, cartilage (blue arrow) appears bright green due to collagen autofluorescence, while various types of internal bone tissue (red arrow), including bone marrow, can be distinguished using fluorescence. The latter observation further suggests the use of handheld optical imaging devices for real-time fluorescence image guidance during orthopedic surgery in human and veterinary patients, as described above (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).

[0190] Figure 27 shows an example of the use of the imaging device for real-time inspection of meat products in the food supply. Here, a) white light imaging and b) corresponding autofluorescence imaging of a piece of pork meat maintained at 37°C for 2 days are shown. The autofluorescence imaging indicates the presence of mixed bacterial contamination, including, for example, Staphylococcus aureus and Escherichia coli, on the surface of the meat (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).

[0191] Surface contamination The imaging device may be useful for detecting surface contamination, such as detecting "surface bacterial contamination" in healthcare environments. The device may be used to detect and image the presence of bacteria / microorganisms and other pathogens on various surfaces / substances / instruments (especially those related to surgical procedures) in hospitals, long-term care facilities, and elderly care facilities where contamination is a major source of infection. The device may be used in conjunction with standard detection, identification, and enumeration of indicator organisms and pathogen countermeasures.

[0192] Figure 28 shows an example of the use of the imaging device for real-time inspection of soil and algae samples in an example of environmental sampling / detection of contaminants. a) White-light image and b) corresponding autofluorescence image of a Petri dish containing soil and mineral samples. c) An example of the imaging device used to detect fluorescent soil contaminants / hazardous substances. Here, for example, a fluorescein-labeled fluid is added to the soil prior to fluorescence imaging to demonstrate the potential use of the imaging device for environmental contaminant detection and monitoring. d) An example of the imaging device used to obtain a white-light image and e) an autofluorescence image of a green algae culture grown under laboratory conditions, demonstrating the applicability of the device for real-time fluorescence image-based monitoring of water conditions (e.g., drinking water purification / safety testing, or algae growth in large manufacturing plants). As an example of an imaging device used to detect plant diseases, f) shows a white light image of a common household plant, while g) shows the corresponding autofluorescence image of a fungal infection affecting a plant leaf, which appears bright green (yellow arrow) compared to healthy leaf tissue, which appears bright reddish-brown (405 nm excitation, 500 nm to 550 nm emission (green), >600 nm emission (red)). Thus, this device may be useful for imaging plant-derived materials.

[0193] Figure 28B shows an example of the use of the imaging device for the detection of body fluid contamination invisible to the naked eye under white light in public and private environments. a) White light and b) corresponding autofluorescence of body fluids contaminating a toilet seat and a bathroom vanity top. These data suggest that the imaging device may be used to detect surface contamination with potentially harmful biological / infectious fluids / samples for image-guided targeted sampling, cleaning, or monitoring (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).

[0194] FIG. 28C shows an example of the use of the device for the detection of bacterial contamination (b; green arrow) of a surgical instrument using fluorescence imaging (405 nm excitation; 490 nm to 550 nm and >600 nm emission channels).

[0195] Forensic use The use of imaging devices to image surface contaminants and targets is a forensic application. For example, the device may be useful in forensic detection of latent fingerprints and body fluids on non-biological surfaces. The device may provide a relatively inexpensive, compact, and portable means for digitally imaging (e.g., in white light, fluorescence, and / or reflectance) latent fingerprints and body fluids, as well as other materials of forensic interest. The former may be made to fluoresce using commercially available fingerprint fluorescent dyes, while the latter may be made to fluoresce using fluid autofluorescence or exogenously applied "targeted" fluorescent dye agents (such as Luminol). ) may be used. Images may be recorded digitally. Additionally, the device may be used during autopsy procedures to detect contusions.

[0196] Figure 29 shows an example of the use of an imaging device for real-time fluorescent detection of liquid leaks using an exogenous fluorescent leak tracer dye. a) White-light image of a typical faucet; b) the corresponding fluorescent image showing the presence of leaking fluid (with fluorescent dye added); and a composite white-light and fluorescent image. Note that the leak (water in this example) is not visible under white light but is easily detected using fluorescence. These data suggest that the imaging device may be useful for relatively fast, image-based tracking and detection of liquid / fluid leaks (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).

[0197] Figure 30 shows an example of the use of an imaging device for real-time fluorescent detection of surface contaminants. a) White light image of a typical laboratory bench surface, and b) shows the area to be imaged using the imaging device. c) Fluorescence imaging may be used to detect contaminants that are not easily visualized under white light (a, b).

[0198] Additionally, imaging devices may be used to detect latent fingerprints, for example, by using fluorescent dyes to enhance fingerprint ridges on table surfaces. This may be done, for example, by including fluorescent dyes in combination with superglues (e.g., cyanoacrylates) to enhance fingerprint contrast against background surfaces. Far-red and near-infrared fluorescent dyes may be used to reduce the possibility of background autofluorescence. These data suggest the use of imaging devices (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)) for relatively rapid, image-based detection of non-biological and biological contaminants and fingerprints, for example, in forensic applications.

[0199] Furthermore, this device may be useful in anti-counterfeiting applications. Figure 31 shows an example in which the imaging device is used to image common currency (in this example, a $20 Canadian bill) in a) white light mode and b, c) autofluorescence mode (a). Special anti-counterfeiting features may be viewed under fluorescence, i.e., embedded fluorescent fibers (b) and embedded watermarks on the bill (c) may be spectrally distinguishable (arrows). These data suggest that this device may be used for anti-counterfeiting purposes (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)).

[0200] Cataloging The imaging device may also allow for the cataloging of animals, such as laboratory animals, based on fluorescence. Figure 32 shows an example of the use of an imaging device for real-time fluorescence detection of identification "barcode" tagging of laboratory animals. The figure shows a) a white light image of a typical laboratory rat, and b) a fluorescent image of a rat tagged with a fluorescent barcode. The use of multiple fluorescent dyes / colors in combination with barcode patterns / bars may be used, for example, for "multiplexed cataloging" of animals for longitudinal research studies. These data may be used, for example, for c) "pathogen contaminant" animal colonies in research laboratories, and for genotyping of animals (e.g., genetically modified animals, c). This paper suggests the use of the imaging device for barcode cataloging of laboratory animals based on relatively rapid, high-power images of the fluorescent protein (405 nm excitation, 500-550 nm emission (green), >600 nm emission (red)). Additionally, the device may be used for imaging of fluorescence-based barcoding or other coding systems in other applications, such as inventory tracking and point-of-sale tracking.

[0201] Kit for the device The imaging device may be provided, for example, in a kit including the device and a fluorescent imaging agent. The imaging agent may be any one or more of those described above. For example, if the kit is for a wound monitoring application, the imaging agent may be for labeling a biomarker in the wound.

[0202] Figure 33 shows an example of a kit including an imaging device. a) shows the handle and touch-sensitive display screen, and b) shows the external housing and excitation light source. The imaging device may be used to scan the body surface of both human and veterinary patients for image-based wound assessment or non-wound imaging applications. The device and any accessories (e.g., electrical / battery power, possible exogenous fluorescent contrast agents, etc.) may be conveniently packaged in a hard-case container for transport within clinical and non-clinical environments, including remote locations, home care environments, and research laboratory environments.

[0203] Cosmetic or dermatological uses Additionally, the imaging device may be used to image cosmetic or dermatological products.

[0204] Figure 34 shows an example of the use of the device for imaging cosmetics. For example, four commercially available cosmetic creams are shown in a) white light imaging mode and b) fluorescence imaging mode, showing the fluorescence contrast between the creams and background skin. These data demonstrate the potential use of the handheld imaging device for use in imaging the presence and possible biological effects of cosmetics (e.g., skin hydration, collagen remodeling, sun damage repair, skin peeling, etc.) and / or dermatological agents or drugs (405 nm excitation; 490 nm to 550 nm and >600 nm emission channels).

[0205] The imaging device may be used in white light and fluorescence modes to improve the administration of these treatments and to non-invasively and quantitatively monitor their effectiveness over time. The device may also be used in combination with other imaging modalities, such as thermal imaging, among others.

[0206] Additionally, the device may be used to test antimicrobial, antibiotic, or antiseptic agents. Fluorescence imaging provided by the device may be used, for example, in combination with white light imaging, to quantitatively detect the effectiveness of drug treatments during drug delivery, optimization, and evaluation in bacterial cultures and other model systems, for example, for wound treatment.

[0207] All examples and embodiments described herein are for illustrative purposes only and are not intended to be limiting. One of ordinary skill in the art will recognize that other variations are possible. All references mentioned are incorporated herein by reference in their entirety.

Claims

1. 1. A handheld, portable system for providing information regarding a wound in tissue, comprising: at least one excitation light source configured to illuminate the wound with excitation light, the excitation light comprising at least one wavelength or wavelength band that causes bacteria in the illuminated wound to fluoresce; a spectral filtering mechanism configured to allow passage of optical signals having wavelengths corresponding to bacterial autofluorescence and / or bacterial fluorescence in response to illumination of the wound, the spectral filtering mechanism including a plurality of selectable filters each corresponding to a different discrete spectral bandwidth, the spectral filtering mechanism including the plurality of selectable filters being separate and independent from the at least one excitation light source; The system further comprises: a mobile communication device having an image sensor for detecting the spectrally filtered optical signal; an element supporting the at least one excitation light source relative to the mobile communication device; a processor configured to receive electrical signals based on the detected spectrally filtered optical signals, spatially and / or temporally register one or more of the in vivo fluorescence data, the ex vivo fluorescence data, the absorbance data, and the reflectance data included in the electrical signals, identify a fluorescence signature of bacteria in the wound based at least in part on one or more of the in vivo fluorescence data, the ex vivo fluorescence data, the absorbance data, and the reflectance data included in the electrical signals, and output information regarding the wound in the treated tissue based on the fluorescence signature of the bacteria; the mobile communications device is a cellular phone, a smartphone, a tablet, or a personal digital assistant; the spectral filtering mechanism is configured to allow passage of optical signals having a plurality of wavelengths different from at least one wavelength or wavelength band of the excitation light emitted by the at least one excitation light source; The spectral filtering mechanism selects the plurality of selectable filters without changing the at least one excitation light source, and selection of the plurality of selectable filters enables detection corresponding to a fluorescent characteristic of the bacteria, while at least one wavelength or wavelength band of the excitation light emitted by the at least one excitation light source remains unchanged.

2. 10. The system of claim 1, further comprising a memory for storing fluorescence, absorbance and / or reflectance of biological components, tissue components, cellular components, molecular components, biomarkers and / or non-biological materials for comparison with fluorescence, absorbance and / or reflectance detected at and / or around the wound.

3. The system of claim 2 , wherein the memory is configured to store a look-up table of fluorescence, absorbance, and / or reflectance of biological components, tissue components, cellular components, molecular components, biomarkers, and / or non-biological materials.

4. 4. The system of claim 1, wherein the spectral filtering mechanism is further configured to allow passage of an optical signal having a wavelength corresponding to tissue autofluorescence in response to illumination of the wound, and wherein the output information about the treated wound includes at least one fluorescence characteristic of bacteria and tissue components present within the wound.

5. 5. The system of claim 4, wherein the processor is configured to analyze the at least one fluorescent characteristic to recognize, classify and / or quantify various components of the wound based on the received electrical signal or the output treated wound information.

6. The system of any one of claims 1 to 5, wherein the image sensor is configured to detect the spectrally filtered signal multiple times at predetermined intervals.

7. 7. The system of claim 1, further comprising a wireless data transfer port configured to transmit the information regarding the wound in the treated tissue based on the fluorescence properties of the bacteria to an external device.

8. The system of claim 7 , wherein the wireless data transfer port is configured to communicate via a Bluetooth protocol.

9. The system of any one of claims 1 to 8, wherein the at least one excitation light source is configured to emit excitation light having a wavelength between 400 nm and 450 nm.

10. 10. The system of claim 1, wherein the spectral filtering mechanism is configured to allow passage of optical signals having wavelengths within one or more of the following ranges: 405 nm±25 nm, 490 nm to 550 nm, and 590 nm to 650 nm.

11. 11. The system of claim 1, further comprising an element for supporting and positioning the at least one excitation light source relative to the mobile communication device, the element comprising a housing configured to receive at least a portion of the mobile communication device.

12. The system of any one of claims 1 to 11, further comprising a white light source for white light imaging.

13. 13. The system of claim 1, wherein the at least one excitation light source comprises a first excitation light source configured to emit excitation light having a first wavelength or wavelength band, and a second excitation light source configured to emit excitation light having a second wavelength or wavelength band, the first wavelength or wavelength band being different from the second wavelength or wavelength band.

14. 14. The system of any one of claims 1 to 13, wherein the processor is further configured to provide an indication of wound infection, and wherein the output information about the treated wound includes one or more of wound contamination information, wound colonization information, wound critical colonization information, wound infection information, bacterial membrane information, and bacterial strain information.

15. The system of any one of claims 1 to 14, wherein the spectral filtering mechanism further comprises one or more polarizing filters.

16. The system of any one of claims 1 to 14, wherein the at least one excitation light source comprises at least one polarizing filter.

17. 1. A method of operating a handheld, portable system for providing information regarding a wound in tissue, comprising: The handheld portable system includes at least one excitation light source that emits excitation light, the at least one excitation light source configured to illuminate the wound and including at least one wavelength or wavelength band that causes bacteria in the illuminated wound to fluoresce, and the method of operating the handheld portable system further includes: filtering the light signal emitted in response to illumination of the wound with the excitation light using a first filter of a plurality of selectable filters included in a spectral filtering mechanism configured to allow light signals having wavelengths corresponding to bacterial autofluorescence and / or bacterial fluorescence to pass through the spectral filtering mechanism, each of the plurality of selectable filters corresponding to a different discrete spectral bandwidth; the spectral filtering mechanism including the plurality of selectable filters is separate and independent from the at least one excitation light source; the spectral filtering mechanism is configured to allow passage of optical signals having a plurality of wavelengths different from at least one wavelength or wavelength band of the excitation light emitted by the at least one excitation light source; the spectral filtering mechanism selects the plurality of selectable filters without changing the at least one excitation light source, and selection of the plurality of selectable filters enables detection corresponding to a fluorescence characteristic of the bacteria, while at least one wavelength or wavelength band of the excitation light emitted by the at least one excitation light source remains unchanged; The method of operating the handheld portable system further comprises: detecting the spectrally filtered optical signal using an image sensor of the mobile communication device; a processor of the handheld portable system receiving an electrical signal based on the detected spectrally filtered optical signal; spatially and / or temporally registering one or more of in vivo fluorescence data, ex vivo fluorescence data, absorbance data, and reflectance data included in the electrical signal; identifying fluorescent characteristics of bacteria in and / or around the wound based at least in part on one or more of the in vivo fluorescence data, ex vivo fluorescence data, absorbance data, and reflectance data included in the electrical signal; and outputting information regarding the wound in the treated tissue based on the fluorescent characteristics of the bacteria.

18. 20. The method of claim 17, further comprising identifying, in the processor, at least one bacterial species based on a fluorescent characteristic of the bacteria.

19. 18. The method of operating a handheld portable system of claim 17, wherein identifying, in the processor, fluorescent signatures of bacteria in and / or around the wound based at least in part on one or more of the in vivo-derived fluorescent data, the ex vivo-derived fluorescent data, the absorbance data, and the reflectance data comprises identifying, in the processor, fluorescent signatures of a plurality of bacteria in and / or around the wound based at least in part on one or more of the in vivo-derived fluorescent data, the ex vivo-derived fluorescent data, the absorbance data, and the reflectance data.

20. 20. The method of operating a handheld portable system of claim 19, further comprising, in the processor, differentiating between bacterial strains present in and / or around the wound based on the fluorescent properties of the bacteria.

21. 20. The method of operating a handheld portable system of claim 17, further comprising, in the processor, providing an indication of wound infection, wherein the output information about the treated wound includes one or more of wound contamination information, wound colonization information, wound critical colonization information, wound infection information, bacterial membrane information, and bacterial strain information.

22. 20. The method of claim 17, wherein outputting the information regarding the wound in the treated tissue based on the fluorescence characteristics of the bacteria includes wirelessly transmitting the fluorescence characteristics of the bacteria to an external device.

23. The method of operating the handheld portable system further comprises: selecting a second filter from the plurality of selectable filters; filtering with the second filter an optical signal emitted in response to illuminating the wound with the excitation light; and detecting the signal spectrally filtered by the second filter with the image sensor of the mobile communication device.

24. 20. The method of claim 17, wherein the excitation light comprises one or more wavelengths in the ultraviolet range.

25. 20. The method of claim 17, wherein each of the plurality of selectable filters is a bandpass filter, a multi-band filter, an acousto-optic tunable filter, a liquid crystal tunable filter, or a continuously variable filter.

26. 20. The method of operating a handheld portable system of claim 17, wherein the excitation light source that emits excitation light for illuminating the wound of the handheld portable system includes at least one polarizing filter that polarizes the excitation light that illuminates the wound.

27. 18. The method of operating a handheld portable system of claim 17, wherein filtering the light signal emitted in response to illumination of the wound with the excitation light using a first of a plurality of selectable filters further comprises filtering the light signal emitted in response to illumination of the wound with the excitation light using a polarizing filter.

28. The at least one excitation light source for emitting excitation light for illuminating a wound of the handheld portable system includes: a first excitation light source configured to emit excitation light for illuminating the wound of the handheld portable system, the first excitation light source configured to emit excitation light having a first wavelength or wavelength band; The at least one excitation light source for emitting excitation light for illuminating a wound of the handheld portable system further comprises:

18. The method of operating a handheld portable system of claim 17, including a second excitation light source that emits excitation light for irradiating a wound of the handheld portable system, the second excitation light source configured to emit excitation light having a second wavelength or wavelength band, and the first wavelength or wavelength band is different from the second wavelength or wavelength band.

29. 1. A system for acquiring and displaying fluorescence-based information about a target, comprising: at least one light source configured to illuminate the target with excitation light, the emitted excitation light comprising a wavelength or wavelength band that causes bacteria associated with the target to fluoresce; a spectral filtering mechanism configured to detect fluorescence of the bacteria in response to illumination of the target; a processor for comparing the detected fluorescence spectrum of the bacteria associated with the target with a predetermined look-up table of fluorescence spectra of biomarkers; a display for displaying a result based on the comparison; the spectral filtering mechanism is configured to allow passage of an optical signal having a wavelength corresponding to bacterial autofluorescence and / or bacterial fluorescence in response to illumination of the target, the spectral filtering mechanism including a plurality of selectable filters each corresponding to a different discrete spectral bandwidth; the spectral filtering mechanism including the plurality of selectable filters is separate and independent from the at least one excitation light source; the spectral filtering mechanism is configured to allow passage of optical signals having a plurality of wavelengths different from at least one wavelength or wavelength band of the excitation light emitted by the at least one excitation light source; The spectral filtering mechanism selects the plurality of selectable filters without changing the at least one excitation light source, and selection of the plurality of selectable filters enables detection corresponding to a fluorescent characteristic of the bacteria, while at least one wavelength or wavelength band of the excitation light emitted by the at least one excitation light source remains unchanged.

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